Serial enrichment-based quantitative proteomics enables deep and multiplexed profiling of post-translational modifications

Abstract Post-translational modifications (PTMs) play central roles in regulating protein function, localization, stability, and signaling networks. Comprehensive characterization of multiple PTMs, however, remains technically challenging due to limited sample availability, enrichment, incompatibilities, and analytical complexity. Here, we present an integrated serial enrichment workflow that enables quantitative profiling of multiple PTMs from a single biological sample. Using a streamlined strategy that combines StageTip-based fractionation with sequential immunoaffinity and metal oxide affinity enrichment, we achieved robust identification and quantification of lysine acetylation, lysine succinylation, phosphotyrosine, and global phosphorylation within the same sample. Application of this workflow resulted in deep proteome coverage alongside high-confidence PTM site identification with minimal sample loss and high reproducibility. Importantly, serial enrichment preserved PTM specificity and enabled comparative quantitative analysis across modification types. This approach provides a practical and scalable solution for integrated multi-PTM analysis, facilitating comprehensive interrogation of proteome regulation under limited sample conditions and offering broad applicability to systems biology, disease profiling, and translational proteomics studies. Competing Interest Statement The authors have declared no competing interest. Footnotes E-mail addresses: Mohd. Altaf Najar mohammadanajar{at}yenepoya.edu.in, Anjana Aravind anjanaaravind{at}yenepoya.edu.in, T. S. Keshava Prasad Keshava.prasad{at}nitte.edu.in, Prashant Kumar Modi prashantmodi{at}yenepoya.edu.in Data Availability Statement All the data related to the study are provided in the manuscript and supplementary data. Abbreviations - ACN - Acetonitrile - BCA - Bicinchoninic acid - CID - Collision-induced dissociation - CDK - Cyclin-dependent kinase - DDA - Data-dependent acquisition - DTT - Dithiothreitol - EDTA - Ethylenediaminetetraacetic acid - ERK - Extracellular signal-regulated kinase - FA - Formic acid - Fe-NTA - Ferric nitrilotriacetic acid - FR - Enriched and fractionated sample - FT - Final flowthrough - GO - Gene Ontology - HAT - Histone acetyltransferase - IAA - Iodoacetamide - IAP - Immunoaffinity purification - IMAC - Immobilized metal affinity chromatography - LC–MS/MS - Liquid chromatography tandem mass spectrometry - LFQ - Label-free quantification - MAPK - Mitogen-activated protein kinase - MS - Mass spectrometry - MS/MS - Tandem mass spectrometry - PBS - Phosphate-buffered saline - PTM - Post-translational modification - pY - Phosphotyrosine - RT - Room temperature - SDS-PAGE - Sodium dodecyl sulfate polyacrylamide gel electrophoresis - SOP - Standard operating procedure - TEABC - Triethylammonium bicarbonate - TFA - Trifluoroacetic acid - TiO₂ - Titanium dioxide - TMT - Tandem mass tag - UE - Unenriched sample - UF - Enriched but unfractionated sample