Identification of Phosphopeptides by MALDI Q-TOF MS in Positive and Negative Ion Modes after Methyl Esterification

Chong-Feng Xu, Yun Lu, Jinghong Ma, Moosa Mohammadi, Thomas A. Neubert
2005 Molecular & Cellular Proteomics  
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
more » ... 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 ␤-casein and ␣-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. Molecular & Cellular Proteomics 4:809 -818, 2005. Reversible phosphorylation of serine, threonine, and tyrosine residues is one of the most common and important regulatory modifications of proteins and often is a key event in cellular signal transduction (1, 2). In recent years, mass spectrometry has become a key technology for characterization of protein phosphorylation and phosphoproteome analysis. Two complementary ionization techniques, MALDI and ESI, in combination with a variety of mass analyzers, have been used to identify phosphopeptides and determine the phosphorylated amino acids on the peptides (3-16). In most cases, characterization of phosphopeptides by MS requires selection of phosphorylated peptides from complex peptide mixtures resulting from proteolysis of phosphorylated proteins followed by MS/MS to confirm the phosphorylation and to identify the phosphorylated amino acid residues on phosphopeptides containing more than a single serine, threonine, or tyrosine residue. Despite many recent advances in methodology, identification of phosphorylation sites on proteins remains a difficult challenge (17). High sensitivity, resolution, and mass accuracy make Q-TOF MS a powerful tool for the characterization of phosphopeptides. The MALDI Q-TOF allows for increased efficiency and sample throughput because identification of phosphopeptides by MS and characterization by CID MS/MS can be performed on a single sample spot (18). However, a limitation of this type of experiment is the low ionization efficiency of phosphopeptides in positive ion mode, resulting in low sensitivity of phosphopeptide detection and consumption of a large portion of the sample during the search for phosphorylated precursor peptides before MS/MS can be performed (18). Phosphorylated precursor ions can be detected by comparing MALDI spectra of a single sample taken in positive and negative ion modes, with phosphopeptides demonstrating greater relative ion intensities in negative ion mode (19, 20) . However, this approach suffers from poor specificity for phosphopeptides because of the high background of nonphosphorylated acidic peptides in negative ion mode caused by the ability of carboxylate groups on glutamate or aspartate residues to develop negative charges in a manner similar to that of phosphate groups. In this article, we show that removal of these acidic groups by methyl esterification (21, 22) can greatly diminish the ion intensity of these acidic nonphosphorylated peptides in negative ion mode and therefore greatly increase the selectivity of the method for phosphopeptides in peptide mixtures. We used the method to identify 12 phosphopeptides containing 22 phosphorylation sites from low femtomolar amounts of a tryptic digest of a model phospho-
doi:10.1074/mcp.t400019-mcp200 pmid:15753120 fatcat:2fxzffkdn5fvdbxyz4xwelg74y