A bespoke analytical workflow for the identification of sulfopep-tides and confident discrimination from phosphopeptides_C4PR_LIV

ABSTRACT: Protein tyrosine sulfation (sY) is a post-translational modification (PTM) catalysed by Golgi-resident Tyrosyl Protein Sul-foTransferases (TPSTs). Information on protein tyrosine sulfation is currently limited to ~50 human proteins with only a handful of those having verified sites of sulfation. The contribution of this chemical moiety for the regulation of biological processes, both inside and outside the cell, remains poorly defined in large part due to analytical limitations. Mass spectrom-etry-based proteomics is the method of choice for PTM analysis, but has yet to be applied for the systematic investigation of biological sulfation (the ‘sulfome’), primarily due to issues associated with discrimination of sY- from phosphotyrosine (pY)-containing peptides. In this study, we developed a mass spectrometry (MS)-based workflow centred on the characteri-zation of sY-peptides, incorporating optimised Zr4+-IMAC and TiO2 enrichment strategies. Extensive characterization of a panel of sY- and pY-peptides using an array of MS fragmentation regimes (CID, HCD, EThcC, ETciD, UVPD) highlights differences in the ability to generate site-determining product ions, which can be exploited to differentiate sulfated peptides from nominally isobaric phosphopeptides based on precursor ion neutral loss at low collision energy. Application of our ana-lytical workflow to the HEK-293 cell extracellular secretome facilitated identification of 23 new sulfotyrosine-containing peptides, several of which we validate enzymatically using in vitro sulfation assays. Our study demonstrates the applicabil-ity of this strategy for confident, high-throughput, ‘sulfomics’ studies.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: leonard daly  

LAB HEAD: Claire eyers

PROVIDER: PXD043723 | Pride | 2024-01-26

REPOSITORIES: Pride