Session: Advances in Mass Spectrometry:

Proteins are important biological macromolecules that are undeniably fundamental to life. Inspired by Nature, naturally occurring amino acids have been broadly used as additives to improve protein stability and solubility. Amino acids have been named “compatible solutes” because they improve protein solubility and stability without affecting protein function. Protein solubility and stability are major concerns for many industrial and pharmaceutical applications, and bioanalytical techniques. While ESI-MS has proven to be an unparalleled technique for protein analysis, when coupled to desorption (DESI-MS), it is fraught with complications and lacks sensitivity. Protein analysis by DESI-MS is believed to be inefficient due to poor protein dissolution in the micro-localized solvent layer formed by the DESI sprayer during desorption. We demonstrated that amino acids, in particular L-serine, improve protein analysis by DESI-MS. The mechanism for this improvement is yet unknown and is the subject of this study. A similar signal improvement was not observed in ESI-MS, suggesting that the observed signal improvement in DESI-MS is associated with the desorption stage. Different approaches were taken to investigate the mechanism of this signal improvement: (1) To better understand the role of amino acid additives in the desorption stage, L-serine was physically placed in different positions during sample preparation; (2) The effect of spray solvent composition was tested in five different solvent systems; (3) The role of protein conformation prior to analysis on the effect of L-serine was studied through comparison of natively deposited protein and protein denatured by acidification before sample preparation; (4) To evaluate the importance of L-serine molecular structure, different derivatives of L-serine, as well as other naturally occurring amino acids that are similar to serine, were also studied. Outcomes included: (1) Any position of serine except mixed with protein prior to analysis works equally well, but it is especially convenient to add it in the desorption solvent; (2) Signal improvement was only observed with denaturing desorption solvents; (3) The effect of L-serine is more significant with natively deposited proteins; (4) Any modification to L-serine molecular structure reduced effectiveness. These findings point to a direct interaction between L-serine or its clusters with the protein that influence protein solubility and aggregation during unfolding.

Roshan Javanshad

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