Session Type Key

Hybrid Sessions
Virtual Sessions
In-Person Sessions
Reset

Advanced Filters

Food Macromolecules: Functionality, Health Benefits, Delivery Systems:
08:00am - 12:00pm USA / Canada - Pacific - March 20, 2022 | Room: Sapphire A/B (Hilton San Diego Bayfront)
Nitin Nitin, Organizer; Wallace Yokoyama, Organizer; Fang Zhong, Organizer, Presider
Division: [AGFD] Division of Agricultural and Food Chemistry
Session Type: Oral - Hybrid
Division/Committee: [AGFD] Division of Agricultural and Food Chemistry
Sunday
Introductory Remarks
08:00am - 08:05am USA / Canada - Pacific - March 20, 2022 | Room: Sapphire A/B (Hilton San Diego Bayfront)
Division: [AGFD] Division of Agricultural and Food Chemistry
Session Type: Oral - Hybrid

Sunday
3659027 - Industrially-scalable microencapsulation of bioactive ingredients in alginates with controlled-release properties
08:05am - 08:20am USA / Canada - Pacific - March 20, 2022 | Room: Sapphire A/B (Hilton San Diego Bayfront)
Division: [AGFD] Division of Agricultural and Food Chemistry
Session Type: Oral - Hybrid
Alginates are naturally-occurring, Generally Regarded As Safe (GRAS), anionic polysaccharides, that have long been known for its potential to microencapsulate food ingredients. Alginates rapidly gel under benign, ambient conditions, resulting in matrices that have enteric release properties. Commonly crosslinked with calcium, alginates incorporate the food ingredient in the alginate matrix, that can be triggered to fully release the payload with the addition of strong chelating agents. Alginates also form complex coacervation matrices with protein, forming matrix-microcapsules with pH-responsive gelation properties. Microencapsulation in alginates is easily executable at the lab bench scale, but the many steps and processes can be cost prohibitive to scale-up for commercial-scale production. This talk will describe industrially-scalable spray-drying and fluid-bed coating processes to microencapsulate bioactive ingredients in crosslinked alginates and complex coacervated alginate-protein matrices. Several examples of successes and challenges in the encapsulation of protein, peptides, oil emulsions, including volatile ingredients will be described.
One-step spray-dyring process to form crosslinked alginate microcapsules (CLAMs).

One-step spray-dyring process to form crosslinked alginate microcapsules (CLAMs).


Sunday
3651530 - Modulation of gut microbiota of broilers by controlled release of polyphenols and essential oils
08:20am - 08:35am USA / Canada - Pacific - March 20, 2022 | Room: Sapphire A/B (Hilton San Diego Bayfront)
Division: [AGFD] Division of Agricultural and Food Chemistry
Session Type: Oral - Hybrid
In the current study, polyphenols from grape pomace and essential oils, namely thymol and carvacrol, were encapsulated by water soluble yellow mustard mucilage (WSM), maltodextrin and gum Arabic at various ratios. The effect of wall materials on the controlled release of the bioactive core compounds were tested in vitro using simulated poultry intestinal digestion system. The optimal formula was further tested in vivo on broilers by evaluating the the effects on intestinal microbiota and growth performance. The encapsulated compounds were mixed with the bird's diet as feed additives and compared with antibiotic diet and control basal diet. The intestinal content was collected and ilium and cecal microbiota were analyzed using16S rRNA sequencing test. The in vitro study showed that the addition of the novel wall material WSM has significantly modified the releasing pattern of the bioactive compounds under gastrointestinal environment, where over 73% of bioactive compounds were delivered to the lower intestinal tract where most infectious bacteria are preferably located, while the particles without WSM had 49% available bioactive compounds left after exposed to the acidic gastro environment. The in vivo study has revealed that the incorporation of the encapsulated compounds has significantly modified the microbiota after 3 weeks of continuous ingestion, where the population of the infectious bacteria like Escherichia coli, Listeria monocytogenes, Staphylococcus aureus has significantly decreased, and bacterial diversity and the population of some representative probiotic bacteria like Lactobacillus have significantly increased (P< 0.05). Additionally the supplementation of encapsulated particles has significantly decreased the feed conversion rate (P<0.05) and increased the carcass weight. In conclusion, the encapsulated compounds were successfully delivered to the target location at lower intestinal tract thus improved poultry gut health and growth performance.
Sunday
3651848 - Effects of Pleurotus eryngii polysaccharide on gut inflammation and microbiota regulation
08:35am - 08:50am USA / Canada - Pacific - March 20, 2022 | Room: Sapphire A/B (Hilton San Diego Bayfront)
Division: [AGFD] Division of Agricultural and Food Chemistry
Session Type: Oral - Hybrid
Pleurotus eryngii is a widely consumed edible mushroom with high nutritional value, from which the P. eryngii polysaccharide (PEP) has been considered to contribute multiple heal-promoting effects. As research focuses on the relationship between gut microbiota and host health level, the biological activities of PEP through its interaction with gut microbiota have not been adequately studied. Hence, correlations between the health benefits and gut microbiota alteration induced by PEP were carried out in the present study. Firstly, PEP was isolated and chemically characterized. Simulated digestion and fermentation models were utilized and revealed that PEP was not decomposed during digestion in the stomach or small intestine but was degraded and utilized by colonic microbiota to produce a variety of short-chain fatty acids (SCFAs). Moreover, two newly identified PEP named WPEP and NPEP were purified from PEP, from which both these two polysaccharides could significantly inhibit LPS-induced inflammatory responses through the blocking of the activation of MAPK and NF-κB pathways. The inhibiting effects and the potential underlying mechanisms of WPEP were investigated. Results showed that WPEP significantly alleviated colitis symptoms and gut microbiota dysbiosis, such as decreased abundance of Akkermansia muciniphila and Clostridium cocleatum and increased abundance of Bifidobacterium pseudolongum, Lactobacillus reuteri, Lactobacillus salivarius, and Ruminococcus bromii. In summary, our results demonstrated the anti-inflammatory potential of PEP and the related mechanisms, indicating that PEP could be utilized as a functional food component in colitis management as well as a potential prebiotic agent to treat inflammation-related disorders.
Sunday
3651148 - Curcumin loaded metal-organic framework delivery systems
08:50am - 09:05am USA / Canada - Pacific - March 20, 2022 | Room: Sapphire A/B (Hilton San Diego Bayfront)
peihua ma, Presenter; Qin Wang
Division: [AGFD] Division of Agricultural and Food Chemistry
Session Type: Oral - Hybrid
Metal-organic frameworks (MOFs), a group of cutting-edge designable porous scaffolding materials attracted attention in reticular chemistry, which has potentials to satisfy fundamental demands for delivery systems. Here, three types of curcumin-loaded UiO-66 (representative high biocompatibility and water-stable MOFs) delivery systems, curcumin-loaded UiO-66, curcumin-loaded UiO-66 high internal-phase Pickering emulsions (HIPPE) and curcumin loaded UiO-66 functional film were prepared, named as curcmin@UiO-66, curcumin@UiO-66 HIPPE, and curcumin@UiO-66/Chitosan, respectively. The loading capacity for these three delivery systems was reached 3.45% w/w, 7.33%, and 26.18%, respectively. All systems were characterized using X-ray diffraction (XRD), physisorption analyzer, scanning electron microscopy (SEM), and energy-dispersive X-ray spectrometer (EDS), for crystallography, morphology, physicochemical properties, with computer assistant optimization with DFT and GCMC simulation for maximum loading capacity. The result showed that these systems all exhibit extremely high surface area and porosity, as well as strong thermal stability as evidenced by TGA results, which demonstrated great potentials for application as a food delivery system. These novel MOF nanoparticle stabilized delivery systems are expected to be useful for other bioactive components and antimicrobial agents, which would find applications in functional food, food safety, and biomedical areas in the future.
Sunday
3655987 - Ultrasonic encapsulation and delivery of functional foods
09:05am - 09:20am USA / Canada - Pacific - March 20, 2022 | Room: Sapphire A/B (Hilton San Diego Bayfront)
Division: [AGFD] Division of Agricultural and Food Chemistry
Session Type: Oral - Hybrid
The interaction between sound waves and microbubbles in solutions generates acoustic cavitation, the growth and collapse of bubbles, under certain experimental conditions. The near adiabatic collapse of microbubbles during acoustic cavitation generates extreme reaction conditions in liquids. Application of ultrasound in food and bioprocessing has attracted significant attention from food scientists and food industries. Ultrasonic encapsulation of nutrients and functional foods in a biopolymeric shell has been developed recently. The physical forces generated during acoustic cavitation process help to emulsify hydrophobic and hydrophilic nutrients and functional foods, stabilized by biopolymers. The oxidative radicals generated within cavitation bubbles are then used to crosslink the biopolymers forming a thin shell around the functional core. For example, the images of ultrasonically synthesized chitosan microspheres are shown. A non-polar liquid nutrient, ultrasonically encapsulated in the core of chitosan-shelled microspheres, can be seen in one of these images. The image of a hollow microsphere can be seen in the 2nd image. Such an encapsulation process results in protecting the core materials from degradation by oxygen and light during storage. This approach has generated the possibility of delivering nutrients using a variety of food matrix (example, yoghurt, fruit juice, milk, etc.). The fundamental aspects of ultrasonics and sonochemistry and their application for the encapsulation and delivery of functional foods will be discussed.
Scanning electron microscopic images of ultrasonically synthesized chitosan microspheres used for the encapsulation of functional foods.

Scanning electron microscopic images of ultrasonically synthesized chitosan microspheres used for the encapsulation of functional foods.


