Phosphoric acid as a matrix additive for MALDI MS analysis of phosphopeptides and phosphoproteins

Phosphopeptides are often detected with low efficiency by MALDI MS analysis of peptide mixtures. In an effort to improve the phosphopeptide ion response in MALDI MS, we investigated the effects of adding low concentrations of organic and inorganic acids during peptide sample preparation in 2,5-dihydroxybenzoic acid (2,5-DHB) matrix. Phosphoric acid in combination with 2,5-DHB matrix significantly enhanced phosphopeptide ion signals in MALDI mass spectra of crude peptide mixtures derived from the phosphorylated proteins α-casein and β-casein. The beneficial effects of adding up to 1% phosphoric acid to 2,5-DHB were also observed in LC-MALDI-MS analysis of tryptic phosphopeptides of B. subtilis PrkC phosphoprotein. Finally, the mass resolution of MALDI mass spectra of intact proteins was significantly improved by using phosphoric acid in 2,5-DHB matrix.