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Purification of recombinant proteins is a necessary step for functional or structural studies and other applications. Immobilized metal affinity chromatography is a common recombinant protein purification method. Mass spectrometry (MS) allows for confirmation of identity of expressed proteins and unambiguous detection of enzymatic substrates and reaction products. We demonstrate the detection of enzymes purified on immobilized metal affinity surfaces by direct or ambient ionization MS, and follow their enzymatic reactions by direct electrospray ionization (ESI) or desorption electrospray ionization (DESI).

A protein standard, His‐Ubq, and two recombinant proteins, His‐SHAN and His‐CS, expressed inEscherichia coliwere immobilized on two immobilized metal affinity systems, Cu–nitriloacetic acid (Cu‐NTA) and Ni‐NTA. The proteins were purified on surface, and released in the ESI spray solvent for direct infusion, when using the 96‐well plate form factor, or analyzed directly from immobilized metal affinity‐coated microscope slides by DESI‐MS. Enzyme activity was followed by incubating the substrates in wells or by depositing substrate on immobilized protein on coated slides for analysis.

Small proteins (His‐Ubq) and medium proteins (His‐SAHN) could readily be detected from 96‐well plates by direct infusion ESI, or from microscope slides by DESI‐MS after purification on surface from clarifiedE. colicell lysate. Protein oxidation was observed for immobilized proteins on both Cu‐NTA and Ni‐NTA; however, this did not hamper the enzymatic reactions of these proteins. Both the nucleosidase reaction products for His‐SAHN and the methylation product of His‐CS (theobromine to caffeine) were detected.

The immobilization, purification, release and detection of His‐tagged recombinant proteins using immobilized metal affinity surfaces for direct infusion ESI‐MS or ambient DESI‐MS analyses were successfully demonstrated. Recombinant proteins were purified to allow identification directly out of clarified cell lysate. Biological activities of the recombinant proteins were preserved allowing the investigation of enzymatic activity via MS.

 

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.