Sunday
3638797 - Development of wet media milled purple sweet potato particle-stabilized Pickering emulsions: the synergistic role of bioactives, starch and cellulose
09:20am - 09:35am USA / Canada - Pacific - March 20, 2022 | Room: Sapphire A/B (Hilton San Diego Bayfront)
Xuanxuan Lu, Presenter
Division: [AGFD] Division of Agricultural and Food Chemistry
Session Type: Oral - Hybrid
Most food-grade Pickering particles nowadays are prepared using purified single-compound substances as starting materials. However, to obtain these substances, complex extraction and purification procedures are often needed. Meanwhile, natural bioactive compounds existing in raw materials are lost. Herein, it is essential to develop a new strategy which could effectively fabricate sustainable and multifunctional particles directly from raw biomass materials. Purple sweet potatoes, rich in bioactive compounds, were used to produce purple sweet potato particles (PSPPs) via wet media milling. The effect of milling time on particle size, morphology, the content and antioxidative activity of bioactive compounds in PSPPs were investigated. Wet media milling could effectively decrease the particle size of PSPPs from 3371 ± 109.98 nm to 312 ± 18.4 nm after 240 min of milling. And significant amounts of bioactive compounds and their antioxidative activity were retained. Pickering emulsions (PSPP-Es) were successfully formed using PSPPs as Pickering stabilizers. The resulting PSPP-Es exhibited good freeze-thaw and heating stability. Low pH (3-6) and strong ionic strength (150 mM-600 mM) conditions contributed to smaller droplet size (10-12 µm) and better storage stability of PSPP-Es due to better complexation between phenolics and major components in PSPPs. Besides, PSPP-Es possessed better lipid oxidative stability than Tween 80-stabilized emulsion due to the presence of intrinsic antioxidants. This work would facilitate the development of Pickering stabilizers with outstanding emulsifying ability and functional properties directly from whole grain materials, and wide application of whole grain particle-stabilized Pickering emulsions in food, cosmetics and pharmaceutical industry.
Abstract figure

Abstract figure


Sunday
3644082 - Characterizing the role of network structure and filler/matrix interactions for tailored functionality in composite protein gels: Emulsion-filled gelatin as a case study
09:35am - 09:50am USA / Canada - Pacific - March 20, 2022 | Room: Sapphire A/B (Hilton San Diego Bayfront)
Division: [AGFD] Division of Agricultural and Food Chemistry
Session Type: Oral - Hybrid
Many food matrices can be viewed as emulsion-filled protein hydrogel composites, including various cheeses, comminuted meats, and yogurts. The functional and textural attributes of such products are strongly impacted by the properties of the embedded droplets, their distribution in space, and their interaction with the continuous protein matrix. This work addresses the effect of network microstructure, lipid physical state, and filler/matrix electrostatic interactions on the linear elastic properties of a model composite food system; emulsion-filled gelatin gels. Solid fat- and liquid oil-based whey protein isolate-stabilized emulsion droplets were used as filler particles (average droplet size 1-2 μm). The gels were prepared with varying pH, either above (pH 6; Gel-6) or below (pH 4; Gel-4) the isoelectric point of whey protein. Confocal micrographs demonstrated the emulsion droplets were homogeneously distributed throughout the Gel-6 composites, whereas the Gel-4 gels were dominated by heterogeneously distributed droplet-rich, protein-dense domains. For both systems, the elastic modulus increased with filler loading, with fat having a greater effect than oil; however, the reinforcement was substantially more pronounced in the heterogeneous Gel-4 composites. Increasing gelatin content (2-8wt% protein) reduced the extent of filler-induced reinforcement in both the fat- and oil-filled Gel-4 composites. This behaviour is not expected for rigid fillers, but could be rationalized based on stress-translation through the heterogeneous network. In contrast, the relative reinforcement of the Gel-6 gels was independent of gelatin content, which was attributed to site-specific filler/matrix interactions. We have proposed a micromechanistic model which more accurately describes the experimentally observed reinforcement in emulsion-filled gels than traditional particle-reinforcement theories. This model provides an intuitive, mechanistic reasoning for the observed mechanical response. Further developing these relationships will be critical for using structural design approaches to modify and design functional food systems, and understanding the role of filler/matrix interactions will be advantageous for adapting new and emerging protein sources.
Sunday
3650680 - Simultaneous loading and protection of bioactive compounds using protein-based assemblies
09:50am - 10:05am USA / Canada - Pacific - March 20, 2022 | Room: Sapphire A/B (Hilton San Diego Bayfront)
Li Liang, Presenter
Division: [AGFD] Division of Agricultural and Food Chemistry
Session Type: Oral - Hybrid
Bioactive compounds (BCs) possess health benefits, but they are not stable upon exposure to sensitive environments. It is thus necessary to encapsulate and protect BCs for their application in functional foods. There appear to be interesting market opportunities for functional foods fortified with a range of BCs and offering multiple health benefits. Co-administering different BCs may produce synergistic effect on their bioactivity and improved stability. These provide motivation to develop the carrier systems that can simultaneously load a plurality of BCs. BCs with different solubility were simultaneously bound to ligand-binding proteins, since the proteins have multiple binding sites. The co-loaded BCs were more stable than the single-loaded bioactive compound. Furthermore, BCs were co-encapsulated at the oil-water interface of emulsions and in the protein matrix in emulsion gels. In addition, it was also found that the BCs with different solubility could be co-loaded in protein-based homogenous particles, due to the smart partition of BCs. Food proteins were used as wall materials to encapsulate and protect multiple BCs, and the effects were improved by their combination with polysaccharides.
Sunday
Intermission
10:05am - 10:15am USA / Canada - Pacific - March 20, 2022 | Room: Sapphire A/B (Hilton San Diego Bayfront)
Division: [AGFD] Division of Agricultural and Food Chemistry
Session Type: Oral - Hybrid

Sunday
3657897 - Micro and nanoscale core-shell carriers for co-delivery of synergistic bioactives
10:15am - 10:30am USA / Canada - Pacific - March 20, 2022 | Room: Sapphire A/B (Hilton San Diego Bayfront)
Division: [AGFD] Division of Agricultural and Food Chemistry
Session Type: Oral - Hybrid
In recent years, the approach towards health and food has drastically changed with an enormous gain of knowledge about nutrition and medicine. The growing demand for customized functional foods and dietary supplements drives the market size for nutraceutical products which is poised to grow at a CAGR of 8.9% from 2020 to 2028. Nutraceutical compounds are nature-derived and are valued for their ability to promote quality of life, health, and longevity. Interestingly, the combination of certain nutraceuticals gives synergistic benefit for bioavailability enhancement, and to reduce the outset of inflammation and oxidation. The development of co-delivery carriers for these synergistic nutraceuticals is important to enhance their stability, processability, and bioavailability. Co-delivery carriers can be designed to alter the release profile of individual nutraceuticals to derive specific health benefits from synergism. The novel encapsulation approaches at scalable operations for co-delivery of synergistic compounds at micro and nanoscale is the demand for both and nutraceutical and pharma industry. Also, encapsulation approaches like matrix, multi-compartment, and core-shell structure are important to alter the release profile and stability of encapsulated bioactives. This talk will provide insight on micro and nanoscale core-shell carrier development using food macromolecules for two potent synergistic nutraceuticals (curcumin and resveratrol). Core-shell alginate microparticles of ~ 8µm using three fluid nozzle spray drying approach and zein-PEG and Zein-ethyl cellulose core-shell nanoparticles (~300 nm) by the electrohydrodynamic operation were developed. The characteristics of core-shell encapsulate to alter the properties of encapsulated bioactives, release profile, bioavailability enhancement and the scalability of the process will be discussed. These particles enhanced the intestinal permeability of resveratrol by 2.2-fold and curcumin by 3.5-fold, compared to its native form. Moreover, customization of these nutraceuticals carriers in a 3D printed food matrix for personalized delivery of synergistic nutraceuticals will be presented.
Sunday
3653092 - Chitin nanofiber stabilized Pickering emulsions with potential in encapsulation of antibacterial essential oils
10:30am - 10:45am USA / Canada - Pacific - March 20, 2022 | Room: Sapphire A/B (Hilton San Diego Bayfront)
Division: [AGFD] Division of Agricultural and Food Chemistry
Session Type: Oral - Hybrid
With increasing consumption demand on products with lower synthetic contents and less environmental risk, novel preservation techniques utilizing natural antimicrobials have become the new trend in the food industry. In the present work, surfactant-free essential oil o/w emulsions were successfully fabricated from Cinnamon cassia oil with partially deacetylated chitin nanofiber (ChNF) adopted as a Pickering stabilizer. The emulsions displayed high stability against coalescence during a storage period of 90 days. The possible mechanisms for the emulsion stabilization were suggested to be the ChNF adsorption at the oil-water interfaces and the subsequent formation of an inter-droplet network by self-assembly of ChNF in the continuous phase, which was further strengthened by the formation of imine bond by the reaction between aldehyde groups from the cinnamon oil and amino groups on ChNF. Furthermore, the in vitro experiment confirmed the antimicrobial performance of the emulsions against E. coli. Although the addition of ChNF seemed to reduce the bactericidal kinetics of the emulsion, enhanced diffusion efficiency of the emulsions were observed on agar disc when compared to that of the pure cassia oil, which would be beneficial for its application in aqueous medium. Meanwhile, controllable release of the cassia oil with extended shelf time was also achieved through the ChNF encapsulation. The present work evidences the potential of biopolymer nanoparticles as both emulsion stabilizers and encapsulation materials for essential oil delivery, which might find their application in food preservation, drug delivery and related field.
Sunday
3656217 - Storage stability and loss of resveratrol in protein particles: loading, antioxidant activity and oxidation sensitivity
10:45am - 11:00am USA / Canada - Pacific - March 20, 2022 | Room: Sapphire A/B (Hilton San Diego Bayfront)
Xin Yin, Presenter; Li Liang
Division: [AGFD] Division of Agricultural and Food Chemistry
Session Type: Oral - Hybrid
Whey protein isolate (WPI), sodium caseinate (SC) and soy protein isolate (SPI) were used as model carriers to complex with resveratrol in the form of nanoparticles. Their effects on the storage stability of resveratrol were studied in terms of antioxidant activity, polyphenol loading capacity and protein oxidation. The antioxidant activity of proteins and load efficiency of resveratrol ranked in the order of SC>SPI>WPI, which were inconsistent with their impact on the polyphenol storage stability. The effect of proteins on the storage stability of resveratrol was mainly dependent on their oxidation. The oxidation degree of proteins ranked in order SPI>SC>WPI. The co-oxidation of resveratrol with SPI and SC occurred during storage. The oxidation of WPI was the least and not affected by resveratrol. WPI improved the storage stability resveratrol, but SPI accelerated the loss of resveratrol, while the impact of SC on resveratrol stability basically changed from protective to harmful effect. The data gathered here should help guide the design of protein-based carriers for the protection of polyphenols.

