Development of a Sample-Preparation Workflow for Sulfopeptide Enrichment: From Target Analysis to Challenges in...

Protein tyrosine O-sulfation is an important post-translational modification, as it has been correlated to inflammation, virus infection, and signal pathways. Nevertheless, methods for the characterization of protein sulfation by sulfopeptide enrichment are currently limited. In this Article, two standard compounds, representative of mono- and disulfated peptides, were used to compare the enrichment capabilities of five sorbent materials: two commercial weak anion-exchange mixed-mode sorbents (Strata X-AW and Oasis WAX) and three phosphopeptide enrichment materials based on affinity chromatography to either immobilized metals (IMAC) or metal oxides, i.e., Fe³⁺, TiO₂, or Ti⁴⁺. The sulfopeptides were analyzed by ultrahigh-performance liquid chromatography (UHPLC) multiple-reaction monitoring analysis and were stable under all the tested experimental conditions. Recoveries of the enrichment step from spiked bovine serum albumin digests were >80% for the commercial Fe-IMAC kit and the Strata X-AW sorbent. Shotgun proteomics was used on the same samples to evaluate the selectivity, calculated as the number of coenriched peptides, and it was found to be better for the Fe-IMAC kit. Therefore, the Fe-IMAC protocol was embedded in a shotgun-proteomics workflow and applied to serum spiked with the sulfopeptides before protein dephosphorylation and digestion. The recovery of the entire analytical workflow was 20%, which was compatible with previous data on TiO₂ phosphopeptide enrichment. Despite the potential, no sulfopeptide was confidently identified in serum digests by conventional shotgun proteomics, probably due to very low abundance of native sulfoproteins, poor ionization efficiency of sulfopeptides in the positive mode, and lack of unambiguous sulfopeptide identification by bioinformatics software. In this context, the use of negative-ionization mode with high-resolution mass spectrometry appeared promising to improve the sensibility and allow sulfopeptide identification in complex samples.