Characterization of a phosphorylated peptide and peptoid and peptoid–peptide hybrids by mass spectrometry

Abstract

Nano-electrospray tandem mass spectrometry (nano-ES-MS/MS) was used to record collision-induced dissociation (CID) spectra of a set of peptoid-peptide hybrids and the complete peptoid derived from the phosphopeptide Ac-pTyr-Glu-Thr-Leu-NH(2) (1). The presence of B and Y''-type fragment ions in the tandem mass spectra of the protonated molecular ions [M + H](+) allowed confirmation of sequence similar to mass spectrometric sequence analysis in peptides. In the isomeric peptoid compounds studied, one or several amino acid residues were replaced by peptoid residues (N-substituted glycine residues), which resulted in characteristic tandem mass spectra with differently increased relative abundances of Y''-and B-type fragment ions. The increment of a particular Y''-ion was directly correlated to the position of a peptoid residue present. In addition to these increased peak intensities, other characteristic peaks were also observed compared with the spectrum of reference peptide 1. When a peptoid phosphotyrosine was incorporated, the presence of this residue was apparent from the occurrence of a relatively intense peak at m/z 187 representing the positively charged side-chain of phosphotyrosine, which was almost absent in the spectrum of the reference peptide 1. Since the threonine side-chain had to be translated into the homo peptoid analog this substitution was apparent from the presence of [M + H](+) and fragment ions 14 mass units higher than observed in the spectrum of the reference phosphopeptide 1. The presence of an NLeu peptoid residue could be confirmed by the specific fragmentation of the immonium ion showing an intense peak in its tandem mass spectrum at m/z 57, which results from the loss of an neutral imine molecule leading to a positively charged [C(4)H(9)](+) ion. By means of these mass spectrometric characteristics, all isomeric peptoid compounds could be distinguished from each other and characterized. The methods used appear to be very useful in future studies of peptoids and peptoid-peptide hybrids.

Fragmentation Patterns and Mechanisms of Singly and Doubly Protonated Peptoids Studied by Collision Induced Dissociation

“…The absence of the B-ions was probably due to the low mass cut-off in the ion trap and, consequently, the lowmass B-ions were not detected. The preference for the formation of Y-type ions has also been observed by other researchers [44]. The fragmentation patterns of protonated peptoids appeared quite different from those of most protonated peptides.…”

“…However, we use capital letters, B or Y, to refer to peptoid ions instead of lower case letters, b or y, which are used for peptide ions. This capital letter convention was also used to refer to peptoid fragment ions by Liskamp and coworkers in their early publications [43,44]. The fragmentation scheme and sample structures are shown in Scheme 2.…”

“…Peptoids fragment in a manner similar to that of peptides under CID conditions [41,42,44,47]. The main fragment ions observed correspond to the dissociation of the amide bonds of the peptoid backbone, which produce B-and Y-series ions (following the Roepstorff and Fohlman nomenclature [49]).…”

“…Tandem mass spectrometry (MS/MS) techniques coupled with either electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI) are now routinely used for peptide and protein sequencing [34][35][36][37]. These tandem mass spectrometry based techniques have also shown promise as the method of choice for peptoid sequencing [29,[38][39][40][41][42][43][44][45][46]. We have previously studied the fragmentation patterns of a group of protonated and alkali-metallated peptoids under collision-induced dissociation (CID) as well as under electron capture dissociation (ECD) conditions [47,48].…”

Gas phase derivatization in peptide analysis I: the utility of trimethyl borate in identifying phosphorylation sites

“…Protein phosphorylation plays an important role in many biological processes [1][2][3][4] and there has been much interest in the development of mass spectrometric methods for identifying phosphorylation sites [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24]. Because phosphorylation is a post-translational modification, the site cannot be inferred by analysis of the DNA coding of the protein.…”