Sunday
3657668 - Fabrication of hollow zein composite particles with sodium tripolyphosphate for the encapsulation of quercetin
11:00am - 11:15am USA / Canada - Pacific - March 20, 2022 | Room: Sapphire A/B (Hilton San Diego Bayfront)
Muhammad Aslam Khan, Presenter; Li Liang, Presenter
Division: [AGFD] Division of Agricultural and Food Chemistry
Session Type: Oral - Hybrid
Hollow zein particles emerged as an alternative to solid particles for the encapsulation and delivery of food bioactives due to the remarkable encapsulation and sustained release capabilities of bioactives. To date, hollow zein particles were fabricated with sodium carbonate templet and employed to encapsulate and deliver bioactives, which are more resistant to degradation in the high alkaline condition caused by the disintegrated sodium carbonate templet. Therefore, there is an urgent need to explore an alternative to sodium carbonate templet. Herein, we proposed sodium tripolyphosphate (STP) as a sacrificial templet to fabricate bare and composite hollow zein particles. TEM micrograph showed a distinct lighter hollow core with a darker shell. The size of hollow zein particles developed using STP was concentration-dependent, and the particles having the smallest diameter of 105 nm were obtained at 1.5 mg/mL of STP with a ζ-potential of -27 mV compared to solid particles with 172 nm and -35 mV prepared under identical conditions. Upon coating with casein/chitosan complex, the size of both hollow and solid particles increased to 142 and 196 nm, with a reversal of ζ-potential to 43 and 40 mV, respectively. The encapsulation efficiency of quercetin in bare and coated hollow zein particles was about 29 and 24% higher than that of solid ones. Moreover, the loading capacity of quercetin in hollow and solid zein particles was 3.8 and 2.5 %, respectively. These findings suggest that the hollow zein particles fabricated employing novel sacrificial templet expand their application as carriers for the encapsulation and delivery of delicate bioactive compounds in functional foods.
Sunday
3661780 - Rapid visible light-mediated crosslinking of casein-based hydrogels to facilitate wound healing
11:15am - 11:30am USA / Canada - Pacific - March 20, 2022 | Room: Sapphire A/B (Hilton San Diego Bayfront)
Division: [AGFD] Division of Agricultural and Food Chemistry
Session Type: Oral - Hybrid
At present, wound infection is a crucial health problem for people. Wound infection can not only hinder healing but also result in serious complications. Hydrogel dressings normally provide an appropriate and humid environment to expedite cell thriving and wound healing, while the wound can still keep active and exudation can be exhausted. In this study, casein, which was be biocompatible, biodegradable, renewable, readily available, inexpensive and non-toxic, was chosen as the raw material for the synthesis of hydrogel. The di-tyrosine bond was formed in casein to synthesize hydrogels by a visible light-mediated photoredox system. The physicochemical properties of the casein-based hydrogels were investigated by gelation kinetics, scanning electron microscopy (SEM) observation, infrared spectra analysis, mechanics analysis, adhesion test and swelling ratio test. This photomediated system rapidly crosslinks casein (<1 min), allowing 3D printing of constructs with high resolution features, in the range of 50-100 μm. The results showed that the hydrogels can quickly stanch after short tail and shorten the wound healing time of skin defect in mice. This demonstrates that the hydrogels hold potential for hemostasis and wound healing therapy.
Sunday
3667355 - Casein-propylene glycol alginate complexes: Formation, stability, and properties for preparation of high internal phase emulsions
11:30am - 11:45am USA / Canada - Pacific - March 20, 2022 | Room: Sapphire A/B (Hilton San Diego Bayfront)
Nan Li, Presenter; Qixin Zhong
Division: [AGFD] Division of Agricultural and Food Chemistry
Session Type: Oral - Hybrid
Protein-polysaccharide nanocomplexes are commonly studied to improve the acidic stability and have recently been studied for formation of high internal phase emulsions with an oil volume fraction of higher than 73%. In the present paper, we present recent studies on casein-propylene glycol alginate (PGA) complexes formed with both non-covalent and covalent interactions. A pH-cycle method was used to prepare casein- PGA complexes by first increasing pH to 11-12, followed by acidification to pH 7.0 or 4.5, near pH 4.6 - the isoelectric point of caseins. At alkaline pH, hydrolysis of ester bonds of PGA led to formation of alginate and covalent casein-alginate conjugates via the transacylation reaction. The covalent conjugate and remainder casein and alginate formed nanocomplexes, and the properties of nanocomplexes were a function of the casein:PGA mass ratio studied from 4:1 to 1:2. At 1% w/v casein, a greater extent of conjugation was observed at a higher content of PGA, and the increased dispersion clarity was observed at both pH 7.0 and 4.5. The hydrodynamic diameter at pH 7.0 was smaller at a lower content of PGA, whereas the hydrodynamic diameter at pH 4.5 was the smallest when the casein:PGA mass ratio was 2:1. Morphology analysis further suggested the importance of particle mass density to dispersion clarity. Molecular force analyses suggested that both covalent and non-covalent (mainly electrostatic and hydrophobic) interactions contributed to complex formation and dispersion stability. The complexes formed with casein:PGA mass ratio of 2:1 were capable of stabilizing emulsions with 80% (v/v) soybean oil without macroscopic phase separation during 7-d storage at room temperature. The high internal phase emulsion had the gel-like structure visually which was supported by rheological and microscopy data. Casein-PGA complexes may find unique applications in protein beverages and emulsion products such as mayonnaises.
Sunday
3667379 - High protein content casein-alginate conjugates produced by transacylation reaction as novel emulsifiers
11:45am - 12:00pm USA / Canada - Pacific - March 20, 2022 | Room: Sapphire A/B (Hilton San Diego Bayfront)
Nan Li, Presenter; Qixin Zhong
Division: [AGFD] Division of Agricultural and Food Chemistry
Session Type: Oral - Hybrid
Polypeptide-containing polysaccharides such as gum arabic are emulsifiers but have the limited polypeptide content (typically <10%). Although Maillard-type conjugates with a relatively high content of proteins have shown the improved emulsifying properties, Maillard reaction is time-consuming and produces numerous undesirable byproducts, even after the conjugation. In our work, covalent casein-alginate conjugates were synthesized by the transacylation reaction, and the purified conjugates had exceptional properties emulsifying and stabilizing corn oil. Conjugation was done by mixing 1% w/v propylene glycol alginate (PGA) and different amounts of sodium caseinate (NaCas) for 2 h at pH 11.0. After dialysis to remove free molecules, NaCas-alginate conjugates with 52.8%, 66.2%, and 76.5% NaCas were prepared at NaCas:PGA mass ratios of 1:2, 1:1, and 2:1, respectively. The purified conjugates were capable of emulsifying corn oil at a low surfactant-to-oil ratio (SOR) of 0.75:100 (w:v), and the emulsions were stable at a wide range of pH and ionic strength, as well as after thermal pasteurization. A higher content of NaCas in the conjugate resulted in smaller oil droplets, whereas the alginate content was critical to the stability of oil droplets through electrostatic and steric mechanisms. To further illustrate structure-function correlations, individual α-, β-, and κ-caseins were conjugated with alginate, corresponding to protein content of 39.17%, 37.78%, and 23.14%, respectively. Similar to the NaCas conjugates, emulsions with a low SOR of 1:100 (w:v) were stabilized by individual casein conjugates, with smaller droplets at a higher protein content of conjugates. The emulsions had much lower SORs than those utilizing Maillard-type conjugates (SOR = 1:21-1:4) and did not have the undesirable color. Since the transacylation reaction is fast, is easy to scale-up, and does not produce byproducts, the casein-alginate conjugates have the potential for use as novel emulsifiers with tunable properties.
ACS Award in Analytical Chemistry: Héctor D. Abruña:
08:00am - 11:55am USA / Canada - Pacific - March 20, 2022 | Room: Room 30D (San Diego Convention Center)
Danielle Hanes, Organizer; Carlos Cabrera, Presider
Division: [ANYL] Division of Analytical Chemistry
Session Type: Oral - Hybrid
Division/Committee: [ANYL] Division of Analytical Chemistry

Symposium to recognize the contributions of Prof. H. D. Abruña

Sunday
Introductory remarks
08:00am - 08:05am USA / Canada - Pacific - March 20, 2022 | Room: Room 30D (San Diego Convention Center)
Division: [ANYL] Division of Analytical Chemistry
Session Type: Oral - Hybrid

Sunday
3694987 - Energy conversion and storage: Novel materials and operando methods
08:05am - 08:35am USA / Canada - Pacific - March 20, 2022 | Room: Room 30D (San Diego Convention Center)
Hector Abruna, Presenter
Division: [ANYL] Division of Analytical Chemistry
Session Type: Oral - Hybrid
This presentation will deal with the development of new materials and operando methods for energy conversion and storage with emphasis on fuel cells and battery materials and technologies. The presentation will begin with a brief overview of the methods employed. Particular emphasis will be placed on the use of X-ray diffraction (XRD), X-ray absorption spectroscopy (XAS) X-ray microscopy and tomography and transmission electron microscopy (TEM) under active potential control. The utility of these methods will be illustrated by selected examples including electrocatalysts for the oxygen reduction reaction (ORR), hydrogen oxidation reaction (HOR) and spectroscopic studies of Li/S batteries and Li metal deposition and dendritic growth and the use of organic materials in electrical energy storage applications. The presentation will conclude with an assessment of future directions.
Sunday
3703135 - Electrochemical biosensing strip for telomerase activity detection
08:35am - 09:00am USA / Canada - Pacific - March 20, 2022 | Room: Room 30D (San Diego Convention Center)
Division: [ANYL] Division of Analytical Chemistry
Session Type: Oral - Hybrid
Electrochemical impedance spectroscopy (EIS) is a widely proposed technology in biosensors, but its use in cancer cell detection has been limited. BIDEA LLC’s biosensing strip (BBS) is an innovative technology capable of detecting the presence of telomerase activity (TA) using EIS. Telomerase is an epithelial cancer cell biomarker, which can be used in early cancer detection, prognosis and/or subsequent monitoring of residual cancer disease. This enzyme elongates telomeres during cell division, restoring their length and avoiding cell death. Telomerase is also responsible for the immortalization of cancer cells. The BBS was modified by immobilizing a telomere-like DNA single-strand (ssDNA) for TA detection in uterine biopsy samples. This ssDNA sequence is designed to be recognized explicitly by telomerase and reduces the probability of interference. When BBS is exposed to a telomerase-positive extract, telomerase binds and elongates the probe, blocking electronic and capacitive processes on its interdigtal electrode surface. This resistance to charge transfer (Rct) change is detected by measuring the impedance using EIS, and it is correlated to TA, which indicates the presence of cancer cells. Data obtained in this study demonstrate significant changes in Rct after exposure to telomerase-positive extract in contrast to minor changes exhibited in a telomerase-negative extract. A limit of detection of 2.94 x 104 cells/mL was reached using a whole-cell telomerase extraction protocol. A preliminary study, with a small set of uterine biopsy samples suggests the feasibility of using the changes in Rct, at BBS, to distinguish positive from negative cancer samples based of TA levels.
Sunday
3695146 - 25 Years of in situ/operando studies of electrochemical systems and interfaces
09:00am - 09:25am USA / Canada - Pacific - March 20, 2022 | Room: Room 30D (San Diego Convention Center)
Joel Brock, Presenter
Division: [ANYL] Division of Analytical Chemistry
Session Type: Oral - Hybrid
The penetrating power of high-energy x-rays and the brightness of modern synchrotron sources makes them uniquely suited for atomic-scale structural characterization of in situ/operando systems. Over the past 25 years, advances in x-ray technology (sources, optics, detectors) coupled with careful, systematic development of electrochemical cells and advances in realistic simulations of electrochemical interfaces have positioned researchers to make and test precise predictions and to develop new materials and devices. In this talk, I will review several of the key milestones and Professor Abruña’s pivotal role in these developments.
Sunday
3695217 - Architected control of local effects in electrochemistry
09:25am - 09:50am USA / Canada - Pacific - March 20, 2022 | Room: Room 30D (San Diego Convention Center)
Debra Rolison, Presenter
Division: [ANYL] Division of Analytical Chemistry
Session Type: Oral - Hybrid
Pore–solid architectures in which the redox-active solid and void are co-continuous amplify the addressable electrified interface per geometric area, simplifying analytical characterization. Hundreds of square centimeters per square centimeter of footprint makes that an inevitability, but it also innately distributes reaction fronts in space imparting more uniform current distribution. We use such architected electrodes because their structural characteristics ensure (i) interconnected pathways that maintain long-range electronic conductivity within the electrode volume; (ii) ionic and molecular flux approaching open-medium diffusion through the pore network; and (iii) confined internal void volume that controls precipitation/dissolution dynamics and product redistribution. Such architected electrodes physically impose a means to control undesirable morphological changes that too often accompany local electrochemistry during charge/discharge cycling of batteries. As examples of this architectural approach to control local electrochemistry, formulating zinc in a sponge form factor creates an alkaline anode that cannot form zinc dendrites while painting layered MnOx as a nanometric coating throughout a carbon nanofoam improves the rate performance of zinc-ion cathodes.
Sunday
Intermission
09:50am - 10:10am USA / Canada - Pacific - March 20, 2022 | Room: Room 30D (San Diego Convention Center)
Division: [ANYL] Division of Analytical Chemistry
Session Type: Oral - Hybrid

