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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: Recently, rearrangement processes that result in scrambling of the original peptide sequence during CID have been reported for these ions.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.

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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Related in: MedlinePlus

Comparison of QTOF MS/MS spectra resulting from (a) CID of [M+H]+ KLVTDLT at m/z 789.47, and (b) CID of [M+H]+ derivatized peptide K(guanidinyl)LVTDLT at m/z 831.49. Although a large number of scrambled species were present (a), only those of significant abundance are labeled
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Fig3: Comparison of QTOF MS/MS spectra resulting from (a) CID of [M+H]+ KLVTDLT at m/z 789.47, and (b) CID of [M+H]+ derivatized peptide K(guanidinyl)LVTDLT at m/z 831.49. Although a large number of scrambled species were present (a), only those of significant abundance are labeled

Mentions: Further examples of the enhanced formation of b(n-1)+H2O species following guanidination of the N-terminal lysine residue are shown in KNVPLY (Figure 2a) and KLVTDLT (Figure 3a); both decompose during CID to produce minor b(n-1)+H2O species corresponding to loss of the C-terminal amino acid [40], in addition to generating ion series that are indicative of sequence rearrangement. In each example following derivatization, the peptide ion containing the N-terminal homoarginine residue dissociates to produce a significantly more abundant b(n-1)+H2O species, with no sequence scrambled species being observed (Figures 2b and 3b). In the case of KNVPLY (Figure 2), the native peptide generates scrambled products from b5 (including a pseudo C- terminal product ion designated [b52]y′3), whereas the derivatized peptide yields the b5+H2O (and b4+H2O) product ion upon CID. Likewise for KLVTDLT (Figure 3), formation of the b6+H2O and b5+H2O product ions upon N-terminal derivatization appear to preclude the generation of b5- and b6-derived scrambled products.Figure 2


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)

Comparison of QTOF MS/MS spectra resulting from (a) CID of [M+H]+ KLVTDLT at m/z 789.47, and (b) CID of [M+H]+ derivatized peptide K(guanidinyl)LVTDLT at m/z 831.49. Although a large number of scrambled species were present (a), only those of significant abundance are labeled
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4197365&req=5

Fig3: Comparison of QTOF MS/MS spectra resulting from (a) CID of [M+H]+ KLVTDLT at m/z 789.47, and (b) CID of [M+H]+ derivatized peptide K(guanidinyl)LVTDLT at m/z 831.49. Although a large number of scrambled species were present (a), only those of significant abundance are labeled
Mentions: Further examples of the enhanced formation of b(n-1)+H2O species following guanidination of the N-terminal lysine residue are shown in KNVPLY (Figure 2a) and KLVTDLT (Figure 3a); both decompose during CID to produce minor b(n-1)+H2O species corresponding to loss of the C-terminal amino acid [40], in addition to generating ion series that are indicative of sequence rearrangement. In each example following derivatization, the peptide ion containing the N-terminal homoarginine residue dissociates to produce a significantly more abundant b(n-1)+H2O species, with no sequence scrambled species being observed (Figures 2b and 3b). In the case of KNVPLY (Figure 2), the native peptide generates scrambled products from b5 (including a pseudo C- terminal product ion designated [b52]y′3), whereas the derivatized peptide yields the b5+H2O (and b4+H2O) product ion upon CID. Likewise for KLVTDLT (Figure 3), formation of the b6+H2O and b5+H2O product ions upon N-terminal derivatization appear to preclude the generation of b5- and b6-derived scrambled products.Figure 2

Bottom Line: Recently, rearrangement processes that result in scrambling of the original peptide sequence during CID have been reported for these ions.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.

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