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Peptide scrambling during collision-induced dissociation is influenced by N-terminal residue basicity.

Chawner R, Holman SW, Gaskell SJ, Eyers CE - J. Am. Soc. Mass Spectrom. (2014)

Bottom Line: Here, analysis of peptide species produced by Lys-N proteolysis of standard proteins is performed and sequences that exhibit such rearrangement processes identified.The effect of increasing the gas-phase basicity of the N-terminal lysine residue through derivatization to homoarginine toward such sequence scrambling is then assessed.Finally, further analysis of a sequence produced by Lys-C proteolysis provides evidence toward a potential mechanism for the apparent inhibition of sequence scrambling during resonance excitation CID.

View Article: PubMed Central - PubMed

Affiliation: Michael Barber Centre for Mass Spectrometry, School of Chemistry, Manchester Institute of Biotechnology, University of Manchester, Manchester, M1 7DN, UK.

ABSTRACT
'Bottom up' proteomic studies typically use tandem mass spectrometry data to infer peptide ion sequence, enabling identification of the protein whence they derive. The majority of such studies employ collision-induced dissociation (CID) to induce fragmentation of the peptide structure giving diagnostic b-, y-, and a- ions. Recently, rearrangement processes that result in scrambling of the original peptide sequence during CID have been reported for these ions. Such processes have the potential to adversely affect ion accounting (and thus scores from automated search algorithms) in tandem mass spectra, and in extreme cases could lead to false peptide identification. Here, analysis of peptide species produced by Lys-N proteolysis of standard proteins is performed and sequences that exhibit such rearrangement processes identified. The effect of increasing the gas-phase basicity of the N-terminal lysine residue through derivatization to homoarginine toward such sequence scrambling is then assessed. The presence of a highly basic homoarginine (or arginine) residue at the N-terminus is found to disfavor/inhibit sequence scrambling with a coincident increase in the formation of b(n-1)+H(2)O product ions. Finally, further analysis of a sequence produced by Lys-C proteolysis provides evidence toward a potential mechanism for the apparent inhibition of sequence scrambling during resonance excitation CID.

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CID of [M+2H]2+ FGERALK with the collision energy set at either (a) 19.5 eV or (b) 26 eV using a QTOF instrument results in formation of a y6 ion at m/z 673.39. Distortion of the isotope distribution in (a) suggests a contribution from the b6 ion at m/z 674.36. Shown in the inset is the theoretical isotope distribution of the b6 ion which was modeled using the MS-Isotope program within Protein Prospector [47]. (c) Equivalent CID analysis of [M+2H]2+ FGERALK using a QIT instrument results in formation of an abundant b6 ion at m/z 674.36. N.B. Slight calibration differences between the QTOF and QIT instruments used for analysis means there is a small discrepancy in the observed m/z.
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Fig6: CID of [M+2H]2+ FGERALK with the collision energy set at either (a) 19.5 eV or (b) 26 eV using a QTOF instrument results in formation of a y6 ion at m/z 673.39. Distortion of the isotope distribution in (a) suggests a contribution from the b6 ion at m/z 674.36. Shown in the inset is the theoretical isotope distribution of the b6 ion which was modeled using the MS-Isotope program within Protein Prospector [47]. (c) Equivalent CID analysis of [M+2H]2+ FGERALK using a QIT instrument results in formation of an abundant b6 ion at m/z 674.36. N.B. Slight calibration differences between the QTOF and QIT instruments used for analysis means there is a small discrepancy in the observed m/z.

Mentions: Further inspection of the relative abundances of the peaks associated with the overlapping isotope patterns of the b6 (m/z 674.36) and y6 (m/z 673.39) ions in each spectrum shows that the b6 population is depleted during analysis in the multiple collision regime of the QTOF collision cell (Figure 6a and b), whereas the singly charged a6 ion (Figure 5) is observed with significant abundance during each analysis. It has previously been demonstrated that N-terminal product ions are less stable toward secondary fragmentation events than their y-ion counterparts under such conditions and as a consequence are often under-represented in QTOF MS/MS spectra compared with those obtained using a QIT [48]. During CID on a QTOF instrument, peptide ions are subject to multiple collisions with the neutral buffer gas causing preferential secondary decomposition of b-ion species to lower members of the ion series. In this instance, it would appear that during analysis on the QTOF platform, once initial decomposition has occurred, the b6 and b5 product ions undergo further activation, promoting rearrangement and secondary fragmentation leading to formation of the [b63]b5 and [b53]b4 species; there is also an increase in the relative abundance of the smaller b4 (and b5) ion(s), as might be expected following multiple dissociation events from larger species (Figure 5). This observation is highlighted by analysis of the y6 ion isotope ratio observed from analysis on the QTOF instrument at high and low collision energy. At lower collision energy (Figure 6a, collision energy set at 19.5 eV) there is a distortion of the distribution of ion current, suggesting a contribution to this isotopic envelope from the b6 ion.Scheme 1


Peptide scrambling during collision-induced dissociation is influenced by N-terminal residue basicity.