Sunday
3695109 - Going with the glow: High-throughput screening and automated data-driven analysis of the photophysical and photochemical properties of phosphorescent Ir(III) Complexes
10:10am - 10:35am USA / Canada - Pacific - March 20, 2022 | Room: Room 30D (San Diego Convention Center)
Stefan Bernhard, Presenter
Division: [ANYL] Division of Analytical Chemistry
Session Type: Oral - Hybrid
Parallel syntheses were used to access 1,440 structurally highly diverse heteroleptic [Ir(C^N)2(N^N)]+ complexes where C^N stands for a large set of cyclometalating ligands and N^N for a family of 1,2-diimine chelators. Most of the complexes were found to be strongly luminescent (Figure), and the emission maxima of the library spanned the visible spectrum (652–459 nm) while the measured excited state lifetimes ranged between ∼0.1–14 μs. Electronic structure features obtained from DFT calculations performed on these Ir(III) chromophores offered clear insights into the excited state properties and allowed the prediction of structure/luminescence relationships in this class of widely used photocatalysts. Models with high prediction accuracy (R2 = 0.89) for emission color were developed on the basis of experimental data. Later, newly developed, massively parallelized reactors allowed the measurement of the photocatalytic activity of the studied [Ir(C^N)2(N^N)]+ complexes in the photoreduction of Sn(II) and Zn(II) cations to their corresponding neutral metals. Automated, kinetic data collection of these solar fuels relevant-processes gathered initial rates for the 1440 photocatalysts ranging between 0 – 120 μM/s for Sn(0) deposition and 0 – 90 μM/s for Zn(0) deposition. Interestingly, no correlation was found between the chromophore’s photophysical properties and its photocatalytic activity even when modern machine learning techniques were employed. A formal photochemical rate law was then developed to help elucidate the observed reactivity. Initial rates were found to be directly correlated to the product of incident photon flux with three reaction elementary efficiencies: (1) the fraction of light absorbed by the photocatalyst, (2) the fraction of excited-state species that are quenched by the electron donor, and (3) the cage escape efficiency.
Sunday
3695026 - Operando X-ray spectroscopy of synergistic co−Mn spinel oxides as oxygen reduction electrocatalysts in alkaline fuel cells
10:35am - 11:00am USA / Canada - Pacific - March 20, 2022 | Room: Room 30D (San Diego Convention Center)
Division: [ANYL] Division of Analytical Chemistry
Session Type: Oral - Hybrid
As high-efficiency energy conversion devices, proton exchange membrane fuel cells (PEMFCs) have been recognized as crucial technologies for electric vehicles. However, PEMFCs still rely on expensive platinum-based electrocatalysts for the sluggish oxygen reduction reaction (ORR). As an emerging alternative, anion exchange membrane fuel cells (AEMFCs) have drawn increasing attention because they can enable the use of non-precious metal electrocatalysts, in particular 3d metal oxides. Here, we demonstrated that Co-Mn spinels achieved a benchmark peak power density of 1.2 W/cm2 at 2.5 A/cm2 in membrane electrode assembly (MEA) measurements, which outperformed their monometallic oxide counterparts and rivaled Pt/C in alkaline fuel cells. Analytical scanning transmission electron microscopy (STEM) with electron energy loss spectroscopy (EELS) probed the heterogeneous crystal and electronic structures at the atomic scale, and indicated that MnCo2O4/C has Co2.7+ in the core and Co2+ in the shell.
However, the origin of such electrocatalytic activity remains elusive, and necessitates the use of in situ/operando techniques to identify the catalytically active (and relevant) sites under real-time electrochemical conditions. Operando X-ray absorption near-edge structure (XANES) was employed to track/monitor the oxidation state changes of Co and Mn, not only under steady state (constant applied potential), but also under non-steady state (potentiodynamic cyclic voltammetry) conditions. The periodic conversion between Mn(III,IV), Co(III) and Mn(II,III), Co(II) during the CV scans suggested that Co and Mn redox couples serve as co-active sites (co-catalyst) for the ORR. These observations strongly suggest a potential (and very likely) synergistic effect between Co and Mn. We propose the following proton-coupled electron transfer (PCET) mechanism in which Mn prefers to bind O2 while Co prefers to bind and activate H2O, which may explain that Mn-Co spinels even outperform Pt/C cathodes under a more realistic lower relative humidity conditions.

Sunday
3694974 - Adventures in proton-coupled electron transfer at gold-solution interfaces
11:00am - 11:25am USA / Canada - Pacific - March 20, 2022 | Room: Room 30D (San Diego Convention Center)
Division: [ANYL] Division of Analytical Chemistry
Session Type: Oral - Hybrid
Gold has long been one of the preferred electrode materials because of its chemical inertness. Yet gold at the nanoscale has been found to bind hydrogen and have much more catalytic activity. Two aspects of hydrogen on gold will be discussed. First, the product isotope effect for hydrogen evolution on polycrystalline gold — H2 vs. HD vs. D2 from mixed H2O/D2O solutions — has been examined using differential electrochemical mass spectrometry (DEMS) with ultrapure D2O. A substantial and potential-independent H/D isotope effect was observed, which has some mechanistic implications. In parallel, we are examining the chemical oxidation and reduction of aqueous colloidal gold nanocrystals, as monitored principally by the gold surface plasmon resonance (SPR). For example, the Au nanocrystals are reduced with borohydrides and then re-oxidized by the nitroxyl radical TEMPO. The gold colloids change color when exposed to H2 gas, a process that is reversible in a number of ways. The chemical nature of these reactions, all of which likely fall under the umbrella of proton coupled electron transfer (PCET), will be discussed.
Sunday
3695159 - Peroxydisulfate-oxalate ping-pong reaction
11:25am - 11:50am USA / Canada - Pacific - March 20, 2022 | Room: Room 30D (San Diego Convention Center)
Division: [ANYL] Division of Analytical Chemistry
Session Type: Oral - Hybrid
Aqueous solutions containing both the oxidant, peroxydisulfate (S2O82-), and reductant, oxalate (C2O42-), are thermodynamically unstable due to the highly exothermic homogeneous redox reaction, S2O82- + C2O42- ® 2SO42- + 2CO2. However, at room temperature, the reaction does not occur to any significant extent due to slow electron-transfer kinetics. We demonstrate that the S2O82-- C2O42- redox reaction can be initiated by electrochemical injection of an electron to generate S2O83-, which then undergoes bond cleavage to form the highly oxidizing SO4-. The SO4- then oxidizes C2O42- to generate CO2 and the highly reducing CO2-. In turn, CO2- then reduces another molecule of S2O82- to S2O83-, resulting in a self-sustaining cyclic redox that consumes both S2O82- and C2O42-. Theoretically, a single electron is capable of initiating the homogeneous process. Using outer-sphere redox mediators (e.g., Ru(NH3)63+/2+) as electrocatalysts to initiate the reaction, we show that, within error of the measurement, the reaction becomes self-sustaining with the injection of a single electron.
Sunday
Concluding remarks
11:50am - 11:55am USA / Canada - Pacific - March 20, 2022 | Room: Room 30D (San Diego Convention Center)
Division: [ANYL] Division of Analytical Chemistry
Session Type: Oral - Hybrid

Advances in Spectroscopy:
08:00am - 11:40am USA / Canada - Pacific - March 20, 2022 | Room: Room 31A (San Diego Convention Center)
Allison Squires, Organizer; Sara Sohail, Presider; Alan McCartt, Presider
Division: [ANYL] Division of Analytical Chemistry
Session Type: Oral - In-person
Division/Committee: [ANYL] Division of Analytical Chemistry

Recent advances in spectroscopy research

Sunday
Introductory Remarks
08:00am - 08:05am USA / Canada - Pacific - March 20, 2022 | Room: Room 31A (San Diego Convention Center)
Division: [ANYL] Division of Analytical Chemistry
Session Type: Oral - In-person

Sunday
3652458 - Direct observation of the assembly of mixed Amyloid β 40 and 42 fibrils using FRET imaging and single-molecule fluorescence spectroscopy
08:05am - 08:30am USA / Canada - Pacific - March 20, 2022 | Room: Room 31A (San Diego Convention Center)
Division: [ANYL] Division of Analytical Chemistry
Session Type: Oral - In-person
Protein aggregation into amyloid fibrils, the hallmark of several neurodegenerative diseases, is a highly specific self-assembly process. Gaining an understanding of disease etiology hinges on our ability to understand the molecular mechanics of how soluble monomers assemble to form insoluble fibrils consisting of thousands of constituent monomers. However, even a single amyloid-forming polypeptide can exist in several forms distinguished by sequence length or post-translational modification, which can drastically alter the physicochemical behavior of the polypeptide. The Alzheimer’s Disease related peptide amyloid β (Aβ) exists primarily as a mixture of its 40- and 42-residue isoforms, which differ by two hydrophobic residues at the C-terminus. Bulk measurements offer conflicting reports on the physical interaction, or lack thereof, between Aβ40 and Aβ42. Here, we investigate the co-aggregation of Aβ40 and Aβ42 at the single fibril level using Förster Resonance Energy Transfer (FRET) imaging coupled with fluorescence lifetime imaging (FLIM) and single molecule fluorescence spectroscopy to monitor the entire aggregation pathway in real-time. Using donor-labeled Aβ42 and acceptor-labeled Aβ40, we observe that fibrillation nucleates from mixed Aβ40/Aβ42 oligomers. Aβ42 monomers primarily add to the oligomers during fibril elongation. We use deep learning to separate overlapping fibrils and to quantitatively analyze the aggregation process for each fibril imaged. FRET imaging enables us to determine the evolution of the Aβ40:Aβ42 ratio within each aggregate as aggregation progresses from small oligomers to mature fibrils. Results of these quantitative analyses will be presented.

