Improved detection of intact tyrosine sulfate-containing peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in linear negative ion mode

Sulfation of tyrosine residues is a common post-translational modification, but detecting and quantitating this modification poses challenges due to lability of the sulfate group. The goal of our studies was to determine how best to detect and to assess the stoichiometry of this modification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS). Sulfated and nonsulfated forms of peptides—hirudin(55–65), caerulein, and cholecystokinin octapeptide and phosphorylated and nonphosphorylated pp60-c-src (521–533)—were analyzed using several matrices: sinapinic acid (SA), 2,5-dihydroxybenzoic acid (DBA), and cyano-4-hydroxycinnamic acid (CHCA). Intact sulfated peptides were difficult to detect using positive ion mode; peptides were observed as desulfated ions. Phosphorylated peptide was stable and was detected in positive and negative ion modes. Detection of sulfated peptides improved with: (1) Analysis in negative ion mode, (2) Decreased laser power, (3) Matrix selection: DBA≥SA>CHCA. In negative ion mode, desorption/ionization of sulfated peptide was equivalent or more efficient than nonsulfated peptide, depending on conditions of analysis. Examination of a tryptic digest of α2-antiplasmin detected the single site of sulfation in negative ion mode but not in positive ion mode. We conclude that improved detection of sulfated peptides can be achieved in negative ion mode. Dual analysis in positive and negative ion modes serves as a potential means of identifying peptides with labile modifications such as sulfation and distinguishing them from phosphorylation.