Sulfoproteomics Workflow with Precursor Ion Accurate Mass Shift Analysis Reveals Novel Tyrosine Sulfoproteins in the Golgi

Abstract

Tyrosine sulfation in the Golgi of secreted and membrane proteins is an important post-translational modification (PTM). However, its labile nature has limited analysis by mass spectrometry (MS), a major reason why no sulfoproteome studies have been previously reported. Here, we show that a phosphoproteomics experimental workflow, which includes serial enrichment followed by high resolution, high mass accuracy MS, and tandem MS (MS/MS) analysis, enables sulfopeptide coenrichment and identification via accurate precursor ion mass shift open MSFragger database search. This approach, supported by manual validation, allows the confident identification of sulfotyrosine-containing peptides in the presence of high levels of phosphorylated peptides, thus enabling these two sterically and ionically similar isobaric PTMs to be distinguished and annotated in a single proteomic analysis. We applied this approach to isolated interphase and mitotic rat liver Golgi membranes and identified 67 tyrosine sulfopeptides, corresponding to 26 different proteins. This work discovered 23 new sulfoproteins with functions related to, for example, Ca2+-binding, glycan biosynthesis, and exocytosis. In addition, we report the first preliminary evidence for crosstalk between sulfation and phosphorylation in the Golgi, with implications for functional control.

Electron Capture vs Transfer Dissociation for Site Determination of Tryptic Peptide Tyrosine Sulfation: Direct Detection of Fibrinogen Sulfation Sites and Identification of Novel Isobaric Interferences

“…This neutral loss differentiates tyrosine sulfation from tyrosine phosphorylation because only partial loss of HPO 3 (79.9663) is observed from phosphotyrosine. In addition, mostly H 3 PO 4 loss (97.98 Da) is observed from serine- and threonine-phosphorylated peptides . Thus, the sulfate modification is confirmed (Figure c).…”

“…Although Tyr sulfation is anticipated to occur in about 1% of eukaryotic proteins, it is an understudied post-translational modification (PTM) with only a few sulfoproteins identified . Recent advances in enrichment strategies, data acquisition strategies, and bioinformatics utilities allowed the first sulfoproteomic analyses of Golgi resident proteins as well as secreted proteins from HEK-293 cells . Tyr sulfation can play a critical role as a part of the “interactome” (i.e., the interaction of a protein with other biomolecules) by regulating extracellular protein–protein interactions to, e.g., harmonize hemostasis, and play roles in leukocyte adhesion, chemokine signaling, as well as inflammatory response. − In addition, there is evidence for cross talk with other PTMs, for example, colocalization of sulfotyrosine and O -glycosylation in crucially functional domains, and sulfo- and phospho-tyrosine in Golgi proteins …”

“…Despite the absence of sulfate-retaining fragments in conventional CID/HCD-based MS/MS analysis, sulfopeptide candidates can be identified via characteristic SO 3 loss upon low energy HCD or via MSFragger-based precursor ion accurate mass shift analysis as compared with an identified, unmodified tyrosine-containing peptide sequence . However, these CID/HCD-based approaches only indirectly assign tyrosine sulfation and cannot identify the sulfation site if more than one tyrosine residue is present.…”

Golgi defect as a major contributor to lysosomal dysfunction

“…Upon the arrival of proteins from the ER into the Golgi, a diverse array of post-translational modifications occurs, including the addition or removal of sugar residues in carbohydrate chains (glycosylation), phosphorylation, sulfation, as well as lipid metabolism and the synthesis of complex lipids such as glycolipids and sphingolipids ( Huang and Wang, 2017 ; Li et al, 2019a ; Kweon et al, 2024 ). In addition to these modifications, the Golgi serves as the focal point for sorting and directing proteins and lipids to their intended destinations within the cell, such as the plasma membrane, lysosomes, or various intracellular organelles, where they can carry out their functions ( Bankaitis et al, 2012 ; Pothukuchi et al, 2021 ).…”

Negative-Ion Electron Capture Dissociation of MALDI-Generated Peptide Anions

“…Although much less common, negative-ion-mode analysis can provide complementary peptide identifications and improved ionization as well as stability of acidic PTMs such as phosphorylation and sulfation . These common PTMs perform various biological roles, e.g., facilitating protein–protein interactions, − but can be difficult to characterize with conventional positive-ion mode MS/MS activation methods . Specifically, vibrationally activating methods like collision-induced dissociation and infrared multiphoton dissociation may yield high sequence coverage but often cause preferential ejection or migration of labile PTMs. − …”