Submitted to: Journal of American Society for Mass Spectrometry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/23/2005
Publication Date: 12/9/2005
Citation: Smith, L.L., Herrmann, K.A., Wysocki, V.H. 2005. Investigation of gas phase ion structure for proline-containing B2 ion. Journal of American Society for Mass Spectrometry. 17:20-28.
Interpretive Summary: Elucidation of gene function from protein expression profiles is routinely performed using mass spectrometry combined with separation technologies and sequence database searching algorithms. Tandem mass spectrometry (MS/MS), in which peptide ions are most commonly collided with neutral gas atoms to induce fragmentation, is playing a key role in high-throughput protein identification experiments. In the past decade, the bottleneck for high-throughput analysis of MS/MS data was manually interpreting the data to sequence the peptides and ultimately, identify the protein of origin. Since the development of automated sequencing algorithms, this interpretive process has been accelerated. Many of the sequencing algorithms rely on the presence of spectral information from fragmentation occurring between every amino acid residue in the peptide sequence to predict MS/MS spectral appearance. Statistical analysis of large databases of actual MS/MS spectra have shown that this is not always the case and is dependent on amino acid residue composition and fragmentation mechanisms occurring in the gas phase of the mass spectrometer. Specifically, it has been shown that cleavage is favored N-terminal to proline residues and very little other sequencing information may be present in the MS/MS spectrum for a peptide containing proline. This work reports the phenomenon that as proline is located closer to the N-terminus of the peptide sequence, cleavage C-terminal to proline occurs readily. Through our investigation, the gas phase structure of the resulting b2 ion when proline is the second residue from the N-terminus is likely to be a protonated diketopiperazine while larger bn (n > 3) ions are usually thought to be oxazolones. This added insight into gas phase structures of peptide fragment ions and the corresponding mechanisms of formation could be incorporated into automated sequencing algorithms to better predict MS/MS spectral appearance and increase overall success rates for protein identification.
Technical Abstract: Unusual fragmentation was observed for doubly charged VPDPR in which cleavage C-terminal to proline and N-terminal to aspartic acid yielded b2 (+ a2) / y3 complementary ions. This unique fragmentation is contradictory to trends previously established by statistical analysis of peptide tandem mass (MS/MS) spectra. Substitution of alanine for aspartic acid (i.e. VPAPR) did not change the fragmentation, indicating the cleavage was not directed by aspartic acid. Fragmentation patterns for VPAPR and V(NmA)APR (NmA = N-methyl alanine) were compared to determine whether conformational constraints from proline’s cyclic side chain contribute to b2 ion formation. While both peptide sequences fragmented to yield b2 / y3 ions, only VPAPR produced a2 ions, suggesting the VP b2 ion is structurally different from the V(NmA) b2 ion. Instead, the V(NmA) b2 ion was accompanied by an ion corresponding to formal loss of 71. The suspected structural differences were confirmed by isolation and fragmentation of the respective b2 ions (i.e. MS3 spectra). Evidence supporting a diketopiperazine structure for the VP b2 ion is reported. Fragmentation patterns for the VP b2 ion and a synthetic VP diketopiperazine showed great similarity. N-terminal acetylation of VPAPR prevented the formation of the VP b2 ion, presumably by blocking nucleophilic attack by the N-terminal amine on the carbonyl oxygen of the protonation site. Acetylation of the N-terminus for V(NmA)APR did not prevent the formation of the V(NmA) b2 ion, indicating the V(NmA) b2 ion has a structure, presumably that of an oxazolone, which requires no attack by the N-terminus for formation. Finally, high resolution, accurate mass measurements determined that the V(NmA) (b2 – 71) ion results from losing a portion of valine from oxazolone V(NmA) b2 ion, rather than cross-ring cleavage of the alternate diketopiperazine.