Sunday
3659354 - Temperature-dependent fluorescence recovery after photobleaching (FRAP) of micro-confined alkyl-imidazolium chloride ionic liquids
08:30am - 08:55am USA / Canada - Pacific - March 20, 2022 | Room: Room 31A (San Diego Convention Center)
Division: [ANYL] Division of Analytical Chemistry
Session Type: Oral - In-person
In recent years, ionic liquids have shown promising applications in the field of separations in micro-confined separation systems and microfluidic devices. In both these systems, ionic liquids are confined to geometries on the magnitude of hundreds of micrometers or less. Both in separation and microfluidic systems, ionic liquids can undergo localized or general heating to control various processes. To effectively apply ionic liquids in these systems, it is imperative that the diffusional properties of molecules through the media, as a function of sample thickness and temperature of molecules through the media, is known. We report the first use of fluorescence recovery after photobleaching (FRAP) for the determination of temperature-dependent (20-100 °C) diffusional properties in alkyl-imidazolium-based ionic liquids. FRAP experiments were conducted with a zwitterionic, hydrophilic dye, ATTO 647, in glycerol, [C6mim][Cl], and [C8mim][Cl] with varying sample thickness (1-120 µm) across the temperature range of interest. From our studies, we note both a thickness- and temperature-dependent diffusion coefficient. In addition, we also note a time-dependent mean square displacement that is also conditional on thickness and temperature.
Sunday
3657096 - Spatially dynamics of self-assembling materials revealed by 2D VSFG microscope
08:55am - 09:20am USA / Canada - Pacific - March 20, 2022 | Room: Room 31A (San Diego Convention Center)
Division: [ANYL] Division of Analytical Chemistry
Session Type: Oral - In-person
Systems with spatial heterogeneity represent a challenge for spectroscopic techniques, as the signal may be generated over an area containing several domains or regions each with distinct properties. In order to address this issue, various nonlinear spectroscopies have been modified to yield novel microscopy methods. Here we have converted two-dimensional sum frequency generation (2D SFG) spectroscopy into an optical microscopy method, and have demonstrated the utility of the method on self-assembled sheets composed of beta cyclodextrin (b-CD) and sodium dodecyl sulfate (SDS). Previous studies revealed that self-assembled sheets of b-CD exhibit a high degree of spatial heterogeneity. We found that the spatial heterogeneity contributes to markedly different dynamics at different spots in the sheet. We analyzed the spectral diffusion using center line slope(CLS) method and found that CLS at different spots are different. These findings inform our understanding of cyclodextrin self-assembly and demonstrate the usefulness of spatial resolution in tandem with time resolved spectroscopy.
Sunday
3645958 - Novel detection of cancer biomarkers using nonlinear multi-photon laser wave-mixing detector interfaced to microfluidics
09:20am - 09:45am USA / Canada - Pacific - March 20, 2022 | Room: Room 31A (San Diego Convention Center)
Division: [ANYL] Division of Analytical Chemistry
Session Type: Oral - In-person
Ultrasensitive and selective laser wave-mixing detector interfaced to microfluidics greatly assist detection of biomarkers used to aid the early detection and diagnosis of cancers. Currently detection methods lack sensitivity, require large sample volume or time-consuming labeling steps. The wave-mixing signal is a coherent laser-like signal and can be collected with high signal-to-noise ratios against 100% dark background. Therefore, zeptomole-level detection sensitivity is achieved. High spatial resolution is another significant advantage of our technique because the probe volume is small (picolitre). Hence, it presents as a detector coupled with microfluidics, microarrays and capillary-based separation systems. Also, laser wave mixing can detect label-free analytes and analytes labeled with fluorophores or chromophores because it is an optical absorption-based method. Pancreatic cancer biomarker carbohydrate antigen 19-9 (CA19-9) and breast cancer biomarker human epidermal growth factor receptor 2 (HER2) were used as proof-of-concept. A solid state 532nm laser is used to obtain excellent detection limits of 0.9 zeptomole and 30 zeptomole for CA 19-9 and HER2, respectively. CA 19-9 labeled with Chromeo P540 is detected using capillary-based separation system; HER2 reacted with BCA assay reagent is sandwiched between two glass slides and detected using surface-based detection systems. Chemical selectivity and sensitivity can be achieved by capillary electrophorese; high throughput and convenient analyses can be obtained by microarray-based wave-mixing detection systems. Lastly, portable solid state laser wave-mixing – microchip detectors is suitable for field use.
Sunday
Intermission
09:45am - 09:55am USA / Canada - Pacific - March 20, 2022 | Room: Room 31A (San Diego Convention Center)
Division: [ANYL] Division of Analytical Chemistry
Session Type: Oral - In-person

Sunday
3654169 - Real time monitoring of paraquat photodegradation using colloidal gold surface enhanced Raman spectroscopy
09:55am - 10:20am USA / Canada - Pacific - March 20, 2022 | Room: Room 31A (San Diego Convention Center)
Division: [ANYL] Division of Analytical Chemistry
Session Type: Oral - In-person
Monitoring chemical reactions in aqueous solution is a challenge because most instrumental techniques either are not suited for the rapid timescales, are not sensitive enough to detect products at low concentrations, or do not have sufficient structure-to-spectrum relationships. Raman spectroscopy is a promising method to monitor reactions, as it is fast, dependent on chemical structure, and has little interference from water. However, Raman scattering is generally very weak. Surface-enhanced Raman spectroscopy (SERS) improves the signal strength of Raman spectroscopy by using a metal surface plasmon, or oscillation of the surface’s electrons, to allow for highly selective and sensitive detection and characterization of analyte molecules. An aqueous colloid of partially aggregated gold nanoparticles is an accessible substrate to generate plasmons necessary for SERS. Analyte is adsorbed to the nanoparticles, initiating aggregation. A surfactant (sodium dodecyl sulfate) is added at an optimal time to arrest aggregation, leading to a highly stable substrate. Because of this, SERS enhancement is present over multiple hours under powerful UV light. As peaks in the Raman spectra evolve over time, structural changes can be detected. Paraquat, a common yet dangerous herbicide that is banned in the EU, China, and many other countries, was monitored with SERS in real time as it degrades when exposed to intense light, analogous to sunlight. The photodegradation products have not been well characterized in the past, but methylpyridinium is expected. A reaction mechanism and kinetics can then be determined for this reaction. Gathering better data about paraquat and its degradation in the environment may help users apply paraquat in a manner that minimizes harm to humans and the ecosystem.
Sunday
3660954 - Plasmon-modified fluorescence spectroscopy of nanostructures in bulk solution
10:20am - 10:45am USA / Canada - Pacific - March 20, 2022 | Room: Room 31A (San Diego Convention Center)
Division: [ANYL] Division of Analytical Chemistry
Session Type: Oral - In-person
It is well-known that plasmonic nanostructures can modify the fluorescence emission of nearby molecules. Although single particle measurements of plasmon-modified fluorescence provide rich data sets, acquisition is often time-consuming and requires specialized instrumentation. By contrast, when sufficiently homogeneous samples are available, performing ensemble measurements in bulk solution would appear to be a more convenient way to routinely characterize new nanomaterials. In addition to its convenience, bulk spectroscopy can also be readily compared to theoretical predictions because, unlike in single-particle measurements, many variables which determine the effect of plasmonic modificiation can be replaced by their reliable, ensemble-averaged values. Despite these apparent benfits, however, it has proven difficult to reliably measure plasmonic modification of fluorescence emission in bulk solution. Here, we identify key factors that commonly corrupt plasmon-modified fluorescence spectra and present an experimental framework to overcome them. Central to this framework is the use of dynamic DNA nanotechnology to control the association of a fluorophore with a nanostructure, permitting accurate plasmon-coupled and reference spectral measurements to be performed in the same solution with minimal perturbation. As a demonstration, we apply this framework to reproducibly measure the modification of fluorescence emission—encompassing both the magnitude of enhancement as well as spectral reshaping—of dye molecules associated with gold nanospheres and gold nanosphere dimers. We anticipate that routine and accurate bulk spectroscopic measurements such as these can guide the development of fluorescence-enhancing nanomaterials as well as deepen our fundamental understanding of plasmonic phenomena.
Sunday
3660675 - Two-color, cavity ringdown spectroscopy measurements of room temperature 14CO2 with sensitivity below the natural abundance
10:45am - 11:10am USA / Canada - Pacific - March 20, 2022 | Room: Room 31A (San Diego Convention Center)
Division: [ANYL] Division of Analytical Chemistry
Session Type: Oral - In-person
Two-color, cavity ring-down spectroscopy (2C-CRDS) utilizes pump and probe lasers which are locked to a three-mirror, traveling wave resonator in a counter-propagating configuration. By probing elevated quantum states that are cyclically pumped, the net, two-color signal can directly measure the analyte and compensate for background variations from the cavity and non-target species. The result is a generally-applicable, high-sensitivity, high-selectivity detection method. The main impetus for 2C-CRDS development was the detection of trace-gases in crowded spectroscopic regions. Here we present the application of 2C-CRDS to the detection of 14CO2. Over the past decade several groups have reported cavity enhanced measurements of 14CO2 below the natural abundance. These measurements all required cooling of the test gas (-20 to -100 °C) to remove interference from CO2 isotopologue hot bands. The figure below shows preliminary room-temperature measurements of 14CO2 which relies on the selectivity of 2C-CRDS for isotopologue hot band compensation. We will discuss the different two-color spectroscopic features observed with our 2C-CRDS setup—doppler-free coherent two-quantum resonances, directly populated stepwise resonances, and collision populated resonances. The precision and accuracy of 2C-CRDS will also be characterized with carbon-14 standards. With its improved sensitivity, room-temperature operation, and robust noise rejection, 2C-CRDS shows promise for carbon-14 measurements in the climate and environmental sciences.
2C-CRDS spectra of CO<sub>2</sub> containing varying amounts of carbon-14 as is indicated by the legend. Red points represent CO<sub>2</sub> from combusted leaves that were collected near the vicinity of a nuclear reactor. The remaining points are from commercial CO<sub>2</sub> derived from either ethanol (green) or petrogenic (blue) sources. Carbon-14 concentrations were verified by accelerator mass spectrometry. All measurements are made at 20 torr and room temperature. Error bars represent the 95% confidence interval of the net two-color signal. When the probe was centered on the <sup>14</sup>CO<sub>2 </sub>2—1 ν<sub>3</sub> R(13) transition, measurements were taken for longer averaging times which results in smaller confidence intervals.

