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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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Comparison of QTOF MS/MS spectra resulting from (a) CID of [M+H]+ peptide KGVLHAV at m/z 723.45, and (b) CID of [M+H]+ peptide K(guanidinyl)GVLHAV at m/z 765.47
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Fig1: Comparison of QTOF MS/MS spectra resulting from (a) CID of [M+H]+ peptide KGVLHAV at m/z 723.45, and (b) CID of [M+H]+ peptide K(guanidinyl)GVLHAV at m/z 765.47

Mentions: To study the effect of increasing the gas-phase basicity of the N-terminal residue on sequence scrambling, it was first necessary to identify those peptides produced by digestion with Lys-N that exhibited evidence of significant rearrangement during CID. A selection of native Lys-N peptides and their corresponding derivatized sequence, now with an N-terminal homoarginine residue, were isolated and subjected to CID in the trap collision cell of a Synapt HDMS instrument (Waters). As expected, increased collision energy was required to induce dissociation of the derivatized analogue because of reduced mobility of the proton now sequestered by the homoarginine residue. Figure 1 compares the CID product ion spectra produced from analysis of the native and lysine-derivatized forms of the singly protonated peptide KGVLHAV. The spectrum derived from the native peptide ion (Figure 1a) contains several species attributable to scrambling of the original primary sequence. However, these rearranged product ions are not observed in the MS/MS spectrum of the derivatized peptide (Figure 1b). The b6 ion of the derivatized peptide (K*GVLHA) does not appear to be generated during CID and, hence, the precursor to formation of many of the observed rearranged product ions is not present. However, there is an abundant peak at m/z 666.40 that corresponds to the b6+H2O ion; such species have previously been shown by Thorne et al. to have a linear structure analogous to that of an intact peptide [40]. As the oxazolone moiety is required for the macrocyclic intermediate to form, scrambled species cannot, consequently, be generated from this linear ion. Moreover, the [b53]b4 scrambled species in the native peptide product ion mass spectrum derived following rearrangement of the primary b5 product ion (labeled according to the Chawner et al. nomenclature [23]) is also not observed upon CID of the derivatized peptide. These differences can only be explained by the induced change in the N-terminal residue side chain. Curiously, a number of product ions were observed from these Lys-N-generated peptides that could not be assigned after consideration of either linear or b-ion rearranged product ions, and their likely identity has yet to be determined. The observation of unassigned product ions is in contrast to the typical ion trap CID spectra of Lys-N peptides exemplified in Taouatas et al. [41], but apparent in the few QTOF CID spectra presented by Rao et al. [42], suggesting that these ions may in part be an effect of the N-terminal basic residue on secondary fragmentation pathways occurring during beam-type CID.Figure 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)

Comparison of QTOF MS/MS spectra resulting from (a) CID of [M+H]+ peptide KGVLHAV at m/z 723.45, and (b) CID of [M+H]+ peptide K(guanidinyl)GVLHAV at m/z 765.47
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Comparison of QTOF MS/MS spectra resulting from (a) CID of [M+H]+ peptide KGVLHAV at m/z 723.45, and (b) CID of [M+H]+ peptide K(guanidinyl)GVLHAV at m/z 765.47
Mentions: To study the effect of increasing the gas-phase basicity of the N-terminal residue on sequence scrambling, it was first necessary to identify those peptides produced by digestion with Lys-N that exhibited evidence of significant rearrangement during CID. A selection of native Lys-N peptides and their corresponding derivatized sequence, now with an N-terminal homoarginine residue, were isolated and subjected to CID in the trap collision cell of a Synapt HDMS instrument (Waters). As expected, increased collision energy was required to induce dissociation of the derivatized analogue because of reduced mobility of the proton now sequestered by the homoarginine residue. Figure 1 compares the CID product ion spectra produced from analysis of the native and lysine-derivatized forms of the singly protonated peptide KGVLHAV. The spectrum derived from the native peptide ion (Figure 1a) contains several species attributable to scrambling of the original primary sequence. However, these rearranged product ions are not observed in the MS/MS spectrum of the derivatized peptide (Figure 1b). The b6 ion of the derivatized peptide (K*GVLHA) does not appear to be generated during CID and, hence, the precursor to formation of many of the observed rearranged product ions is not present. However, there is an abundant peak at m/z 666.40 that corresponds to the b6+H2O ion; such species have previously been shown by Thorne et al. to have a linear structure analogous to that of an intact peptide [40]. As the oxazolone moiety is required for the macrocyclic intermediate to form, scrambled species cannot, consequently, be generated from this linear ion. Moreover, the [b53]b4 scrambled species in the native peptide product ion mass spectrum derived following rearrangement of the primary b5 product ion (labeled according to the Chawner et al. nomenclature [23]) is also not observed upon CID of the derivatized peptide. These differences can only be explained by the induced change in the N-terminal residue side chain. Curiously, a number of product ions were observed from these Lys-N-generated peptides that could not be assigned after consideration of either linear or b-ion rearranged product ions, and their likely identity has yet to be determined. The observation of unassigned product ions is in contrast to the typical ion trap CID spectra of Lys-N peptides exemplified in Taouatas et al. [41], but apparent in the few QTOF CID spectra presented by Rao et al. [42], suggesting that these ions may in part be an effect of the N-terminal basic residue on secondary fragmentation pathways occurring during beam-type CID.Figure 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