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Arabidopsis > Protocols > Protein identification by mass spectrometry
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Protein identification by mass spectrometry

Protein identification by mass spectrometry

Spots of interest were excised from GelCode-stained 2D gels and digested by sequence grade trypsin (Promega Biotec, Madisson, WI, USA). After digestion, the supernatant containing peptides was concentrated by batch adsorption on POROS 50 R2 beads (Roche Molecular Biochemicals, Basel, Switzerland), and used for MALDI-MS analysis on a Bruker Reflex II MALDI-TOF spectrometer after on-target desorption with matrix solution (Gevaert et al., 1998). Before each analysis, the instrument was externally calibrated using two synthetic peptides spotted as near as possible to the biological sample. Proteins were identified by searching the protein databases using MASCOT (http://www.matrixscience.com). Theoretical masses and isoelectric points of identified proteins were predicted by entering the sequence at http://www.expasy.org/tools/peptide-mass.html. To denote a protein as unambiguously identified, the following criteria were used: coverage of the protein by the matching peptides must reach a minimum of 10% and at least four independent peptides should match within a stringent 10-ppm maximum deviation of mass accuracy. In some cases, protein identities were further confirmed from Post Source Decay (PSD) spectra, generated from selected peptides.
Search for sequence homology was carried out at
http://www.arabidopsis.org/Blast.

  • Gevaert K, Demol H, Sklyarova T, Vandekerckhove J, Houthaeve T (1998) A peptide concentration and purification method for protein characterization in the subpicomole range using matrix assisted laser desorption/ionisation-postsource decay (MALDI-MS) sequencing. Electrophoresis 19, 909-917

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