2C-CRDS spectra of CO2 containing varying amounts of carbon-14 as is indicated by the legend. Red points represent CO2 from combusted leaves that were collected near the vicinity of a nuclear reactor. The remaining points are from commercial CO2 derived from either ethanol (green) or petrogenic (blue) sources. Carbon-14 concentrations were verified by accelerator mass spectrometry. All measurements are made at 20 torr and room temperature. Error bars represent the 95% confidence interval of the net two-color signal. When the probe was centered on the 14CO2 2—1 ν3 R(13) transition, measurements were taken for longer averaging times which results in smaller confidence intervals.


Sunday
3662377 - Noninvasive imaging of pH changes associated with implanted medical devices using X-ray excited luminescence chemical imaging (XELCI)
11:10am - 11:35am USA / Canada - Pacific - March 20, 2022 | Room: Room 31A (San Diego Convention Center)
Division: [ANYL] Division of Analytical Chemistry
Session Type: Oral - In-person
Implanted medical device-associated infections are a major cause of fixation failure. Detection and treatment of these infections remain as a challenge as clinical symptoms are often delayed or completely absent till the infection reaches a later stage and harder to treat with antibiotics. Once diagnosed early, infections can be treated without removal of implants and undergoing a second surgery. Herein, we describe a X-ray Excited Luminescence Chemical Imaging (XELCI) based pH sensor for noninvasive detection and imaging of changes in local pH with high spatial and pH resolution.
The sensor is fabricated as a coating on an implant (orthopedic plates, Intramedullary nails, etc). It has two layers: a layer of Gd2O2S:Eu scintillators that produces 620 nm and 700 nm luminescence upon X-ray irradiation, and a pH-indicating film containing BTB (Bromothymol Blue) which covers the scintillators and alters the luminescence spectrum in a pH dependent manner. The spectrum escaping through the tissue is measured to determine the local pH as a focused X-ray beam irradiates one spot on the implant. The X-ray beam scans across the sensor surface to generate an image and measures pH point by point which enables high spatial resolution, low background measurements of local pH for studying implant associated infections. We are interested in detecting pH changes through bone and infections associated with intramedullary nails. These are a type of orthopedic implants which are commonly forced into the medullary cavity of the bone to fix comminuted femoral and tibial fractures. They are commonly associated with osteomyelitis. As a preliminary study, we have imaged an epoxy-PEG coated stainless-steel rod with two distinct pH regions (pH 4 and pH 8) in a reamed rabbit cadaver tibia. We were successfully able to image the above pH changes through bone in vitro with XELCI imaging. In future studies, we will be applying these sensors in the rabbit model to detect pH changes through bone in vivo.

Sunday
Concluding Remarks
11:35am - 11:40am USA / Canada - Pacific - March 20, 2022 | Room: Room 31A (San Diego Convention Center)
Division: [ANYL] Division of Analytical Chemistry
Session Type: Oral - In-person

Advances in Mass Spectrometry:
08:00am - 10:00am USA / Canada - Pacific - March 20, 2022 | Room: Room 30E (San Diego Convention Center)
Benjamin Garcia, Organizer, Presider
Division: [ANYL] Division of Analytical Chemistry
Session Type: Oral - Hybrid
Division/Committee: [ANYL] Division of Analytical Chemistry

Session will highlight advances in mass spectrometry technology, methodology and associated data analyses spanning across the chemical and biochemical sciences.

Sunday
3641319 - Method validation of gamma hydroxybutyrate detection upon herpes simplex virus type 1 infection using LC-MRM-MS with 3-nitrophenylhydrazine derivatization
08:00am - 08:20am USA / Canada - Pacific - March 20, 2022 | Room: Room 30E (San Diego Convention Center)
Division: [ANYL] Division of Analytical Chemistry
Session Type: Oral - Hybrid
Volatile organic compounds (VOCs) release triggered by infection of DNA virus is not known. Previously, we reported that gamma-butyrolactone (GBL), a VOC, was released upon Herpes Simplex Virus Type-1 (HSV-1) acute infection, in vitro. Based on the metabolic pathway and chemical conversion of GBL, we hypothesized that infected cells produce gamma-hydroxybutyrate (GHB) as a non-volatile key pathway intermediate for the subsequent production of GBL. To improve a chromatographical detection, we developed a sensitive and specific method for GHB and GBL quantitation via a pre-column derivatization. Our data showed that the carboxylic acid functional group of GHB derivatized with 3-nitrophenylhydrazine hydrochloride (3-NPH), selectively. The results further indicated that the product was detectable via LC-MS. This protocol obtained a linearity at the concentration range of 1 μg/mL- 6 μg/mL pre injection, with over 95% reaction completion and the intra- and inter- assay coefficient of variation was less than 10%. Together this method offered a quantitative detection of GHB for HSV-1 acute infection.

Sunday
3655548 - Time resolved measurements using the tTIMS/MS provide insight into the structural heterogeneity of proteins in the gas phase
08:20am - 08:40am USA / Canada - Pacific - March 20, 2022 | Room: Room 30E (San Diego Convention Center)
Division: [ANYL] Division of Analytical Chemistry
Session Type: Oral - Hybrid
The onset of diseases is often related structural changes of proteins and/or the formation of protein assemblies. Therefore, it is crucial to understand the protein structure-function relationship. Ion mobility-mass spectrometry has emerged as a biologically useful structural tool due to its ability to characterize ions by their m/z and cross-sections. Despite the gas phase environment of ion mobility measurements, it is known ions can retain their native-like structure in the gas phase under ‘soft’ experimental settings. However, it remains unclear how long native-like structures can be retained and what interactions contribute the most to the stability of the native-like protein in the gas phase.
In this work, we employ the newly developed tandem-trapped ion mobility spectrometer/mass spectrometer (tTIMS/MS) to determine the unfolding kinetics of wild type and mutant forms of ubiquitin in the gas phase. tTIMS/MS allows for ions to be stored in each of the mobility analyzers for a predetermined amount of time. Activation by high-speed collisions prior to trapping can be used to determine the structural evolution of activated ions. In addition, tTIMS/MS can perform mobility selection prior to trapping, which allows for the unfolding kinetics of different subpopulations to be determined.
We have successfully trapped ubiquitin in the first and second TIMS analyzer for up to 12s. Our preliminary data show compact ubiquitin conformations for charge state 6+ and 7+ are retained for up to 12s in each of the TIMS analyzers. The gas phase lifetime of compact ubiquitin indicates the native-like structure is stable in the gas phase. Time-resolved measurements reveal protein unfolding in the gas phase is a complex reaction. This complexity suggests compact ions do not unfold at the same rate. By mobility-selecting different subpopulations of compact ubiquitin from charge state 7+, we were able to show different unfolding kinetics for different subpopulations. Analysis of our preliminary results suggests the compact ions unfold in a set of consecutive reactions, some being faster than the other. These changes in the unfolding timescale compliment the idea that multiple distinct conformations are present inside a single cross-section feature. Activation prior to trapping reveals activated ions refold in the gas phase. The refolding kinetics of different subpopulations could provide insight into the structural heterogeneity of native-like structures in the gas phase.

Sunday
3644031 - Differentiating isomeric pneumococcal polysaccharides by using partial chemical degradation followed by mass spectrometry
08:40am - 09:00am USA / Canada - Pacific - March 20, 2022 | Room: Room 30E (San Diego Convention Center)
Division: [ANYL] Division of Analytical Chemistry
Session Type: Oral - Hybrid
Novel methods for the quantitation of capsular polysaccharides present in bacterial conjugate vaccines are desired as their complexity and the number of serotypes included in them increases. Chemical degradation followed by mass spectrometry is a promising approach for this analysis. However, current mass spectrometry-based methods cannot be used to quantitate isomeric polysaccharides due to their identical monosaccharide compositions and molecular weight repeating units. In this study, differentiation and quantitation of isomeric pneumococcal polysaccharides 6A and 6B, and 19A and 19F, were investigated based on a partial chemical degradation mass spectrometry approach to generate an oligosaccharide marker for one isomer, and not the other. Hydrophilic interaction chromatography (HILIC) negative-ion mode electrospray ionization mass spectrometry ((-)ESI MS)) was utilized here to detect potential oligosaccharide markers. Mild base degradation conditions were able to generate marker ions of m/z 683 for 6A and not 6B, while strong acid degradation conditions were able to generate ions of m/z 555 for 6B and not 6A. Furthermore, mild base degradation conditions were able to generate ions of m/z 608 for 19A and not 19F, while more harsh base degradation conditions were able to generate ions of m/z 608 for 19F and not 19A. The generation of unique oligosaccharides for each ST via various chemical degradation conditions was possible by taking advantage of the varying stabilities of the phosphodiester and glycosidic bonds of the repeating units of each ST. Linear relationships were established for all STs between the ion abundance measured for the oligosaccharide marker and the starting pneumococcal polysaccharide concentration. Furthermore, all within-day and between-day precision measurements for each method were lower than 15% variability demonstrating good robustness. Therefore, partial chemical degradation followed by mass spectrometry was successful at differentiating and quantifying isomeric pneumococcal polysaccharides and may be adopted for other bacterial types

Sunday
3656717 - High throughput amino acid and acylcarnitine profiling in infant serum by means of LC-MS/MSMRM for the elucidation of early-life obesity risk
09:00am - 09:20am USA / Canada - Pacific - March 20, 2022 | Room: Room 30E (San Diego Convention Center)
Division: [ANYL] Division of Analytical Chemistry
Session Type: Oral - Hybrid
Obesity has become a global pandemic in recent years. Alongside the increasing number of obese adults, also the rate of overweight and obese children has reached alarming levels. These developments pose a significant financial burden on health systems due to the rising incidence of associated non-communicable diseases, such as diabetes and cardiovascular diseases. Therefore, it is essential to understand the metabolic alterations towards overweight development already at an early stage of life. Associations between early-life risk factors such as high pre-conceptional maternal BMI and long-term offspring health outcomes have been identified successfully. However, the metabolic processes underlying these factors have not been clarified to date, mainly due to the low metabolite concentrations and the resulting analytical challenges.
To gain more comprehensive knowledge on obesity and nutrition related pathways, a sensitive and reliable multi-analyte hydrophilic interaction liquid chromatography (HILIC)-MS/MS method was developed for infant serum. A fast and derivatization-free sample preparation was established using minimum amounts of serum from 3 to 4-month-old infants of the prospective mother-child cohort PEACHES (Programming of Enhanced Adiposity Risk in CHildhood–Early Screening) and facilitating high throughput analysis. By means of the multiple reaction monitoring (MRM) mode and isotopically labelled standards, ∑ 62 serum metabolites, in particular 40 amino acids and derivatives as well as 22 acylcarnitines, could be quantified precisely within a single 20 minutes run. Comprehensive method validation utilizing reference control serum and covering parameters such as matrix effects, precision and accuracy, highlighted the applicability of this multi-analyte method. This profiling method enabled the derivation of different infant metabotypes in combination with the characterization of nutrition related metabolites and thus provides a basis for investigating metabolic risk for overweight development already early in life.

