Statistical Characterization of Ion Trap Tandem Mass Spectra from Doubly Charged Tryptic Peptides

Abstract

Collision-induced dissociation (CID) is a common ion activation technique used to energize massselected peptide ions during tandem mass spectrometry. Characteristic fragment ions form from the cleavage of amide bonds within a peptide undergoing CID, allowing the inference of its amino acid sequence. The statistical characterization of these fragment ions is essential for improving peptide identification algorithms and for understanding the complex reactions taking place during CID. An examination of 1465 ion trap spectra from doubly charged tryptic peptides reveals several trends important to understanding this fragmentation process. While less abundant than y ions, b ions are present in sufficient numbers to aid sequencing algorithms. Fragment ions exhibit a characteristic series-specific relationship between their masses and intensities. Each residue influences fragmentation at adjacent amide bonds, with Pro quantifiably enhancing cleavage at its N-terminal amide bond and His increasing the formation of b ions at its C-terminal amide bond. Fragment ions corresponding to a formal loss of ammonia appear preferentially in peptides containing Gln and Asn. These trends are partially responsible for the complexity of peptide tandem mass spectra.Tandem mass spectrometry (MS/MS) of peptides is a central technology for proteomics, enabling the identification of thousands of peptides from a complex mixture. [1][2][3][4] This increasingly widespread technique relies upon the fragmentation of peptides by collisioninduced dissociation (CID), but the chemistry behind the fragmentation process is complex and not comprehensively understood. [5][6][7][8] Peptides undergo CID after they are isolated from other ions by their mass-to-charge (m/z) ratios. Peptides in an acidic solution are introduced to the vacuum of the mass spectrometer via electrospray ionization. 9 The peptide ions are accelerated during CID, leading to more energetic collisions with the ion trap's inert gas molecules. The mobile proton model 10 describes how the added internal energy causes the ionizing proton(s) on each peptide to transfer intramolecularly until one destabilizes a peptide bond, resulting in the cleavage of that bond and the production of two fragments. While more energetic techniques may cleave many classes of bonds within the peptide structure, low-energy CID preferentially breaks the amide bonds. Once the fragment ions are produced, the mass spectrometer records their m/z ratios in a tandem mass spectrum.Determining the sequence of a peptide from its tandem spectrum is complicated by the variety and variability of the fragment ions produced. Cleavage of amide bonds results in b and y Figure 1). b ions may fragment further to produce a ions. 13 If only these three ions were produced for every amide bond in a 10-residue peptide, the fragment ion spectrum would contain 27 peaks. This ideal spectrum differs from experimental spectra as a result of several causes. First, a subset of the expected fragment ions may not be present. Second...

Verification of automated peptide identifications from proteomic tandem mass spectra

“…The 'proline effect' describes the intense fragments resulting from cleavage on the N-terminal side of proline residues, but the magnitude by which this cleavage exceeds others is highly dependent upon the residue on the N-terminal side of the bond 11 . Glycine and serine also enhance N-terminal cleavage (to a lesser extent), whereas valine, leucine and isoleucine produce more fragment ions from the bonds at their C-terminal sides 12 .…”

Influence of Basic Residue Content on Fragment Ion Peak Intensities in Low-Energy Collision-Induced Dissociation Spectra of Peptides

“…The spectra in each subset were analyzed by a modification of the DaughterDB algorithm. 11 The measures reported for each subset were processed by scripts created in the R statistical environment. 19 …”

Fragmentation of protonated ions of peptides containing cysteine, cysteine sulfinic acid, and cysteine sulfonic acid

“…Our data with the [M ϩ H] ϩ ion of the PRCGVPDVA appear not to be consistent with this view because the selective cleavage occurs at the amide bond between valine and proline, which gives the b 5 ion. However, it has been recently shown that cleavage N-terminal to a proline residue is a very facile process especially when the residue at the N-terminal side of the proline is a valine [24,25]. It seems that sequence context plays a more important role in the fragmentation of the [M ϩ H] ϩ ion of PRC(SO 3 H)GVPDVA than intramolecular ionic interaction between the side chains of acidic and basic amino acid residues.…”