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Detection and Identification of Phosphorylation Sites in Proteins Using ,,, LC/MS/MS with Neutral Fragment Loss Mapping

hing of peptide fragmentation spectra.(3) The MS/MS spectra of peptides provide a wealth of information enabling the assignment of peptide sequences and identification of modified residues. We present a simple strategy enabling an unambiguous identification of the phosphorylated residue of a peptide analyzed by LC/MS/MS in a complex peptide mixture. Goal In this report, we use capillary LC/MS/MS and protein database searching techniques to:
  1. Identify a phosphorylated peptide by its neutral loss profile
  2. Unambiguously assign the modified amino acid residue
Experimental Conditions Thermo Finnigan Surveyor pump
Magic flow splitter (Michrom)
Thermo Finnigan LCQ Deca XP mass spectrometer fitted with NanoSpray Ion Source (positive ion mode)
Data Dependent MS/MS
Capillary temp: 150C
Needle voltage: + 1.8 kV
LC column: PicoFrit BioBasic 4.9 cm,
75 um ID (New Objectives)
Injection volume: 2 L
LC solvent program: Figure 1. Phosphorylated peptides exhibit a prominent loss of a phosphate group in their MS/MS spectra. A loss of 49 amu observed in the MS/MS spectrum of a doubly-charged phosphorylated peptide (RT = 27.71 min). Discussion Proteins from a human brain sample were analyzed using SDS-polyacrylamide gel electrophoresis, and the neurofilament proteins were digested in-gel. A mixture of peptides was then analyzed by capillary chromatography LC/MS, utilizing a fully-automated coupling of the Sur
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