Occurrence of C-Terminal Residue Exclusion in Peptide Fragmentation by ESI and MALDI Tandem Mass Spectrometry

Abstract

By screening a data set of 392 synthetic peptides MS/MS spectra, we found that a known C-terminal rearrangement was unexpectedly frequently occurring from monoprotonated molecular ions in both ESI and MALDI tandem mass spectrometry upon low and high energy collision activated dissociations with QqTOF and TOF/TOF mass analyzer configuration, respectively. Any residue localized at the C-terminal carboxylic acid end, even a basic one, was lost, provided that a basic amino acid such arginine and to a lesser extent histidine and lysine was present in the sequence leading to a fragment ion, usually depicted as (b n-1 +H 2 O) ion, corresponding to a shortened nonscrambled peptide chain. Far from being an epiphenomenon, such a residue exclusion from the peptide chain C-terminal extremity gave a fragment ion that was the base peak of the MS/MS spectrum in certain cases. Within the frame of the mobile proton model, the ionizing proton being sequestered onto the basic amino acid side chain, it is known that the charge directed fragmentation mechanism involved the C-terminal carboxylic acid function forming an anhydride intermediate structure.The same mechanism was also demonstrated from cationized peptides. To confirm such assessment, we have prepared some of the peptides that displayed such C-terminal residue exclusion as a C-terminal backbone amide. As expected in this peptide amide series, the production of truncated chains was completely suppressed. Besides, multiply charged molecular ions of all peptides recorded in ESI mass spectrometry did not undergo such fragmentation validating that any mobile ionizing proton will prevent such a competitive C-terminal backbone rearrangement. Among all well-known nondirect sequence fragment ions issued from non specific loss of neutral molecules (mainly H 2 O and NH 3 ) and multiple backbone amide ruptures (b-type internal ions), the described Cterminal residue exclusion is highly identifiable giving raise to a single fragment ion in the high mass range of the MS/MS spectra. The mass difference between this signal and the protonated molecular ion corresponds to the mass of the C-terminal residue. It allowed a straightforward identification of the amino acid positioned at this extremity. It must be emphasized that a neutral residue loss can be misattributed to the formation of a y m-1 ion, i.e., to the loss of the N-terminal residue following the a 1 -y m-1 fragmentation channel. Extreme caution must be adopted when reading the direct sequence ion on the positive ion MS/MS spectra of singly charged peptides not to mix up the attribution of the N-and C-terminal amino acids. Although such peculiar fragmentation behavior is of obvious interest for de novo peptide sequencing, it can also be exploited in proteomics, especially for studies involving digestion protocols carried out with proteolytic enzymes other than trypsin (Lys-N, Glu-C, and Asp-N) that produce arginine-containing peptides.

Laser desorption ionization mass spectrometry of protein tryptic digests on nanostructured silicon plates

“…The dissociated precursor ions (m/z 689.4 for protonated AWSVAR, m/z 1439.8 for protonated RHPEYAVSVLLR and m/z 1479.8 for protonated LGEYGFQNALIVR) were also satisfying the fragmentation rules [49]. An abundant loss of the C-terminal residue (R) was observed for the second peptide (RHPEYAVSVLLR) corresponding to a miscleavage [40] as seen in Fig. S4b together with the loss of a neutral molecule from the arginine sidechain as depicted previously in the literature [39].…”

“…The first peptide of the β chain [461-468] (m/z 1049.6, IRPYFPEQ) was the result of a trypsin miscleavage, an arginine residue being present within the sequence. As recently reported, such arginine-containing sequence is prone to a C-terminal rearrangement leading to the abundant loss of the C-terminal residue providing a truncated peptide ion referred as a 'b n − 1 + H 2 O' fragment ion [40]. This behavior was also here observed from NanoSi deposit in LDI-MS/MS, the Cterminal glutamine (Q) residue being excluded from the protonated precursor ion to give an abundant b 7 + H 2 O (m/z 1049.6 → m/z 921.6) as displayed in Fig.…”

“…Our results were thus confronted to the results recorded from classical MALDI experiments. Furthermore, we exploited the ability of modern MALDI-MS instruments that are now most of the time equipped with TOF/TOF configuration for MS/MS experiments [37][38][39][40] aiming not only at detecting the targeted peptides, but also to subsequently sequence them in a single SALDI-MS analysis. Using this technology, we first studied 49 synthetic peptides mimicking proteolytic sequences issued from both trypsin and Lys-N digestions, then tryptic digests from various proteins presenting molecular weights ranging from 12 to 340 kDa (Cytochrome C, β-Casein, Bovine Serum Albumin, and Fibrinogen).…”

Metastable decomposition at the peptide C‐terminus: Possible use in protein identification

“…Several characteristic PSD ions have been reported for modified peptides, such as peptides that are phosphorylated at Ser or Thr, oxidized at Met, sialylated at a glycosylated residue, etc. Other than these, loss of a C‐terminal AA residue via C‐terminal rearrangement from the PSD ion by metastable decomposition is prominently observed for peptides that contain Arg residues(s) in the sequence, and this is particularly useful for database searching based on the MS/MS spectrum as well as for the determination of the C‐terminal AA. The ions that are produced in the time span of the flights in a field‐free region (the space between the exit of the ion source and the inlet of a reflectron in the instrument used in this study) are observed as broad, poorly resolved peaks with m / z values that are slightly higher than the theoretical values, unless the reflectron is electronically tuned taking the kinetic energies of those ions into consideration.…”

“…Sequence‐specific fragmentation such as the loss of the C‐terminal amino acid (AA) residue from a peptide via an intramolecular rearrangement upon collision‐induced dissociation (CID) or metastable decomposition has been reported . The basicity and the position of a basic AA in the sequence, particularly arginine (Arg), are critical for the rearrangement to occur at the C‐terminus, generating a [ b n −1 + H 2 O] ([ b n −1 + 18]) ion .…”