Searching for Sulfotyrosines (sY) in a HA(pY)STACK

Protein sulfation can be crucial in regulating protein–protein
interactions but remains largely underexplored. Sulfation is nearly
isobaric to phosphorylation, making it particularly challenging to
investigate using mass spectrometry. The degree to which tyrosine
sulfation (sY) is misidentified as phosphorylation (pY) is, thus,
an unresolved concern. This study explores the extent of sY misidentification
within the human phosphoproteome by distinguishing between sulfation
and phosphorylation based on their mass difference. Using Gaussian
mixture models (GMMs), we screened ∼45 M peptide-spectrum matches
(PSMs) from the PeptideAtlas human phosphoproteome build for peptidoforms
with mass error shifts indicative of sulfation. This analysis pinpointed
104 candidate sulfated peptidoforms, backed up by Gene Ontology (GO)
terms and custom terms linked to sulfation. False positive filtering
by manual annotation resulted in 31 convincing peptidoforms spanning
7 known and 7 novel sY sites. Y47 in calumenin was particularly intriguing
since mass error shifts, acidic motif conservation, and MS2 neutral loss patterns characteristic of sulfation provided strong
evidence that this site is sulfated rather than phosphorylated. Overall,
although misidentification of sulfation in phosphoproteomics data
sets derived from cell and tissue intracellular extracts can occur,
it appears relatively rare and should not be considered a substantive
confounding factor in high-quality phosphoproteomics data sets.

蛋白质硫酸化在调控蛋白质-蛋白质相互作用中发挥关键作用，但目前相关研究仍未得到充分探索。硫酸化与磷酸化几乎等压，这使得利用质谱（mass spectrometry）开展相关研究极具挑战性。因此，酪氨酸硫酸化（tyrosine sulfation, sY）被误判为磷酸化（phosphorylation, pY）的程度，仍是一个尚未解决的问题。本研究基于硫酸化与磷酸化的质量差异对二者进行区分，以此探究人类磷酸化蛋白质组中sY被误判的程度。研究使用高斯混合模型（Gaussian mixture models, GMMs），从PeptideAtlas人类磷酸化蛋白质组构建版本的约4500万个肽段-谱图匹配（peptide-spectrum matches, PSMs）中，筛选出带有指示硫酸化的质量误差偏移的肽型（peptidoforms）。本次分析共确定了104个候选硫酸化肽型，相关结果得到了与硫酸化相关的基因本体（Gene Ontology, GO）术语及自定义注释术语的支持。通过人工注释开展假阳性筛选后，最终得到31个可靠的硫酸化肽型，涵盖7个已知酪氨酸硫酸化位点与7个全新酪氨酸硫酸化位点。钙联蛋白（calumenin）的Y47位点尤为引人关注：质量误差偏移、酸性基序保守性，以及硫酸化特征性的MS2中性丢失模式，均为该位点发生硫酸化而非磷酸化提供了强有力的证据。总体而言，尽管在源自细胞及组织细胞内提取物的磷酸化蛋白质组数据集当中，可能存在硫酸化被误判的情况，但这类误判相对罕见，不应将其视为高质量磷酸化蛋白质组数据集的实质性混杂因素。