Sunday
3650808 - Unbiased high-throughput MALDI-TOF MS lipid and metabolite screening assays in primary human cells for drug discovery
09:20am - 09:40am USA / Canada - Pacific - March 20, 2022 | Room: Room 30E (San Diego Convention Center)
Division: [ANYL] Division of Analytical Chemistry
Session Type: Oral - Hybrid
MALDI-TOF MS has become a powerful tool for high-throughput screening (HTS) approaches in drug discovery, overcoming the shortcomings of conventional fluorescence label-based technologies. Most of the MALDI-TOF based HTS approaches have focused on in vitro assays with simple readouts, and have been limited mainly to peptide/protein-centric activity assays. Although, phenotypic cellular assays using MALDI-TOF MS are possible using higher molecular masses, the capability of MALDI-TOF to detect compounds in the low mass range is generally considered limited due to interference peaks brought by the matrix. Metabolomics-based drug discovery presents therefore an exciting challenge for MS analysis as the system becomes inherently more complex. Herein, we apply this technology for cellular assays, specifically to detect metabolites and lipids in a comprehensive, untargeted, and unbiased HTS approach for drug discovery in idiopathic pulmonary fibrosis (IPF).

Primary human nasal epithelial cells were used to develop a cellular assay pipeline for untargeted metabolite phenotypic identification using MALDI-TOF MS. Multiple IPF-relevant stimuli and inhibitors were tested to see if stimulation and inhibition could be distinguished in the assay. Next, different sample preparation conditions, such as testing different matrices, additives, derivatization reagents, and extraction protocols, were investigated to ensure the most effective analysis for metabolites and lipids. All the parameters were optimized using Mosquito-TTP Labtech liquid handling robot to allow a systematic screening of a large number of combinations to find the best conditions. Moreover, all the protocols were simplified and adapted to HTS compatible platforms to ensure a smooth translation from an academic laboratory to industry.

Preliminary testing revealed spectra that could be distinguished between the unstimulated, stimulated cells, and stimulated cells with inhibitor, in both the low and high mass region (m/z 200-1000 and 2,000-20,000). Using principal component analysis, hierarchical clustering, and machine learning strategies, a subset of peaks was identified to be unique to each condition. These data suggests that it is possible to elucidate important metabolic features of cells in modelled pathophysiology. This approach has the potential to be further optimized as an automated HTS drug discovery assay in the industrial setting.

Sunday
3638226 - Quatitative mass spectrometry identifies histone H2A proteolysis which destabilizes nucleosomes to oppose gene activation
09:40am - 10:00am USA / Canada - Pacific - March 20, 2022 | Room: Room 30E (San Diego Convention Center)
Benjamin Garcia, Presenter
Division: [ANYL] Division of Analytical Chemistry
Session Type: Oral - Hybrid
Histone proteolysis is a poorly understood phenomena by which the N-terminal tail of histones are irreversible cleaved by intracellular proteases. During development histone PTMs are known orchestrate gene expression and ultimately cell fate decision, therefore it will be key to decipher underlying mechanisms that lead to proteolysis during cellular differentiation. Here we found that the N-terminus of histone H2A is proteolytically clipped and removed by the lysosomal protease Cathepsin L. Using quantitative mass spectrometry (MS), we identified L23 to be the primary site for this protease, with this clipped H2A (cH2A) form representing ~1% of the total H2A reaching a maximum after 4 days of differentiation. Using ChIP-Seq, we found reduced proteolysis, lead to increase acetylated H2A at promoter regions in differentiated ES cells. We also report novel readers of acetylated H2A during in pluripotency. All in all, our data suggest that proteolysis serve as a quick mechanism to silence genes involve in pluripotency and destabilize the nucleosome core particle as indicated by protein degradation studies.
Fundamental Biological Chemistry :
08:00am - 12:00pm USA / Canada - Pacific - March 20, 2022 | Room: Sapphire O/P (Hilton San Diego Bayfront)
Phoebe Glazer, Organizer, Presider; Ekaterina Pletneva, Organizer
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - In-person
Division/Committee: [BIOL] Division of Biological Chemistry
Sunday
3658863 - Allosteric networks regulate function in multi-enzyme complexes
08:00am - 08:20am USA / Canada - Pacific - March 20, 2022 | Room: Sapphire O/P (Hilton San Diego Bayfront)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - In-person
Networks of noncovalent amino acid interactions propagate allosteric signals throughout proteins. Tryptophan synthase (TS) is an allosterically controlled bienzyme in which the indole product of the alpha subunit (αTS) is transferred through a 25 Å hydrophobic tunnel to the active site of the beta subunit (βTS). Previous nuclear magnetic resonance and molecular dynamics simulations identified allosteric networks in αTS important for its function. Here, we show that these allosteric networks change across the catalytic cycle of αTS, such that chemical catalysis is associated with specific conformational dynamics and networks not observed in the substrate- and products-bound states. Moreover, substitution of a distant, surface-exposed network residue in αTS enhances tryptophan production, not by activating αTS function, but through dynamically controlling the opening of the indole channel and stimulating βTS activity. While stimulation is modest, the substitution (A198W) also enhances cell growth in a tryptophan-auxotrophic strain of Escherichia coli compared to complementation with wild-type αTS, emphasizing the biological importance of the network. Network residues thus represent ideal targets to optimize mutagenesis strategies in protein engineering studies directed towards changing enzyme function and regulation, especially in multi-enzyme complexes.
Figure. Structure of tryptophan synthase, showing alpha (white) and beta (pink) subunits, and the dynamic allosteric network in alpha that connects with the beta subunit. Substitutions at position 198 change enzyme activity both in vitro and in vivo.

Figure. Structure of tryptophan synthase, showing alpha (white) and beta (pink) subunits, and the dynamic allosteric network in alpha that connects with the beta subunit. Substitutions at position 198 change enzyme activity both in vitro and in vivo.


Sunday
3661555 - Natural product pathway discovery through activity–guided single–cell genomics
08:20am - 08:40am USA / Canada - Pacific - March 20, 2022 | Room: Sapphire O/P (Hilton San Diego Bayfront)
Dr. Michael Burkart, Presenter
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - In-person
We have developed a new strategy to identify natural product biosynthetic pathways from complex microbial ecosystems that combines chemical biology tools with single cell sequencing. This methodology leverages a fluorescent in situ enzyme assay that targets carrier proteins common to polyketide (PKS) and non–ribosomal peptide synthetases (NRPS). By applying fluorescence–activated cell sorting to a treated microbiome, microbes with active secondary metabolic capabilities can be enriched and subjected to single–cell genomics. As an example, we demonstrated the genetic basis for biosynthetic diversity in complex marine organisms like tunacates. Here, the enzyme–active cells revealed a member of marine Oceanospirillales harboring a novel NRPS gene cluster with high similarity to phylogenetically distant marine and terrestrial bacteria. Interestingly, this synthase belongs to a larger class of siderophore biosynthetic gene clusters commonly associated with pestilence and disease. This strategy demonstrates activity–guided single–cell genomics as a tool to guide novel biosynthetic discovery.
Activity–guided microbial single–cell genomics of <i>Ciona intestinalis</i>

