Issue 21, 2021

Effects of amino acid additives on protein solubility – insights from desorption and direct electrospray ionization mass spectrometry

Abstract

Naturally occurring amino acids have been broadly used as additives to improve protein solubility and inhibit aggregation. In this study, improvements in protein signal intensity obtained with the addition of L-serine, and structural analogs, to the desorption electrospray ionization mass spectrometry (DESI-MS) spray solvent were measured. The results were interpreted at the hand of proposed mechanisms of solution additive effects on protein solubility and dissolution. DESI-MS allows for these processes to be studied efficiently using dilute concentrations of additives and small amounts of proteins, advantages that represent real benefits compared to classical methods of studying protein stability and aggregation. We show that serine significantly increases the protein signal in DESI-MS when native proteins are undergoing unfolding during the dissolution process with an acidic solvent system (p-value = 0.0001), or with ammonium bicarbonate under denaturing conditions for proteins with high isoelectric points (p-value = 0.001). We establish that a similar increase in the protein signal cannot be observed with direct ESI-MS, and the observed increase is therefore not related to ionization processes or changes in the physical properties of the bulk solution. The importance of the presence of serine during protein conformational changes while undergoing dissolution is demonstrated through comparisons between the analyses of proteins deposited in native or unfolded states and by using native state-preserving and denaturing desorption solvents. We hypothesize that direct, non-covalent interactions involving all three functional groups of serine are involved in the beneficial effect on protein solubility and dissolution. Supporting evidence for a direct interaction include a reduction in efficacy with D-serine or the racemic mixture, indicating a non-bulk-solution physical property effect; insensitivity to the sample surface type or relative placement of serine addition; and a reduction in efficacy with any modifications to the serine structure, most notably the carboxyl functional group. An alternative hypothesis, also supported by some of our observations, could involve the role of serine clusters in the mechanism of solubility enhancement. Our study demonstrates the capability of DESI-MS together with complementary ESI-MS experiments as a novel tool for understanding protein solubility and dissolution and investigating the mechanism of action for solubility-enhancing additives.

Graphical abstract: Effects of amino acid additives on protein solubility – insights from desorption and direct electrospray ionization mass spectrometry

Supplementary files

Article information

Article type
Paper
Submitted
02 Aug 2021
Accepted
22 Sep 2021
First published
27 Sep 2021

Analyst, 2021,146, 6592-6604

Effects of amino acid additives on protein solubility – insights from desorption and direct electrospray ionization mass spectrometry

R. Javanshad and A. R. Venter, Analyst, 2021, 146, 6592 DOI: 10.1039/D1AN01392K

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