Site-specific characterization of (D)-amino acid containing peptide epimers by ion mobility spectrometry

Abstract

Traditionally, the d-amino acid containing peptide (DAACP) candidate can be discovered by observing the differences of biological activity and chromatographic retention time between the synthetic peptides and naturally occurring peptides. However, it is difficult to determine the exact position of d-amino acid in the DAACP candidates. Herein, we developed a novel site-specific strategy to rapidly and precisely localize d-amino acids in peptides by ion mobility spectrometry (IMS) analysis of mass spectrometry (MS)-generated epimeric fragment ions. Briefly, the d/l-peptide epimers were separated by online reversed-phase liquid chromatography and fragmented by collision-induced dissociation (CID), followed by IMS analysis. The epimeric fragment ions resulting from d/l-peptide epimers exhibit conformational differences, thus showing different mobilities in IMS. The arrival time shift between the epimeric fragment ions was used as criteria to localize the d-amino acid substitution. The utility of this strategy was demonstrated by analysis of peptide epimers with different molecular sizes, [d-Trp]-melanocyte-stimulating hormone, [d-Ala]-deltorphin, [d-Phe]-achatin-I, and their counterparts that contain all-l amino acids. Furthermore, the crustacean hyperglycemia hormones (CHHs, 8.5 kDa) were isolated from the American lobster Homarus americanus and identified by integration of MS-based bottom-up and top-down sequencing approaches. The IMS data acquired using our novel site-specific strategy localized the site of isomerization of l- to d-Phe at the third residue of the CHHs from the N-terminus. Collectively, this study demonstrates a new method for discovery of DAACPs using IMS technique with the ability to localize d-amino acid residues.

Figure 1

Workflow of the proposed strategy for localization of d-amino acids in peptides. The analysis can be performed in a single LC–MS/MS–IMS run. The two peptide epimers are separated by RPLC and respectively fragmented by CID. Their fragment ions are then submitted to IMS for arrival time measurement. By comparing the arrival time distributions between the two sets of fragment ions, the position of d-amino acid can be determined: √, arrival time shift; ×, no shift. For illustration purpose, only y ions are listed in this workflow. Note that other fragment ions can also be used as indicators for localization of d-amino acids.

Figure 2

Site-specific characterization of d/l-MSH peptide epimers. (A) Extracted ion chromatogram of LC–MS analysis of d/l-MSH peptides. (B) Molecular ions and (C) the corresponding IMS distributions of d/l-MSH peptides. (D) IMS distributions of fragment ions of d/l-MSH peptides. (E) Localization of d-amino acid residue position by comparison of arrival time shift: √, arrival time shift; ×, no shift. (F) CCS differences (ΔCCS, absolute values) of peptide precusor and fragment ions. Error bars stand for standard deviations.

Figure 3

Site-specific characterization of d/l-DTP and d/l-achatin-I peptide epimers. (A) Extracted ion chromatogram of LC–MS analysis of d/l-DTP peptides. (B) Molecular ions and (C) the corresponding IMS distributions of d/l-DTP peptides. (D) IMS distributions of fragment ions of d/l-DTP peptides. (E) Localization of d-amino acid residue position by comparison of arrival time shift. (F) IMS analysis of d/l-achatin-I and localization of d-amino acid position: √, arrival time shift; ×, no shift; ∗, interference ions.

Figure 4

Identification of CHH_-A and CHH_-B peptides (isolated from the sinus glands of American lobsters) by top-down MS/MS. (A) Isotopic distributions of intact and DTT-reduced CHH_-A. (B) HCD MS/MS spectrum of DTT-reduced CHH_-A. (C) Top-down fragmentation maps of CHH_-A and CHH_-B:⌉, b ions; ⌊, y ions. The different residues between the two CHH peptides are highlighted in blue. The d-Phe residue is localized in the third position from the N-terminus, highlighted in green shading.

Figure 5

Localization of d-amino acid in tryptic peptides, pQV_d/lFDQAC*K. (A) Extracted ion chromatogram and (B) MS/MS of the tryptic peptide epimers I and II. (C) IMS distributions of fragment ions from tryptic peptide epimers I and II. (D) Localization of d-amino acid residue by comparison of arrival time shift: √, arrival time shift; ×, no shift. It should be noted that the elution order of the two d/l-peptide epimers cannot be determined by our current method, so we use epimer I and II for annotation.

Figure 6

IMS distributions of fragment ions from tryptic peptide epimers, pQV_d/lFDQAC*KGVYDRNFLK. Tryptic peptide epimers I and II were eluted from RPLC at 33.21 and 35.70 min, respectively: √, arrival time shift; ×, no shift.