Verification of the position of the phosphate group in some synthetic phosphopeptides by fast‐atom bombardment and tandem mass spectrometry

Abstract

Some synthetically obtained linear and cyclic phosphopeptides of low molecular weight have been studied by fast-atom bombardment and tandem mass spectrometry to verify the position of the phosphate group in these compounds. Based upon the occurrence/non-occurrence of loss of phosphoric acid from low abundance fragment ions induced by low-and high-energy collisions with target gases, it is shown that the position of the phosphate group in the phosphopeptides studied can be determined unequivocally.The development of soft ionization methods such as field desorption (FD),' fast-atom bombardment (FAB) ,* laser desorption (LD)3 and electrospray (ES)4has enabled the analysis of peptide? in the solid phase or in solution without the need for prior derivatization. The corresponding mass spectra usually contain large peaks due to the protonated or cationized molecules and therefore are very suited for providing the molecular weights of peptides. However, the reduced extent of fragmentation and, as a consequence, small fragmention peaks in such spectra make it more difficult to obtain the amino acid sequence of the peptides. The latter problem can be overcome by the use of tandem mass spectrometry (MS/MS) in conjunction with collision-induced dissociation (CID) to promote fragmentation useful for the structure elucidation of peptides.6 In this paper the analyses of several synthetic linear and cyclic phosphopeptides by FAB-MS and FAB-MS/MS are reported as an extension of our earlier papers7,' on the sequence analysis of synthetic nucleopeptides and deuterium-labelled peptides. It will be shown that it is possible to determine unequivocally the position of the phosphate group in the synthetic phosphopeptides by application of FAB-MS/MS. EXPERIMENTAL InstrumentsThe spectra were recorded by use of a ZAB-HFqQ mass spectrometer (VG Analytical, Manchester, UK) with the configuration BEqQ (B =magnetic sector, E = electric sector, q = R F only quadrupole, Q = quadrupole mass filter) which was coupled to a VG 11/250 data system. For obtaining the full-scan FAB spectra, the phosphopeptides were dissolved in thioglycerol (concentration 1 mmol/L), loaded on a stainless steel probe and bombarded with xenon atoms having 8 keV kinetic energy. For the acquisition of the FAB/MIKE (mass-analyzed ion kinetic energy) and

Identification of phosphorylation sites in phosphopeptides by positive and negative mode electrospray ionization-tandem mass spectrometry

“…Fragmentation information from TSQVAPA is summarized in Table 1. ion, which corresponds to a loss of H 3P04 , is seen [17]. Similar to the spectrum for the nonphosphorylated Figure 3a.…”

“…For structure elucidation of phosphopeptides, fragmentation upon FAB ionization has been of some utility [5,[11][12][13]. Similarly, a number of applications of fast-atom bombardment tandem mass spectrometry (FAB-MS/MS) for determination of sites of phosphorylation have been reported [1,9,[14][15][16][17]. Collisional activation also has been utilized with ESI-generated phosphopeptide ions.…”

Studies on the dephosphorylation of phosphotyrosine-containing peptides during post-source decay in matrix-assisted laser desorption/ionization

“…Mass spectrometric methods that ensure the selective identification and sequence analysis of phosphopeptides in complex protein digests or peptide mixtures include fast atom bombardment mass spectrometry (FABMS),17–19 plasma desorption time‐of‐flight mass spectrometry (PD‐TOFMS),20 positive‐ and negative‐mode electrospray ionization tandem mass spectrometry (ESI‐MS/MS)21–24 and matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) 23–28. Post‐source decay (PSD) in MALDI‐MS29 has mostly been used to identify globally phosphoserines and phosphothreonines based on the mass losses of 80 and 98 Da of the parent ions, corresponding to [M + H − HPO 3 ] + and [M + H − H 3 PO 4 ] + ions.…”

Four decades of joy in mass spectrometry

“…The same strategy was applied in a subsequent study to verify the position of the phosphate group in some cyclic and linear synthetic phosphopeptides. Based upon the occurrence/non‐occurrence of loss of phosphoric acid from low abundant fragment ions, induced by low‐ and high‐energy collisions, the position of the phosphate group could be determined unequivocally (Nijenhuis et al, 1993). High‐resolution fast atom bombardment and tandem mass spectrometry were also used to sequence the heptapeptide‐ p‐ coumaric acid chromophore conjugate obtained after the combined pepsin/proteinase K digestion of the photoactive yellow protein from Ectothiorhodospira halophila .…”