Abstract
We have developed an efficient, sensitive, and specific method for the detection of phosphopeptides present in peptide mixtures by MALDI Q-TOF mass spectrometry. Use of the MALDI Q-TOF enables selection of phosphopeptides and characterization by CID of the phosphopeptides performed on the same sample spot. However, this type of experiment has been limited by low ionization efficiency of phosphopeptides in positive ion mode while selecting precursor ions of phosphopeptides. Our method entails neutralizing negative charges on acidic groups of nonphosphorylated peptides by methyl esterification before mass spectrometry in positive and negative ion modes. Methyl esterification significantly increases the relative signal intensity generated by phosphopeptides in negative ion mode compared with positive ion mode and greatly increases selectivity for phosphopeptides by suppressing the signal intensity generated by acidic peptides in negative ion mode. We used the method to identify 12 phosphopeptides containing 22 phosphorylation sites from low femtomolar amounts of a tryptic digest of beta-casein and alpha-s-casein. We also identified 10 phosphopeptides containing five phosphorylation sites from an in-gel tryptic digest of 100 fmol of an in vitro autophosphorylated fibroblast growth factor receptor kinase domain and an additional phosphopeptide containing another phosphorylation site when 500 fmol of the digest was examined. The results demonstrate that the method is a fast, robust, and sensitive means of characterizing phosphopeptides present in low abundance mixtures of phosphorylated and nonphosphorylated peptides.
Highlights
We have developed an efficient, sensitive, and specific method for the detection of phosphopeptides present in peptide mixtures by MALDI Q-TOF mass spectrometry
We have shown that the method can be used to rapidly monitor all previously identified phosphorylation sites on subpicomolar amounts of the medically important N549H mutant FGFR2 tyrosine kinase domain
Based on a previous study of the highly homologous FGFR1 kinase domain, we expected to find six tyrosine phosphorylation sites on the kinase domain of FGFR2
Summary
Materials—2,6-Dihydroxyacetophenone (DHAP), ␣-cyano-4-hydroxycinnamic acid (CHCA), dihydroxybenzoic acid (DHB), diammonium hydrogen citrate, and acetyl chloride were purchased from Sigma-Aldrich Two picomoles of the phosphorylated kinase domain was diluted in this sample buffer at a ratio of 1:1 (v/v), denatured at 95 °C for 5 min. In-solution Digestion and in-Gel Digestion of Proteins—For in-solution digestion, proteins were dissolved in 25 mM ammonium bicarbonate and denatured by heating in 95 °C water for 5 min and digested in 25 mM ammonium bicarbonate by sequence-grade trypsin at a ratio of 1:50 (enzyme/protein) for 5 h at 37 °C. Preparation of Matrix for MALDI Q-TOF Mass Spectrometry—A 50 mM solution of the DHAP matrix was prepared by dissolving 15.2 mg of 2,6-dihydroxyacetophenone in 1 ml water/methanol (10:90, v/v) followed by the addition of 100 mM diammonium hydrogen citrate in water at a ratio of 1:1 (v/v)..
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