Daly, Leonard A ORCID: 0000-0001-9712-9676, Byrne, Dominic P, Perkins, Simon, Brownridge, Philip J, McDonnell, Euan, Jones, Andrew R ORCID: 0000-0001-6118-9327, Eyers, Patrick A ORCID: 0000-0002-9220-2966 and Eyers, Claire E ORCID: 0000-0002-3223-5926
(2023)
Custom Workflow for the Confident Identification of Sulfotyrosine-Containing Peptides and Their Discrimination from Phosphopeptides.
Journal of proteome research, 22 (12).
pp. 3754-3772.
Abstract
Protein tyrosine sulfation (sY) is a post-translational modification (PTM) catalyzed by Golgi-resident tyrosyl protein sulfo transferases (TPSTs). Information on sY in humans is currently limited to ∼50 proteins, with only a handful having verified sites of sulfation. As such, the contribution of sulfation to the regulation of biological processes remains poorly defined. Mass spectrometry (MS)-based proteomics is the method of choice for PTM analysis but has yet to be applied for systematic investigation of the "sulfome", primarily due to issues associated with discrimination of sY-containing from phosphotyrosine (pY)-containing peptides. In this study, we developed an MS-based workflow for sY-peptide characterization, incorporating optimized Zr<sup>4+</sup> immobilized metal-ion affinity chromatography (IMAC) and TiO<sub>2</sub> enrichment strategies. Extensive characterization of a panel of sY- and pY-peptides using an array of fragmentation regimes (CID, HCD, EThcD, ETciD, UVPD) highlighted differences in the generation of site-determining product ions and allowed us to develop a strategy for differentiating sulfated peptides from nominally isobaric phosphopeptides based on low collision energy-induced neutral loss. Application of our "sulfomics" workflow to a HEK-293 cell extracellular secretome facilitated identification of 21 new sulfotyrosine-containing proteins, several of which we validate enzymatically, and reveals new interplay between enzymes relevant to both protein and glycan sulfation.
Item Type: | Article |
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Uncontrolled Keywords: | Humans, Tyrosine, Phosphotyrosine, Phosphopeptides, Proteins, Workflow, HEK293 Cells |
Divisions: | Faculty of Health and Life Sciences Faculty of Health and Life Sciences > Institute of Systems, Molecular and Integrative Biology |
Depositing User: | Symplectic Admin |
Date Deposited: | 20 Nov 2023 15:53 |
Last Modified: | 05 Jan 2024 10:24 |
DOI: | 10.1021/acs.jproteome.3c00425 |
Open Access URL: | https://doi.org/10.1021/acs.jproteome.3c00425 |
Related URLs: | |
URI: | https://livrepository.liverpool.ac.uk/id/eprint/3176911 |