Electrospray tandem mass spectrometric studies of phosphopeptides and phosphopeptide analogues

A set of synthetic phosphopeptides and phosphopeptide analogues was studied by tandem nano-electrospray mass spectrometry. The influence of the collis ion offset and of the charge state of the molecular ion on phosphate-specif ic fragmentation processes was investigated in detail. H-D exchange experim ents and structural considerations support a six-centered transition being present in the neutral loss of H3PO4 from serine, threonine and homoserine phosphopeptides, where the C-alpha hydrogen of serine or threonine or the C -beta hydrogen of homoserine is transferred to the protonated phosphate gro up. Neutral loss of H3PO4 at moderate collision offset potential represents a very abundant fragmentation process for serine, threonine and homoserine phosphopeptides. The most specific feature for discrimination of these pho sphopeptides from tyrosine phosphopeptides is the m/z 79:97 ratio in the ne gative ion product spectra, which is consistently elevated in tyrosine phos phopeptides as compared with serine, threonine and homoserine phosphopeptid es. The fragment ions of methylphosphono- and H-phosphonopeptides can be ex plained by the same mechanisms as are applicable to phosphopeptides. Copyri ght (C) 1999 John Wiley & Sons, Ltd.