Phosphopeptide Analysis by Matrix-Assisted Laser Desorption Time-of-Flight Mass Spectrometry

• Published in: Analytical chemistry (Washington) Vol. 68; no. 19; pp. 3413 - 3421
• Main Authors: Annan, Roland S, Carr, Steven A
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01-10-1996
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chemistry; Chromatography, High Pressure Liquid; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; Ions; Peptides; Phosphopeptides - analysis; Proteins; Scientific imaging; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods; Investigation method; Phosphoproteins; Time of flight spectrometer; Mass spectrometry; Laser desorption; Structural analysis
• DOI: 10.1021/ac960221g
• EISSN: 1520-6882
• ISSN: 0003-2700

In this paper we present methods for identifying and sequencing phosphopeptides in simple mixtures, such as HPLC fractions, at the subpicomole level by (+) ion matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-MS). Data are presented which indicate that when a reflectron time-of-flight mass spectrometer is used, MALDI can distinguish tyrosine phosphorylation from serine and threonine phosphorylation for peptides containing a single phosphate group. Phosphopeptides are identified in the (+) ion MALDI reflector spectrum by the presence of [MH − H3PO4]+ and [MH − HPO3]+ fragment ions formed by metastable decomposition. An abundant [MH − H3PO4]+ ion, accompanied by a weaker [MH − HPO3]+ ion indicates that the peptide is most likely phosphorylated on serine or threonine. In contrast, phosphotyrosine-containing peptides generally exhibit [MH − HPO3]+ fragment ions and little, if any [MH − H3PO4]+. Ambiguities do arise, most often with phosphopeptides that contain residues which readily lose water (such as unmodified serine), but these can often be resolved by recording a complete metastable fragment ion (postsource decay) spectrum. Postsource decay is shown here to be a viable technique for sequencing phosphopeptides. It can be used to distinguish between serine/threonine and tyrosine phosphorylation and in many cases can be used to determine the exact site of phosphorylation in a peptide sequence. Nearly complete sequence coverage and phosphorylation site mapping is generally possible using ∼300 fmol of peptide.