Liquid chromatography with mass spectrometry (LC–MS/MS) analysis was performed using a Fusion-Orbitrap mass spectrometer (Thermo Scientific) and a U-3000 nanoflow-HPLC system (Thermo Scientific) as described previously46 (link). The entire TAIR10 database was searched (www.Arabidopsis.org ) using Mascot (v.2.3.02, Matrix Science) (with the inclusion of sequences of common contaminants, such as keratins and trypsin). Parameters were set for 10 ppm peptide mass tolerance and allowing for Met oxidation and two missed tryptic cleavages. Carbamidomethylation of Cys residues was specified as a fixed modification, and oxidation of Met and phosphorylation of Ser, Tyr or Thr residues were allowed as variable modifications. Scaffold (v.3; Proteome Software) was used to validate MS/MS-based peptide and protein identifications and annotate spectra. The position and quality of spectra for phosphopeptides were also manually examined before acceptance.
Protocol detail
Phosphoproteomics of Arabidopsis by LC-MS/MS
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Arabidopsis
Cleavages
Hplc
Keratins
Liquid chromatography
Mass spectrometry
Ms ms
Peptide
Phosphopeptides
Phosphorylation
Protein
Proteome
Tolerance
Tryptic
Corresponding Organization :
Other organizations : Sainsbury Laboratory, University of East Anglia, Norwich Research Park, University of Maryland, College Park, University of Würzburg, University of Zurich, John Innes Centre, University of Helsinki
Top 5 similar protocols
Variable analysis
independent variables
- Liquid chromatography with mass spectrometry (LC–MS/MS) analysis
- TAIR10 database search
- Mascot search parameters (10 ppm peptide mass tolerance, Met oxidation, two missed tryptic cleavages, Carbamidomethylation of Cys, oxidation of Met, phosphorylation of Ser/Tyr/Thr)
- Peptide and protein identifications
- Phosphopeptide spectra
- Fusion-Orbitrap mass spectrometer
- U-3000 nanoflow-HPLC system
- Inclusion of sequences of common contaminants (keratins and trypsin)
- Scaffold software for validation of MS/MS-based peptide and protein identifications
- Manual examination of the position and quality of spectra for phosphopeptides before acceptance
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