Rapid Quantification of Peptide Oxidation Isomers From Complex Mixtures

Abstract

Hydroxyl radical protein footprinting (HRPF) is a powerful technique for probing changes in protein topography, based on quantifying the amount of oxidation of different regions of a protein. While quantification of HRPF oxidation at the peptide level is relatively common and straightforward, quantification at the residue level is challenging because of the influence of oxidation on MS/MS fragmentation and the large number of complex and only partially chromatographically resolved isomeric peptide oxidation products. HRPF quantification of isomeric peptide oxidation products (where the peptide sequence is the same but isomeric oxidation products are formed at different sites) at the residue level by electron transfer dissociation tandem mass spectrometry (ETD MS/MS) has been demonstrated in both model peptides and HRPF products, but the method is hampered by the partial separation of oxidation isomers by reversed phase chromatography. This requires custom MS/MS methods to equally sample all isomeric oxidation products across their elution window, greatly increasing method development time and reducing the oxidation products quantified in a single LC-MS/MS run. Here, we present a zwitterionic hydrophilic interaction capillary chromatography (ZIC-HILIC) method to ideally coelute all isomeric peptide oxidation products while separating different peptides. This allows us to relatively quantify peptide oxidation isomers using an ETD MS/MS spectrum acquired at any point across the single peptide oxidation isomer peak, greatly simplifying data acquisition and data analysis.

Figure 1.. Representation of making mass inclusion list of a myoglobin peptide for a C18 RP ETD custom method.

Each blue line represents an ETD MS/MS scan. This process has to be done for all the peptides in all of their oxidation states for each sample. It takes several hours to create a custom method with a full targeted mass inclusion list for each replicate.

Figure 2.. Chromatographic separation of synthetic isomeric peptide oxidation products.

A. Capillary ZIC-HILIC yields co-elution of all three isomeric oxidized peptides. B. Isomeric oxidized peptides are separated by nano-C18 RP.

Figure 3.. Average measured oxidation of three synthetic peptide isomer set RPMFAIWK, calculated using ETD spectra at various ZIC-HILIC retention times.

(A) Extracted product ion chromatogram of c2 product ion from oxidized peptide precursor. Black trace shows the unoxidized c2 ion, specific for the two isomers containing oxygen on F or A, while the red trace shows the oxidized c2 ion, specific for oxidation of P. Two traces are overlapping, indicating co-elution of the oxidation isomers. Markers on the peak indicate ETD spectra used for quantification. (B) Average quantification (Mean ± SD) of each oxidation isomer at each of five different elution times in triplicate, using ETD product ion abundances.

Figure 4.. Measured oxidation of RPMFAIWK oxidation isomers at varying relative concentrations calculated using ZIC-HILIC ETD spectra at five different retention times.

(A) Representative ZIC-HILIC selected ion chromatogram of the peptide oxidation isomers. Each shape represents a different retention time sampled by ETD for quantitation. (B) Measurement of oxidation of three peptide oxidation isomers mixed in six different relative ratios. The x-axis represents the oxidation of each amino acid as measured by ZIC-HILIC ETD, while the y-axis represents the theoretical oxidation of that amino acid in the synthetic mixture. The diagonal line represents x=y. Different marker shapes represent different times for ETD MS/MS spectrum acquisition as shown in A; different colors represents different ratios of synthetic oxidation products listed as Hyp2:Tyr4:Ser5. No systematic error is apparent; the measurements all cluster around the theoretical value.

Figure 5.. Separation of non-isomeric tryptic peptides.

Selected ion chromatograms for various peptides are shown for ZIC-HILIC separation of tryptic digests of (A) BSA and (B) myoglobin.

Figure 6.. Extracted product ion chromatogram of c10 product ion for myoglobin peptide (VEADIAGHGQEVLIR) M+16 isomers via ZIC-HILIC and C18 RPLC.

Black trace shows selected ion chromatogram of singly oxidized VEADIAGHGQEVLIR isomer peptides. Red trace shows the unoxidized c10 ion, while the green trace shows the oxidized c10 ion. (A) In ZIC-HILIC chromatography, the three traces are overlapping, indicating co-elution of peptide oxidation isomers. Accurate quantification of oxidation at the residue level could be obtained by a data-dependent ETD MS/MS spectrum obtained at any point along the single peak. (B) In C18 RPLC, there is a significant spread in the elution times of various peptide oxidation isomers. Accurate quantification of these isomers would require scheduling of ETD MS/MS across an almost four minute elution time window.

Figure 7.. Peptide level comparison of Myoglobin peptides using ZIC-HILIC (blue) VS. C18 chromatography (orange).

Both methods generated statistically indistinguishable quantification for each peptide (α = 0.05).

Figure 8.. Amino acid level analysis of myoglobin peptides using ZIC-HILIC.

Error bars represent one standard deviation from a triplicate analysis.