Fragmentation of cationized phosphotyrosine containing peptides by atmospheric pressure MALDI/Ion trap mass spectrometry

Abstract

An investigation of phosphate loss from sodium-cationized phosphotyrosine containing peptide ions was conducted using liquid infrared (2.94 m) atmospheric pressure matrixassisted laser desorption/ionization (AP MALDI) coupled to an ion trap mass spectrometer (ITMS). Previous experiments in our laboratory explored the fragmentation patterns of protonated phosphotyrosine containing peptides, which experience a loss of 98 Da under CID conditions in the ITMS. This loss of 98 Da is unexpected for phosphotyrosine, given the structure of its side chain. Phosphate loss from phosphotyrosine residues seems to be dependent on the presence of arginine or lysine residues in the peptide sequence. In the absence of a basic residue, the protonated phosphotyrosine peptides do not undergo losses of HPO 3 (⌬ 80 Da) nor HPO 3 ϩ H 2 O (⌬ 98 Da) in their CID spectra. However, sodium cationized phosphotyrosine containing peptides that do not contain arginine or lysine residues within their sequences do undergo losses of HPO 3 (⌬ 80 Da) and HPO 3 ϩ H 2 O (⌬ 98 Da) in their CID spectra.

The impact of chromatography and mass spectrometry on the analysis of protein phosphorylation sites

“…A 98 Da loss for H 3 PO 4 is usually observed for pS and pT, while pY tends to lose 80 Da for HPO 3 [46,47,48]. Spectral interpretation might be complicated by the fact that the loss of 98 Da from a phosphorylated fragment ion is indistinguishable from the loss of 18 Da (H 2 O) from a non-phosphorylated ion.…”

“…Spectral interpretation might be complicated by the fact that the loss of 98 Da from a phosphorylated fragment ion is indistinguishable from the loss of 18 Da (H 2 O) from a non-phosphorylated ion. pY is stabilised by resonance, and the tendency to lose the phosphate moiety decreases in the order pS>pT>>pY [48,49,50,51], although a neutral loss of 98 Da was observed for pS and pY using a custom-built MALDI-IT instrument [52]. Phosphopeptides can be missed in that scan mode when the peptide additionally loses water upon fragmentation, mimicking a neutral loss of 116 Da [47].…”

“…The example of the phosphoprotein standard β-casein demonstrates some of the difficulties one might have to expect in phosphorylation site analysis. Only the smaller phosphopeptide (amino acid residues [33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48] FQpSEEQQQTEDELQDK, m/z=2061.8290; SwissProt P02666) was detected, but was then missed by data-dependent HPLC-MS/MS in positive ion mode. Only SIM on the pre-determined m/z finally produced a suitable spectrum (Fig.…”

Formation of [nM−nH+(n+1)Na]+ cluster ions from amino acid by electrospray ionization

“…Besides ESI, sodiated amino acids were also generated by MALDI [24] and FAB [25]. The [nM−nH+(n + 1)Na] + ions of amino acids have not been reported, but the [nM−nH+(n + 1)Na] + ions of peptides such as achatin-I [26] and phosphotyrosine peptide [27] were reported. Protonated cluster ions, [nM+H] + of amino acids generated by ESI were reported [21,22].…”

The fragmentation of ethoxylated surfactants by AP-MALDI-QIT

“…A quadrupole ion trap (QIT) mass spectrometer is an excellent choice in this regard. Some early work showed examples of high quality MS/MS experiments on MALDI generated peptides [13][14][15][16]. The ability to analyze synthetic polymers using AP MALDI methods on a QIT have also been demonstrated [17].…”