The effect of phosphorylation on the electron capture dissociation of peptide ions

Abstract

The effect of site and frequency of phosphorylation on the electron capture dissociation of peptide ions has been investigated. The ECD of a suite of synthetic peptides (APLSFRGSLPKSYVK; one unmodified, three singly-phosphorylated, three-doubly phosphorylated, and one triply-phosphorylated); two tryptic phosphopeptides (YKVPQLEIVPN(p)SAEER, alpha-casein and FQ(p)SEEQQQTEDELQDK, beta-casein) and their unmodified counterparts, were determined over a range of ECD cathode potentials. The results show that, for doubly-charged precursor ions, the presence of phosphorylation has a deleterious effect on ECD sequence coverage. The fragmentation patterns observed suggest that for peptides with multiple basic residues, the phospho-groups exist in their deprotonated form and form salt-bridges with protonated amino acid side chains. The fragmentation observed for the acidic tryptic peptides suggested the presence of noncovalent interactions, which were perturbed on phosphorylation. Increasing the ECD electron energy significantly improves sequence coverage. Alternatively, improved sequence coverage can be achieved by performing ECD on triply-charged precursor ions. The findings are important for the understanding of gas-phase fragmentation of phosphopeptides.

Graphical abstract

Figure 1

ECD mass spectra of doubly-charged ions of four synthetic peptides (APLS1FRGS2LPKS3YVK). ECD of (a) unmodified peptide at cathode potentials of (left) −3.34 V (standard) and (right) −13.34 V (cathode potential which gave the greatest sequence coverage) (6 scans); (b) Ser1 phosphopeptide at cathode potentials of (left) −3.34 V and (right) 14.34 V (4 scans); (c) Ser2 phosphopeptide at cathode potentials of (left) −3.34 V and (right) −14.34 V (7 scans); (d) Ser3 phosphopeptide at cathode potentials of (left) −3.34 V and (right) −14.84 V (9 scans).

Figure 2

Summary of fragments observed following ECD of doubly-charged precursor ions at ‘standard’ cathode potential and at the cathode potential which gave greatest sequence coverage.

Figure 3

Normalized relative abundance of peptide fragment ions versus ECD cathode potential for (a) the unmodified peptide (APLSFRGSLPKSYVK); (b) the singly modified phosphopeptide (APLSFRGSLPK_PSYVK).

Figure 4

ECD mass spectra of triply-charged synthetic (APLS1FRGS2LPKS3YVK) peptide ions obtained at “standard” (−3.34 V) ECD cathode potential. (a) Unmodified peptide (12 scans); (b) Ser1 phosphopeptide (10 scans); (c) Ser2 phosphopeptide (12 scans); (d) Ser3 phosphopeptide (12 scans); (e) Ser1, Ser2 phosphopeptide (11 scans); (f) Ser1, Ser3 phosphopeptide (9 scans); (g) Ser2, Ser3 phosphopeptide (10 scans); (h) Ser1, Ser2, Ser3 phosphopeptide (9 scans).

Figure 5

(Top) ECD mass spectra (4 scans) of doubly-charged ions of the β-casein tryptic peptide FQSEEQQQTEDELQDK obtained at ECD cathode potentials of (a) −3.34 V (standard) and (b) −9.34 V. (Bottom) ECD mass spectra (7 scans) of doubly-charged ions of FQ_pSEEQQQTEDELQDK obtained at ECD cathode potentials of (c) −3.34 V (standard) and (d) −12.84 V.

Figure 6

(Top) ECD mass spectra (3 scans) of doubly-charged ions of the α-S1-casein tryptic peptide YKVPQLEIVPNSAEER obtained at ECD cathode potentials (a) −3.34 V (standard) and (b) −11.84 V. (Bottom) ECD mass spectra (5 scans) of doubly-charged ions of YKVPQLEIVPN_pSAEER obtained at cathode potentials of (c) −3.34 V (standard) and (d) −12.34 V.