Activity–guided microbial single–cell genomics of Ciona intestinalis


Sunday
3660590 - in vitro glycosylation of membrane proteins involved in human disease
08:40am - 09:00am USA / Canada - Pacific - March 20, 2022 | Room: Sapphire O/P (Hilton San Diego Bayfront)
Gabriel Cook, Presenter
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - In-person
Glycoproteins take part in nearly every biological process and make up a large percent of the proteome. N-glycosyltransferase (NGT) from Actinobacillus pleuropneumoniae, which recognizes the consensus amino acid sequence, -Asn-X-Ser/Thr- (NXT) within the protein, has been shown to successfully glycosylate peptides and proteins in vitro. The enzyme catalyzes glycosidic bond formation between the oligosaccharide donor, containing nucleoside phosphatase, and the amide nitrogen of the asparagine residue. We are specifically interested in the properties of membrane glycoproteins, which are key components in a number of different disease states. In vitro studies of N-linked glycosylation were done in a step-wise fashion in a membrane mimetic environment to confirm the methods for glycosylating soluble proteins could be applicable to membrane proteins. Detergent and lipid systems were used to solubilize the hydrophobic peptides and membrane proteins for glycosylation. Gel electrophoresis, mass spectrometry, and NMR studies were used for the detection and quantification of glycosyltransferase activity. Our experiments demonstrated that full-length membrane proteins that contain a N-glycosylation consensus sequence, can be glycosylated by NGT, even in the presence of membrane mimetic environments. These methods will be used as the starting point for further oligosaccharide attachment and characterization of structure, dynamic and interactions of these important proteins.
Sunday
3657488 - New fluorophore scaffolds for chemical biology
09:00am - 09:20am USA / Canada - Pacific - March 20, 2022 | Room: Sapphire O/P (Hilton San Diego Bayfront)
Clifford Stains, Presenter
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - In-person
Small molecule fluorophores provide valuable tools for visualizing biological species. Fluorophores that absorb and emit light within the far-red to near-infrared (NIR) window are particularly desirable for monitoring biological events since light at these wavelengths has deeper tissue penetration and mitigates issues associated with autofluorescence of natively occurring fluorophores. We are endowing fluorophores with new chemical functionalities in order to obtain NIR reagents to probe biologically-relevant signaling molecules as well as new tools for perturbing living systems. Our recent efforts to measure and modulate biological processes using these new fluorophore scaffolds will be discussed.
Sunday
3661915 - Activity-based profiling of RNA modifying enzymes
09:20am - 09:40am USA / Canada - Pacific - March 20, 2022 | Room: Sapphire O/P (Hilton San Diego Bayfront)
Ralph Kleiner, Presenter
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - In-person
Epitranscriptomic RNA modifications can regulate RNA activity, however there remains a major gap in our understanding of the scope of RNA chemistry present in biological systems, the enzymes responsible for regulating modification levels, and the ultimate function of these modifications in biological processes. To address this gap, we have developed RNA-mediated activity-based protein profiling (RNABPP), a chemoproteomic strategy relying upon metabolic RNA labeling with modified nucleoside probes, RNA-protein enrichment, and quantitative proteomics. We apply RNABPP with fluoropyrimidine nucleosides in human cells in order to investigate RNA pyrimidine-modifying enzymes acting upon mRNA. In addition to profiling 5-methylcytidine (m5C) and 5-methyluridine (m5U) methyltransferase activity on mRNA, we identify a novel mechanism-based RNA-protein crosslink between 5-fluorouridine-modified RNA and the uncharacterized human dihydrouridine synthase DUS3L. Further, we study the cellular substrates of DUS3L using quantitative nucleoside LC-MS/MS and cross-linking and immunoprecipitation-based sequencing. Taken together, our work provides a general approach for profiling RNA modifying enzyme activity in vivo, and reveals the existence of new pathways for the epitranscriptomic regulation of RNA behavior in human cells.
Sunday
3660807 - Photoresponsive ligands for dopamine and serotonin receptors: Synthesis and proteomics-based target identification
09:40am - 10:00am USA / Canada - Pacific - March 20, 2022 | Room: Sapphire O/P (Hilton San Diego Bayfront)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - In-person
Dopamine and serotonin receptors are central to brain function, yet are extremely challenging to study, especially in precise regions and narrow timescales. This is because traditional drugs for these receptors are subject to diffusion and other pharmacokinetic and pharmacodynamic factors that limit the spatiotemporal precision of experimental outcomes. Optical control has emerged as an excellent method to control neurons with the spatial and temporal resolution unavailable with drugs, however, methods to create light-controlled non-mutant receptors are lacking. Our ultimate goal is to create new tools to study dopamine and serotonin GPCRs in living mammals, without the need for genetic manipulation. To do this, we are synthesizing both broad (multiple subtypes) and specific modulators of dopamine and serotonin receptors, with an overall scheme of: 1) pharmacophore, 2) photoresponsive group, and 3) covalent handle. Broadly-acting pharmacophores are derivatives of apomorphine and ergoline alkoloids, and specific modulators are derivatives of FDA-approved drugs including ropinirole and pramipexole. Here, we report on the synthesis of new probe molecules, their evaluation in functional assays in human and rodent cell lines and primary cells, and proteomic analysis of probe targets via SILAC (Stable Isotope Labeling by/with Amino acids in Cell culture).
Sunday
Intermission
10:00am - 10:20am USA / Canada - Pacific - March 20, 2022 | Room: Sapphire O/P (Hilton San Diego Bayfront)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - In-person

Sunday
3660349 - Illuminating chromatin complexes with chemical genomics
10:20am - 10:40am USA / Canada - Pacific - March 20, 2022 | Room: Sapphire O/P (Hilton San Diego Bayfront)
Brian Liau, Presenter
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - In-person
Layered on top of the genome, epigenetic modifications comprise a critical mechanism for controlling gene expression and genome function. Somatic mutations often alter the function of chromatin complexes that regulate epigenetic modifications, leading to aberrant chromatin landscapes frequently observed in human disease states. Accordingly, there is immense interest in developing small molecule therapeutics that target chromatin factors for drug discovery. Beyond their potential as therapeutics, these small molecules are powerful reagents to study chromatin complexes, the function and regulation of which remain unclear. By combining genome-editing with chemical inhibitor profiling in an approach called CRISPR-suppressor scanning, we describe the systematic identification of drug resistance-conferring alleles across protein targets, including chromatin modifier complexes. These drug resistance mutations not only confirm on-target engagement but can be used as powerful discovery tools to uncover new principles and aspects of chromatin complex biology and chemistry. We showcase these capabilities through deep mechanistic vignettes exploring several chromatin modifier complexes and the diverse small molecules that modulate them.
Sunday
3648196 - Chemoproteomic tools to characterize the dark matter of the human genome
10:40am - 11:00am USA / Canada - Pacific - March 20, 2022 | Room: Sapphire O/P (Hilton San Diego Bayfront)
Sarah Slavoff, Presenter
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - In-person
Advanced methods in next-generation sequencing and proteogenomics have revealed thousands of previously invisible human genes, increasing the known size of the human genome by at least 10%. This previously unannotated “dark matter” of the human genome includes small open reading frames (smORFs) encoding polypeptides of fewer than 100 amino acids, and alternative open reading frames (alt-ORFs) encoding proteins 100 amino acids or larger. Sm/alt-ORFs previously escaped detection due to their short lengths, overlap with annotated protein coding sequences in different reading frames, and/or initiation with non-AUG start codons. Recent studies have shown that hundreds of smORFs are required for cell growth and survival, and some smORF-encoded polypeptides (SEPs) bind to and regulate the activity of macromolecular complexes involved in critical cellular processes and disease. However, the vast majority of smORFs remain entirely uncharacterized, and it is now imperative to identify their biological functions. We propose that SEPs with similar properties, motifs and modifications to canonical proteins are likely to be functional, and that we can leverage bio-orthogonal labeling and chemoproteomics to identify SEPs with properties of interest. We describe chemoproteomic strategies to identify newly translated SEPs, as well as to map SEPs to specific subcellular localizations, in high throughput, and reveal novel regulators of the critical cellular process of ribosome biogenesis. These studies show that chemical labeling and high-throughput proteomic technologies can reveal a dimension of functional information in tandem with smORF discovery, accelerating our ability to generate hypotheses about the biological roles of the "dark matter" of the human genome.
Sunday
3652808 - Cellular and whole animal quantitative chemiluminescence imaging using 1,2-dioxetanes
11:00am - 11:20am USA / Canada - Pacific - March 20, 2022 | Room: Sapphire O/P (Hilton San Diego Bayfront)
Alex Lippert, Presenter
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - In-person
Triggered chemiluminescence emission from sterically hindered 1,2-dioxetanes provides an analyte specific signal with low background, high sensitivity, and no phototoxicity. These features make chemiluminescence imaging particularly well-suited for quantification and imaging of analytes in living organisms. Our laboratory has pioneered two methods for quantitative chemiluminescence imaging using 1,2-dioxetanes in cells and in mouse models. Ratiometric chemiluminescence imaging has been achieved using a putative chemiluminescence resonance energy transfer (CRET) approach, providing dual wavelength emission that differentially responds to an analyte to provide an internal reference. Quantification with kinetics-based methods uses knowledge of measured kinetics parameters to calculate precise analyte concentrations from the raw chemiluminescence emissions. These two quantitative methods have been used for quantification of pH, oxygen, nitroxyl, and peroxynitrite in vitro and/or in vivo. This presentation will cover our laboratory’s most recent progress in this area.
Sunday
3659194 - Integrative strategy to uncover protein glutathionylation
11:20am - 11:40am USA / Canada - Pacific - March 20, 2022 | Room: Sapphire O/P (Hilton San Diego Bayfront)
Young-Hoon Ahn, Presenter
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - In-person

The reactive oxygen species (ROS) are key signaling molecules that control diverse biological processes, including cell migration. The major molecular targets of ROS are protein cysteines that form various thiol oxoforms, including S-glutathionylated cysteines. Despite the advance in identifying glutathionylated proteins, the identification of glutathionylated cysteines that regulate a specific biological process, such as cell migration, has been limited. We have developed an integrative strategy that combines our clickable glutathione-based chemical proteomic platform with functional biological analyses to streamline the identification of glutathionylated cysteines that control cell migration. Our approach enabled us to find three novel proteins, including PP2Cα, that potentially increase cell migration via glutathionylation. Biochemical and cellular analyses demonstrated that PP2Cα is susceptible to glutathionylation under hydrogen peroxide production and PP2Cα glutathionylation increases cell migration by activating the JNK pathway. Overall, we report a systematic strategy that identifies and validates glutathionylation-susceptible cysteines in regulating specific biological processes.

Sunday
3659696 - Expanding the sulfome: Characterization of hydrogen sulfide (H2S) and small oxoacids of sulfur (SOS) using derivatization and mass spectrometry
11:40am - 12:00pm USA / Canada - Pacific - March 20, 2022 | Room: Sapphire O/P (Hilton San Diego Bayfront)
Division: [BIOL] Division of Biological Chemistry
Session Type: Oral - In-person
Hydrogen sulfide (H2S) is an endogenous signaling molecule which like nitric oxide plays important roles in physiological functions related to cardiovascular health, but it’s mechanism of action remains poorly understood. We have developed methodology to characterize and quantify endogenous hydrogen sulfide (H2S) and small oxoacids of sulfur (SOS = HOSH, HOSOH) in numerous cell lines and mammalian tissue samples via translation of well-studied nucleophilic and electrophilic traps for cysteine and oxidized cysteines into a mass spectrometry methodology to trap small molecular weight species. This method allows quantification of these species giving both intra- and extra-cellular concentrations based on an H2S calibration curve. We propose the term sulfomic index, a ratio of S-O content, as a profile of non-protein sulfide redox status. The production of H2S and SOS is orders of magnitude higher in human melanoma and smooth muscle cells (nanomolar) as compared to other human cells (picomolar) and yeast and bacteria (femtomolar). In many cells the analytes concentrations following pattern of H2S > HOSOH > HOSH, but in primary vascular lines had distributions where HOSOH > H2S > HSOH, with much higher sulfomic indexes than seen in non-vascular derived cell lines. An important observation is that H2S and SOS are effluxed into surrounding media in much higher concentrations than found intracellularly. The efflux was continuous over time and against a concentration gradient, implying an active transport. Human primary aortic smooth muscle cells were used to examine changes in intracellular and efflux concentrations of H2S and SOS under hypoxic growth conditions. Under hypoxia, the smooth muscle cells demonstrated reduced mitochondrial respiration and increased ROS production, with a corresponding reduction in levels of H2S and HOSOH, but large increases in levels of HSOH, which we attribute to retrograde electron transfer to Complex 1 during hypoxia. We believe that measurements and characterizations these analytes under different disease states may help uncover the mechanisms of action attributed to H2S in cardiovascular health and disease.