Localizing Isomerized Residue Sites in Peptides with Tandem Mass Spectrometry

Abstract

Isomerized amino acid residues have been identified in many peptides extracted from tissues or excretions of humans and animals. These isomerized residues can play key roles by affecting biological activity or by exerting an influence on the process of aging. Isomerization occurs spontaneously and does not result in a mass shift. Thus, identifying and localizing isomerized residues in biological samples is challenging. Herein, we introduce a fast and efficient method using tandem mass spectrometry (MS) to locate isomerized residues in peptides. Although MS² spectra are useful for identifying peptides that contain an isomerized residue, they cannot reliably localize isomerization sites. We show that this limitation can be overcome by utilizing MS³ experiments to further evaluate each fragment ion from the MS² stage. Comparison at the MS³ level, utilizing statistical analyses, reveals which MS² fragments differ between samples and, therefore, must contain the isomerized sites. The approach is similar to previous work relying on ion mobility to discriminate MS² product ions by collision cross-section. The MS³ approach can be implemented using either ion-trap or beam-type collisional activation and is compatible with the quantification of isomer mixtures when coupled to a calibration curve. The method can also be implemented in combination with liquid chromatography in a targeted approach. Enabling the identification and localization of isomerized residues in peptides with an MS-only methodology will expand accessibility to this important information.

Keywords: Epimer; Fragmentation; Isoleucine; Isomer; Leucine; isoAsp.

Scheme 1. Isomerization Mechanism for Aspartic Acid (Asp) Involves the Succinimide Ring Formation

Figure 1

MS spectra at annotated levels for FAEDVGSNK [M + 2H]²⁺: (a) All-l and (b) d-Asp. MS¹ spectra are identical, as expected. CID fragmentation reveals patterns that differ between the two isomers. Subsequent HCD fragmentation of the y₇⁺ ions (highlighted in yellow) again yields different fragmentation patterns in MS³ spectra. (c) Boxplots of the intensity ratios for All-l (blue) and d-Asp (orange) peptide isomers differ considerably. (d) Boxplots for replicate data are far more similar.

Scheme 2. (a) Experimental Workflow for Localization of Isomerized Residue Using Tandem MS and (b) Data Analysis Process for Localization of Isomerized Residue Using MS³ Mass Spectra

Figure 2

[FAEDVGSNK + 2H]²⁺ precursor produces several CID fragment ions which were fragmented by HCD in MS³. The fragment ions containing the isomerized residues are labeled in red. (a) The intensity ratios of the most different MS³ fragments between peptide isomers All-l and d-Asp are plotted for each fragment ion along with their corresponding statistical analyses. (b) No statistically significant differences were observed in All-l replicates. Collective results for 10 peptides using CID in MS² followed by either (c) HCD or (d) CID in MS³. The log values (effect sizes) of the intensity ratios from two peptide isomers are categorized into fragments with or without the presence of isomerized residues (colored in green and purple). All fragment ions without the isomerized residue have a log₁₀ effect size of <0, while most isomeric fragments have a log₁₀ effect size of >0.

Figure 3

Ten peptide isomers (a–j), where modified amino acids are highlighted in red, were fragmented using CID and followed by HCD to localize sites of isomerization. White boxes indicate ions for which MS³ data could not be collected. For all other ions, the color derived from the effect size indicates the probability that a fragment contains an isomerized site (values of >0 are consistent with isomerized sites).

Figure 4

The same peptide isomers (a–j) were fragmented using CID-CID for isomer mapping, where modified amino acids are highlighted in red. The intensity ratio changed due to the isomerized residue is indicated by color as seen in the effect size scale.

Figure 5

A mixture of different compositions of two peptide isomers, GGGIGGGK and GGGLGGGK, is compared to a pure GGGIGGGK standard. CID fragment ions containing isomerized residues y₅⁺ (shown in yellow) and y₆⁺ (shown in blue) were fragmented using CID in MS³. A highly correlated, linear relationship between the composition of isomers and the corresponding effect size was observed in both fragment ions. In contrast, CID fragments without the isomerized residue (y₃⁺ and y₄⁺) do not appear to be correlated and yield effect sizes below 1.0 for all compositions (gray shaded area).