Custom Workflow for the Confident Identification of Sulfotyrosine-Containing Peptides and Their Discrimination from Phosphopeptides.
Custom Workflow for the Confident Identification of Sulfotyrosine-Containing Peptides and Their Discrimination from Phosphopeptides.
复制标题
DOI：
10.1021/acs.jproteome.3c00425
复制
发表时间：
2023-12-01
影响因子：
4.4
通讯作者：
Eyers, Claire E.
中科院分区：
文献类型：
--
作者：
Daly, Leonard A.;Byrne, Dominic P.;Perkins, Simon;Brownridge, Philip J.;Mcdonnell, Euan;Jones, Andrew R.;Eyers, Patrick A.;Eyers, Claire E.
关键词：
蛋白酪氨酸硫酸化（sY）是由高尔基体酪氨酸蛋白磺基转移酶（TPSTs）催化的翻译后修饰（PTM）。关于人类sY的信息目前仅限于1050种蛋白质，只有少数几种蛋白质具有经过验证的硫酸化位点。因此，硫酸化对生物过程调节的贡献仍然不明确。基于质谱（MS）的蛋白质组学是PTM分析的首选方法，但尚未应用于“磺基”的系统研究，主要是由于与含sY的肽与含磷酸酪氨酸（pY）的肽的区分相关的问题。在这项研究中，我们开发了一个基于MS的sY-肽表征的工作流程，结合优化的Zr 4+固定化金属离子亲和色谱（IMAC）和TiO 2富集策略。广泛的表征的sY-和pY-肽使用一系列的片段化制度（CID，HCD，EThcD，ETciD，UVPD）突出的差异产生的位点决定的产物离子，并允许我们开发一种策略，区分硫酸化肽从名义上同量异位磷酸肽的基础上低碰撞能量诱导的中性损失。应用我们的“sulfomics”工作流程的HEK-293细胞外分泌组促进鉴定21个新的含磺基酪氨酸的蛋白质，其中几个我们验证酶，并揭示了新的酶之间的相互作用相关的蛋白质和聚糖硫酸化。
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 Zr4+ immobilized metal-ion affinity chromatography (IMAC) and TiO2 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.
登录
查看更多内容
DOI：
10.1042/bcj20180266
发表时间：
2018-08-14
期刊：
The Biochemical journal
影响因子：
--
作者：
Byrne DP;Li Y;Ngamlert P;Ramakrishnan K;Eyers CE;Wells C;Drewry DH;Zuercher WJ;Berry NG;Fernig DG;Eyers PA
通讯作者：
Eyers PA
DOI：
10.1042/bcj20180265
发表时间：
2018-08-14
期刊：
The Biochemical journal
影响因子：
--
作者：
Byrne DP;Li Y;Ramakrishnan K;Barsukov IL;Yates EA;Eyers CE;Papy-Garcia D;Chantepie S;Pagadala V;Liu J;Wells C;Drewry DH;Zuercher WJ;Berry NG;Fernig DG;Eyers PA
通讯作者：
Eyers PA
影响因子：
3.9
作者：
Connor, PA;McQuillan, AJ
通讯作者：
McQuillan, AJ
影响因子：
2
作者：
Boontheung, P;Brinkworth, CS;Baudinette, RV
通讯作者：
Baudinette, RV
影响因子：
7.3
作者：
Daly, Leonard A.;Brownridge, Philip J.;Eyers, Claire E.
通讯作者：
Eyers, Claire E.