Endogenous Protein-Protein Interaction Network of the NPC Cholesterol Transporter 1 in the Cerebral Cortex.

NPC intracellular cholesterol transporter 1 (NPC1) is a multipass, transmembrane glycoprotein mostly recognized for its key role in facilitating cholesterol efflux. Mutations in the NPC1 gene result in Niemann-Pick disease, type C (NPC), a fatal, lysosomal storage disease. Due to the progressively expanding implications of NPC1-related disorders, we investigated endogenous NPC1 protein-protein interactions in the mouse cortex and human-derived iPSCs neuronal models of the disease through coimmunoprecipitation-coupled with LC-MS based proteomics. The current study investigated protein-protein interactions specific to the wild-type and the most prevalent NPC1 mutation (NPC1I1061T) while filtering out any protein interactor identified in the Npc1-/- mouse model. Additionally, the results were matched across the two species to map the parallel interactome of wild-type and mutant NPC1I1061T. Most of the identified wild-type NPC1 interactors were related to cytoskeleton organization, synaptic vesicle activity, and translation. We found many putative NPC1 interactors not previously reported, including two SCAR/WAVE complex proteins that regulate ARP 2/3 complex actin nucleation and multiple membrane proteins important for neuronal activity at synapse. Moreover, we identified proteins important in trafficking specific to wild-type and mutant NPC1I1061T. Together, the findings are essential for a comprehensive understanding of NPC1 biological functions in addition to its classical role in sterol efflux.

Effects of Amino Acid Additives on Protein Stability during Electrothermal Supercharging in ESI-MS.

The surprising formation of highly charged protein ions from aqueous ammonium bicarbonate solution is a fascinating phenomenon referred to as electrothermal supercharging (ETS). Although the precise mechanism involved is not clearly understood, previous studies predominantly suggest that ETS is due to native protein destabilization in the presence of bicarbonate anion inside the electrospray ionization droplets under high temperatures and spray voltages. To evaluate existing hypotheses surrounding the underlying mechanism of ETS, the effects of several additives on protein charging under ETS conditions were investigated. The changes in the protein charge state distributions were compared by measuring the ratios between the intensities of highest intensity charge states of native and unfolded protein envelopes and shifts in the lowest and highest observed charge states. This study demonstrated that source temperature plays a more important role in ETS compared to spray voltage, especially when using a nebulized microelectrospray ionization source. Moreover, the effect of amino acids on ETS were generally in good agreement with the extensive literature available on the stabilization or destabilization of proteins by these additives in bulk solution. Among the natural amino acids, protein supercharging was significantly reduced by proline and glycine; however, imidazole provided the highest degree of noncovalent complex stabilization against ETS, outperforming the amino acids. Overall, our study shows that the simple addition of stabilizing reagents such as proline and imidazole can reduce the extent of apparent protein unfolding and supercharging in ammonium bicarbonate solution and provide evidence against the roles of charge depletion and thermal unfolding during ETS.

Analysis of histidine-tagged recombinant proteins from nickel and copper coated surfaces by direct electrospray ionization and desorption electrospray ionization mass spectrometry.

RATIONALE: 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). METHODS: A protein standard, His-Ubq, and two recombinant proteins, His-SHAN and His-CS, expressed in Escherichia coli were 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. RESULTS: 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 clarified E. coli cell 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. CONCLUSIONS: 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.

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

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.

The Addition of Polar Organic Solvent Vapors During the Analysis of Proteins by DESI-MS.

Exposure of electrospray droplets to organic vapors was shown to dramatically reduce alkali-metal adduction on protein ions and shift protein charge states. Since DESI-MS is affected by similar adduct species as ESI-MS and shares similar ionization mechanisms, polar organic vapor additives should likewise also improve the DESI-MS analysis of proteins. Here the DESI spray was exposed to a variety of polar organic vapor additives. Head space vapors of polar organic solvents were entrained in nitrogen gas and delivered to the atmosphere inside a semi-enclosed plastic enclosure surrounding the spray plume. The vapors of acetone, acetonitrile, ethyl acetate, methanol, and water were investigated. Vapor dependent effects were observed with respect to changes in protein charge state distributions and signal intensities. With ethyl acetate vapor addition, the signal intensities of all proteins investigated were significantly increased, including proteins larger than 25 kDa such as carbonic anhydrase II and bovine serum albumin.

Addition of Serine Enhances Protein Analysis by DESI-MS.

Previous studies have suggested that the loss in sensitivity of DESI-MS for large molecules such as proteins is due to the poor dissolution during the short time scale of desorption and ionization. An investigation into the effect of serine as a solvent additive leads to the interesting observation that there is a concentration-dependent improvement in protein signal intensity when micromolar to low millimolar concentrations of serine is combined with a suitable co-additive in DESI spray. This effect, however, was not observed during similar ESI-MS experiments, where the same solvents and proteins were sprayed directly into the MS inlet. This suggests that the mechanism of signal improvement in DESI is associated with the desorption step of proteins, possibly by facilitating dissolution or improving solubility of proteins on the surface in the solvent micro-layer formed during DESI. Other than poor dissolution, cation adduction such as by sodium ions is also a major contributing factor to the mass-dependent loss in sensitivity in both ESI and DESI, leading to an increase in limits of detection for larger proteins. The adduction becomes a more pressing issue in native-state studies of proteins, as lower charge states are more susceptible to adduction. Previous studies have shown that addition of amino acids to the working spray solution during ESI-MS reduces sodium adduction and can help in stabilization of native-state proteins. Similar to the observed reduction in sodium adducts during native-state ESI-MS, when serine is added to the desorbing spray in DESI-MS, the removal of up to 10 mM NaCl is shown. A selection of proteins with high and low pI and molecular weights was analyzed to investigate the effects of serine on signal intensity by improvements in protein solubility and adduct removal. Graphical Abstract.