Laser-induced dissociation of phosphorylated peptides using matrix assisted laser desorption/ionization tandem time-of-flight mass spectrometry

Dongxia Wang,Robert J Cotter,Philip A Cole

doi:10.1007/bf02752496

Abstract

Abstract Reversible phosphorylation is one of the most important posttranslational modifications of cellular proteins. Mass spectrometry is a widely used technique in the characterization of phosphorylated proteins and peptides. Similar to nonmodified peptides, sequence information for phosphopeptides digested from proteins can be obtained by tandem mass analysis using either electrospray ionization or matrix assisted laser desorption/ionization (MALDI) mass spectrometry. However, the facile loss of neutral phosphoric acid (H3PO4) or HPO3 from precursor ions and fragment ions hampers the precise determination of phosphorylation site, particularly if more than one potential phosphorylation site or concensus sequence is present in a given tryptic peptide. Here, we investigated the fragmentation of phosphorylated peptides under laser-induced dissociation (LID) using a MALDI-time-of-flight mass spectrometer with a curved-field reflectron. Our data demonstrated that intact fragments bearing phosphorylated residues were produced from all tested peptides that contain at least one and up to four phosphorylation sites at serine, threonine, or tyrosine residues. In addition, the LID of phosphopeptides derivatized by N-terminal sulfonation yields simplified MS/MS spectra, suggesting the combination of these two types of spectra could provide an effective approach to the characterization of proteins modified by phosphorylation.

Highlights

Reversible phosphorylation is one of the most common and important regulatory modifications of cellular proteins [1]
For the two peptides with phosphoserine and phosphothreonine (Fig. 2A,B) neutral loss of phosphoric acid from the molecular ion is the dominant process, whereas fragments resulting from the loss of both H3PO4 and HPO3 from the precursor ion of the phosphotyrosinecontaining peptide (Fig. 2C) were observed but not dominant
There is extensive sequence-specific (b- and y-series) fragmentation that includes intact sequence fragments retaining the phosphate group, and sequence fragments showing the loss of 98 U or 80 mass units

Summary

Introduction

Reversible phosphorylation is one of the most common and important regulatory modifications of cellular proteins [1]. This is primarily because the most common method used to induce fragmentation in electrospray ionization (ESI) and matrix assisted laser desorption/ionization (MALDI) mass spectrometers, lowenergy collision-induced dissociation (CID), results in significant loss of neutral phosphoric acid (H3PO4 98 Da) or HPO3 (80 Da) from phosphorylated amino acid residues [3,4,5,6,7].

Results

Conclusion