Gas-phase ion isomer analysis reveals the mechanism of peptide sequence scrambling

Abstract

Peptide sequence scrambling during mass spectrometry-based gas-phase fragmentation analysis causes misidentification of peptides and proteins. Thus, there is a need to develop an efficient approach to probing the gas-phase fragment ion isomers related to sequence scrambling and the underlying fragmentation mechanism, which will facilitate the development of bioinformatics algorithm for proteomics research. Herein, we report on the first use of electron transfer dissociation (ETD)-produced diagnostic fragment ions to probe the components of gas-phase peptide fragment ion isomers. In combination with ion mobility spectrometry (IMS) and formaldehyde labeling, this novel strategy enables qualitative and quantitative analysis of b-type fragment ion isomers. ETD fragmentation produced diagnostic fragment ions indicative of the precursor ion isomer components, and subsequent IMS analysis of b ion isomers provided their quantitative and structural information. The isomer components of three representative b ions (b9, b10, and b33 from three different peptides) were accurately profiled by this method. IMS analysis of the b9 ion isomers exhibited dynamic conversion among these structures. Furthermore, molecular dynamics simulation predicted theoretical drift time values, which were in good agreement with experimentally measured values. Our results strongly support the mechanism of peptide sequence scrambling via b ion cyclization, and provide the first experimental evidence to support that the conversion from molecular precursor ion to cyclic b ion (M → (c)b) pathway is less energetically (or kinetically) favored.

Figure 1

Workflow of the developed strategy for gas-phase ion isomer analysis. On the left panel, under ETD fragmentation each b ion isomer generates certain diagnostic fragment ions and the FH-labeled linear b ion is used for alignment. On the right panel, each isomer is separated by IMS, followed by comparison with the drift time of the FH-labeled b ions. Although ETD fragments multiply charged ions, we show singly charged ions here for simplicity in illustration.

Figure 2

ETD fragmentation spectra and IMS distributions of three b ions. (A,B) b₉²⁺ ion of neurokinin. (C,D) _FH-b₉²⁺ ion of neurokinin. (E,F) b₁₀²⁺ ion of substance P. (G,H) _FH-b₁₀²⁺ ion of substance P. (I,J) b₃₃⁴⁺ ion of CPRP. (K,L) _FH-b₃₃⁴⁺ ion of CPRP. The letter n denotes system noise. The details of ion assignment are listed in Tables S4–9.

Figure 3

IMS dynamics study of neurokinin b₉. (A) Workflow of the experimental design on ESI-QTOF-IMS. (B) Drift time distributions of b₉²⁺ under various collision energy values (CE, eV; bin, 0.069 ms). (C,D) CID spectra of b₉²⁺ from MS³-IMS-MS⁴ experiment. (E) Simulated drift time distributions of the four b₉²⁺ isomers (bin, 0.069 ms). Note that the integrated distributions from simulations are the same for the four isomers because 1500 structures are used for the simulation of each isomer. By contrast, the populations of the four isomers are different in experiment.

Figure 4

MS² (CID) of CPRP peptide (A) and MS³ (CID) of b₃₃ ion (B) acquired on LTQ-FTICR. The ^rb ions were highlighted with red and assigned according to ion nomenclature reported in Ref. The details of ion assignment are listed in Table S10. Note that the [b₃₃19]b₉~[b₃₃19]b₁₄ and [b₃₃8]b₂₅ ions could also be internal fragments produced by backbone cleavage of ^olb₃₃.