Relative Quantification of Sites of Peptide and Protein Modification Using Size Exclusion Chromatography Coupled with Electron Transfer Dissociation

Abstract

One difficult problem in the analysis of peptide modifications is quantifying isomeric modifications that differ by the position of the amino acid modified. HPLC separation using C18 reverse phase chromatography coupled with electron transfer dissociation (ETD) in tandem mass spectrometry has recently been shown to be able to relatively quantify how much of a given modification occurs at each amino acid position for isomeric mixtures; however, the resolution of reverse phase chromatography greatly complicates quantification of isomeric modifications by ETD because of the chromatographic separation of peptides with identical modifications at different sequence positions. Using peptide oxidation as a model system, we investigated the use of size exclusion chromatography coupled with ETD fragmentation to separate peptide sequences. This approach allows for the benefits of chromatographic separation of peptide sequences while ensuring co-elution of modification isomers for accurate relative quantification of modifications using standard data-dependent acquisitions. Using this method, the relative amount of modification at each amino acid can be accurately measured from single ETD MS/MS spectra in a standard data-dependent acquisition experiment. Graphical Abstract ᅟ.

Keywords: Electron transfer dissociation; Fast photochemical oxidation of proteins; Hydroxyl radical protein footprinting; Peptide modification; Size exclusion chromatography.

Figure 1

Chromatography for peptide isomer sets using two different LC methods. (a) C18 RP, most isomers were baseline separated; and (b) TSKgel SEC, peptide sequences were separated, while oxidation isomers co-eluted.

Figure 2

Measured oxidation of two different synthetic peptide isomer sets (a) RPMFAIWK, and (b) FESNFNTQATNR calculated using ETD spectra at various retention times from SEC method. Black trace: MS extracted ion chromatogram (EIC) of the precursor; green trace: MS/MS EIC of unoxidized ETD product ions; red trace: MS/MS EIC of oxidized ETD product ions. Product ions traced for RPMFAIWK and FESNFNTQATNR peptide sets are the c2 ions (specific for oxidation of P2) and the z4 ions (specific for oxidation of A9), respectively. All three traces are thoroughly overlapping for each peptide, indicating coelution of the oxidation isomers.

Figure 3

Relationship for lysozyme oxidized by FPOP between calculated oxidation percentages at each amino acid using RP-ETD method with multiple ETD measurements averaged across all oxidation isomer peaks and SEC-ETD using single spectrum data dependent ETD MS/MS measurements for quantification, based on FPOP of lysozyme. The line represents the ideal 1:1 relationship between RP-ETD and SEC-ETD methods, and r is the sample Pearson correlation coefficient. The SEC-based methods gave quantification results highly comparable to RP-based ETD.