Phosphopeptide detection and sequencing by matrix-assisted laser desorption/ionization quadrupole time-of-flight tandem mass spectrometry.
A prototype matrix-assisted laser desorption/ionization quadrupole time-of-flight (MALDI-TOF) tandem mass spectrometer was used to sequence a series of phosphotyrosine-, phosphothreonine- and phosphoserine-containing peptides. The high mass resolution and mass accuracy of the instrument allowed the localization of one, three or four phosphorylated amino acid residues in phosphopeptides up to 3.1 kDa. Tandem mass spectra of two different phosphotyrosine peptides permitted amino acid sequence determination and localization of one and three phosphorylation sites, respectively. The phosphotyrosine immonium ion at m/z 216.04 was observed in these MALDI low-energy CID tandem mass spectra. Elimination of phosphate groups was evident from the triphosphorylated peptide but not from the monophosphorylated species. The main fragmentation pathway for the synthetic phosphothreonine-containing peptide and for phosphoserine-containing peptides derived from beta-casein and ovalbumin was the beta-elimination of phosphoric acid with concomitant conversion of phosphoserine to dehydroalanine and phosphothreonine to 2-aminodehydrobutyric acid. Peptide fragment ions of the b- and y-type allowed, in all cases, the localization of phosphorylation sites. Ion signals corresponding to (b-17), (b-18) and (y-17) fragment ions were also observed. The abundant neutral loss of phosphoric acid (-98 Da) is useful for femtomole level detection of phosphoserine-peptides in crude peptide mixtures generated by gel in situ digestion of phosphoproteins.
Bennett
Keiryn L
KL
MDS Proteomics A/S, Staermosegaardsvej 6, DK-5230 Odense M, Denmark. kbennett@mdsproteomics.dk
Stensballe
Allan
A
Podtelejnikov
Alexandre V
AV
Moniatte
Marc
M
Jensen
Ole Nørregaard
ON
D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000737:chemistry; D002417:Cattle; D002846:Chromatography, Affinity; D008969:Molecular Sequence Data; D010047:Ovalbumin / Q000737:chemistry; D010748:Phosphopeptides / Q000032:analysis*; D010768:Phosphoserine / Q000737:chemistry; D010769:Phosphothreonine / Q000737:chemistry; D019000:Phosphotyrosine / Q000737:chemistry; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation / Q000379:methods*
D002364:Caseins; D010748:Phosphopeptides; D010769:Phosphothreonine; D010768:Phosphoserine; D019000:Phosphotyrosine; D010047:Ovalbumin