Modified cysteine S-phosphopeptide standards for mass spectrometry-based proteomics

Abstract

The regulatory role of protein cysteine phosphorylation is an under-researched area. The difficulty of accessing reference S-phosphorylated peptides (pCys-peptides) hampers progress in MS-driven cysteine phosphoproteomics, which requires targeted analytical procedures. This work describes an uncomplicated process for the conversion of disulfide-bridged protein into a complex model mixture of combinatorially modified peptides. Hen egg-white lysozyme was reduced with tris(2carboxyethyl)phosphine (TCEP) followed by alkylation of cysteine with (3-acrylamidopropyl)trimethyl-ammonium chloride (APTA) and subsequent beta-elimination in aqueous Ba(OH) 2 to yield modified polypeptides containing multiple dehydroalanine (Dha) residues. The conjugate addition of thiophosphoric acid to Dha residues followed by trypsinolysis led to numerous D/L phosphocysteine-containing peptides, which were identified by higher-energy collisional-dissociation tandem mass spectrometry (HCD-MS/MS). Our results show that some pCys-peptides produce prominent neutral losses of 80 Da, 98 Da and a weak 116 Da loss. These are similar to the neutral-loss triplets generated by phosphohistidine peptides.

Protein cysteine S-glycosylation: oxidative hydrolysis of protein S-glycosidic bonds in aqueous alkaline environments

“…The initial research phase required inventing a feasible procedure for synthesizing S-glycosylated model proteins. The chemical transformation of lysozyme P00698 into multi-dehydroalanine-containing derivatives, earlier described in (Buchowiecka 2019 ), followed by the conjugate addition of 1-thio-β-D-glucose to dehydroalanine (Dha) residues, permitted the incorporation of β-S-glucosylated cysteines into the lysozyme sequence (Fig. 1 ).…”

“…In Step C, β-elimination of Qat-SH upon barium hydroxide led to products of the general formula L-C[S-Qat] 2±1 -[Dha] 6±1 -[Side +]-COOH carrying on average x = (6 ± 1) Dha residues per lysozyme molecule. Unintended side modifications [Side +] also occurred, such as deamidation of Asn and Gln or carbamylation of Lys and N-terminal amine groups (Buchowiecka 2019 ). The final conjugate addition of 1-β-S-Glc to the Dha produced a mixture of polypeptides of the general formula L-C[S-Qat] 2±1 -[Dha] 4±1 -C[S-Glc] 2±1 -[Side +]-COOH—i.e., bearing on cysteine positions C[S-Qat], [Dha], and C D/L [S-Glc] (Step D).…”

“…S-glycosylated protein was obtained by the chemical transformation of hen egg-white lysozyme P00698. The synthetic procedure comprises five basic steps and is analogous to a method described previously (Buchowiecka 2019 ). The three initial steps (A, B, C in.…”

“…After each step of the transformations, the products were precipitated with an excess of isopropanol (5:1/v) and analyzed by MALDI MS. The resulting lysozyme derivatives containing on average 5–7 Dha residues per molecule, denoted as L-C[S-Qat] 2 ± 1 -[Dha] 6 ± 1 -[Side +]-COOH (Buchowiecka 2019 ), were subjected to Step D of the transformation.

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A Novel Adductomics Workflow Incorporating FeatureHunter Software: Rapid Detection of Nucleic Acid Modifications for Studying the Exposome

“…While our results focused on detecting NA modifications using LC-Q-LIT-OT-MS, FeatureHunter ’s filtering of informative features in MS 2 spectra and its ability to classify certain compounds based on the user-defined tags makes it versatile for use with other mass analyzers. Furthermore, numerous classes of toxicologically and pharmacologically relevant compounds are also revealed through neutral loss or fragment ion detection, making FeatureHunter a valuable asset across diverse research domains (e.g., the analysis of protein phosphorylation, glutathione conjugates, and lipid species ,, ). The incorporation of FeatureHunter into the NA adductomics workflow is likely to be an invaluable tool to the successful processing of the extensive and intricate data sets produced by LC-HR-MS/MS.…”