Chawner R, Holman SW, Gaskell SJ, Eyers CE - J. Am. Soc. Mass Spectrom. (2014)

CID of [M+2H]2+ FGERALK with the collision energy set at either (a) 19.5 eV or (b) 26 eV using a QTOF instrument results in formation of a y6 ion at m/z 673.39. Distortion of the isotope distribution in (a) suggests a contribution from the b6 ion at m/z 674.36. Shown in the inset is the theoretical isotope distribution of the b6 ion which was modeled using the MS-Isotope program within Protein Prospector [47]. (c) Equivalent CID analysis of [M+2H]2+ FGERALK using a QIT instrument results in formation of an abundant b6 ion at m/z 674.36. N.B. Slight calibration differences between the QTOF and QIT instruments used for analysis means there is a small discrepancy in the observed m/z.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4197365&req=5

Fig6: CID of [M+2H]2+ FGERALK with the collision energy set at either (a) 19.5 eV or (b) 26 eV using a QTOF instrument results in formation of a y6 ion at m/z 673.39. Distortion of the isotope distribution in (a) suggests a contribution from the b6 ion at m/z 674.36. Shown in the inset is the theoretical isotope distribution of the b6 ion which was modeled using the MS-Isotope program within Protein Prospector [47]. (c) Equivalent CID analysis of [M+2H]2+ FGERALK using a QIT instrument results in formation of an abundant b6 ion at m/z 674.36. N.B. Slight calibration differences between the QTOF and QIT instruments used for analysis means there is a small discrepancy in the observed m/z.
Mentions: Further inspection of the relative abundances of the peaks associated with the overlapping isotope patterns of the b6 (m/z 674.36) and y6 (m/z 673.39) ions in each spectrum shows that the b6 population is depleted during analysis in the multiple collision regime of the QTOF collision cell (Figure 6a and b), whereas the singly charged a6 ion (Figure 5) is observed with significant abundance during each analysis. It has previously been demonstrated that N-terminal product ions are less stable toward secondary fragmentation events than their y-ion counterparts under such conditions and as a consequence are often under-represented in QTOF MS/MS spectra compared with those obtained using a QIT [48]. During CID on a QTOF instrument, peptide ions are subject to multiple collisions with the neutral buffer gas causing preferential secondary decomposition of b-ion species to lower members of the ion series. In this instance, it would appear that during analysis on the QTOF platform, once initial decomposition has occurred, the b6 and b5 product ions undergo further activation, promoting rearrangement and secondary fragmentation leading to formation of the [b63]b5 and [b53]b4 species; there is also an increase in the relative abundance of the smaller b4 (and b5) ion(s), as might be expected following multiple dissociation events from larger species (Figure 5). This observation is highlighted by analysis of the y6 ion isotope ratio observed from analysis on the QTOF instrument at high and low collision energy. At lower collision energy (Figure 6a, collision energy set at 19.5 eV) there is a distortion of the distribution of ion current, suggesting a contribution to this isotopic envelope from the b6 ion.Scheme 1

Bottom Line: Here, analysis of peptide species produced by Lys-N proteolysis of standard proteins is performed and sequences that exhibit such rearrangement processes identified.The effect of increasing the gas-phase basicity of the N-terminal lysine residue through derivatization to homoarginine toward such sequence scrambling is then assessed.Finally, further analysis of a sequence produced by Lys-C proteolysis provides evidence toward a potential mechanism for the apparent inhibition of sequence scrambling during resonance excitation CID.

View Article: PubMed Central - PubMed

Affiliation: Michael Barber Centre for Mass Spectrometry, School of Chemistry, Manchester Institute of Biotechnology, University of Manchester, Manchester, M1 7DN, UK.

ABSTRACT
'Bottom up' proteomic studies typically use tandem mass spectrometry data to infer peptide ion sequence, enabling identification of the protein whence they derive. The majority of such studies employ collision-induced dissociation (CID) to induce fragmentation of the peptide structure giving diagnostic b-, y-, and a- ions. Recently, rearrangement processes that result in scrambling of the original peptide sequence during CID have been reported for these ions. Such processes have the potential to adversely affect ion accounting (and thus scores from automated search algorithms) in tandem mass spectra, and in extreme cases could lead to false peptide identification. Here, analysis of peptide species produced by Lys-N proteolysis of standard proteins is performed and sequences that exhibit such rearrangement processes identified. The effect of increasing the gas-phase basicity of the N-terminal lysine residue through derivatization to homoarginine toward such sequence scrambling is then assessed. The presence of a highly basic homoarginine (or arginine) residue at the N-terminus is found to disfavor/inhibit sequence scrambling with a coincident increase in the formation of b(n-1)+H(2)O product ions. Finally, further analysis of a sequence produced by Lys-C proteolysis provides evidence toward a potential mechanism for the apparent inhibition of sequence scrambling during resonance excitation CID.

Show MeSH
Related in: MedlinePlus