EZYprep LC-coupled MALDI-TOF/TOF MS: An improved matrix spray application for phosphopeptide characterisation

Journal Article

Expression Proteomics

10.1002/pmic.200900800

MALDI-TOF, Ion Trap

2010

ImagePrep Station, Ultraflex III, HCT ion-trap

Mark R. Condina, Johan O.R. Gustafsson, Manuela Klingler-Hoffmann, Christopher J. Bagley, Shaun R. McColl, Peter Hoffmann

Proteomics

Abstract

The quality of MALDI-TOF mass spectrometric analysis is highly dependent on the matrix and its deposition strategy. Although different matrix deposition methods have specific advantages, one major problem in the field of proteomics, particularly with respect to quantitation, is reproducibility between users or laboratories. Compounding this is the varying crystal homogeneity of matrices depending on the deposition strategy utilised. Here we describe a novel optimised matrix deposition strategy for LC-MALDI-TOF/TOF MS using an automated instrument that produces a nebulised matrix mist under controlled atmospheric conditions. Comparisons of this with previously reported strategies showed the method to be advantageous for the atypical matrix, 2,5-DHB and improved phosphopeptide ionisation when compared with deposition strategies for CHCA. This optimised DHB matrix deposition strategy with LC-MALDI-TOF/TOF MS, termed EZYprep LC, was subsequently optimised for phosphoproteome analysis and compared to LC-ESI-IT-MS and a previously reported approach for phosphotyrosine identification and characterisation. These methods were utilised to map phosphorylation on EGF-stimulated epidermal growth factor receptor (EGFR) to gauge the sensitivity of the proposed method. EZYprep DHB LC-MALDI-TOF/TOF MS was able to identify more phosphopeptides and characterise more phosphorylation sites than the other two proteomic strategies, thus proving to be a sensitive approach for phosphoproteome analysis.

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