Funding information NIGMSMethanol-chloroform based protein precipitation is an essential step in many liquid chromatography-tandem mass spectrometry-based cellular proteomics applications.However, re-solubilization of the total protein precipitate is difficult using regular in-solution digestion protocol. Sodium deoxycholate is reported as an efficient surfactant for re-solubilization of membrane fractions. In this study, we demonstrated an application combining methanol-chloroform based protein precipitations and deoxycholic acid assisted re-solubilization of pellets to evaluate the improvement of protein identifications in mass spectrometry-based bottom-up proteomics. We evaluated the modified method using an equal amount of Raw 264.7 mouse macrophage cell lysate.Detailed in-solution trypsin digestion studies were presented on methanol-chloroform precipitated samples with or without deoxycholic acid treatments and compared with popular sample digestion methods. A mass spectrometric analysis confirmed an 82% increase in protein identification in deoxycholic acid-treated samples compared to other established methods. Furthermore, liquid chromatography-tandem mass spectrometry analysis of an equal amount of proteins from methanol-chloroform precipitated, and methanol-chloroform/deoxycholic acid-treated macrophage cell lysate showed a 14% increase and 27% unique protein identifications. We believe this improved digestion method could be a complementary or alternative method for mammalian cell sample preparations where sodium dodecyl sulfate based lysis buffer is frequently used. K E Y W O R D Sdeoxycholic acid, methanol-chloroform precipitation, proteomics sample preparation, raw macrophages, trypsin digestion Article Related Abbreviations: ABC, ammonium bicarbonate; DCA, deoxycholic acid; FA, formic acid; IAA, iodoacetamide; MeOH-Chl, methanol-chloroform; MeOH-Chl-DCA, methanol-chloroform-deoxicholic acid; MeOH-Chl-NaDCO, methanol-chloroform-Na-deoxicholate; SDC/or NaDCO, sodium deoxycholate.
Pyruvate dehydrogenase complex (PDHC) and oxoglutarate dehydrogenase complex (OGDC), which belong to the mitochondrial α-ketoacid dehydrogenase family, play crucial roles in cellular metabolism. These multi-subunit enzyme complexes use lipoic arms covalently attached to their E2 subunits to transfer an acyl group to coenzyme A (CoA). Here, we report a novel mechanism capable of substantially inhibiting PDHC and OGDC: reactive nitrogen species (RNS) can covalently modify the thiols on their lipoic arms, generating a series of adducts that block catalytic activity. S-Nitroso-CoA, a product between RNS and the E2 subunit’s natural substrate, CoA, can efficiently deliver these modifications onto the lipoic arm. We found RNS-mediated inhibition of PDHC and OGDC occurs during classical macrophage activation, driving significant rewiring of cellular metabolism over time. This work provides a new mechanistic link between RNS and mitochondrial metabolism with potential relevance for numerous physiological and pathological conditions in which RNS accumulate.
Chemical crosslinking coupled with mass spectrometry (MS) is becoming a routinely and widely used technique for depicting and constructing protein structures and protein interaction networks. One major challenge for crosslinking/MS is the determination of informative low abundant intercrosslinked products, generated within a sample of high complexity. A C18 stationary phase is the conventional means for reverse-phase (RP) separation of inter-crosslinked peptides. Various RP stationary phases, which provide different selectivities and retentions, have been developed as alternatives to C18 stationary phases. In this study, two phenyl-based columns, biphenyl and fluorophenyl, were investigated and compared with a C18 phase for separating BS3 (bis(sulfosuccinimidyl)suberate) crosslinked bovine serum albumin (BSA) and myoglobin by bottom-up proteomics. Fractions from the 3 columns were collected and analyzed in a linear ion trap (LIT) mass spectrometer for improving detection of low abundant inter-crosslinked peptides. Among these three columns, the fluorophenyl column provides additional ion exchange interaction and exhibits unique retention in separating the crosslinked peptides. The fractioned data was analyzed in pLink, showing fluorophenyl column consistently obtained more inter-crosslinked peptide identifications than both C18 and biphenyl columns. For BSA crosslinked sample, the identified inter-crosslinked peptide number of fluorophenyl to C18 column are 136 to 102 in “low confident” result and 11 to 6 in “high confident” result. Fluorophenyl column could potentially be a better alternative for targeting the low stoichiometric inter-crosslinked peptides.
Protein post‐translational modifications and protein interactions are the central research areas in mass‐spectrometry‐based proteomics. Protein post‐translational modifications affect protein structures, stabilities, activities, and all cellular processes are achieved by interactions among proteins and protein complexes. With the continuing advancements of mass spectrometry instrumentations of better sensitivity, speed, and performance, selective enrichment of modifications/interactions of interest from complex cellular matrices during the sample preparation has become the overwhelming bottleneck in the proteomics workflow. Therefore, many strategies have been developed to address this issue by targeting specific modifications/interactions based on their physical properties or chemical reactivities, but only a few have been successfully applied for systematic proteome‐wide study. In this review, we summarized the highlights of recent developments in the affinity enrichment methods focusing mainly on low stoichiometric protein lipidations. Besides, to identify potential glyoxal modified arginines, a small part was added for profiling reactive arginine sites using an enrichment reagent. A detailed section was provided for the enrichment of protein interactions by affinity purification and chemical cross‐linking, to shed light on the potentials of different enrichment strategies, along with the unique challenges in investigating individual protein post‐translational modification or protein interaction network.
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