Improved identification and relative quantification of sites of peptide and protein oxidation for hydroxyl radical footprinting

Abstract

Protein oxidation is typically associated with oxidative stress and aging and affects protein function in normal and pathological processes. Additionally, deliberate oxidative labeling is used to probe protein structure and protein-ligand interactions in hydroxyl radical protein footprinting (HRPF). Oxidation often occurs at multiple sites, leading to mixtures of oxidation isomers that differ only by the site of modification. We utilized sets of synthetic, isomeric "oxidized" peptides to test and compare the ability of electron-transfer dissociation (ETD) and collision-induced dissociation (CID), as well as nano-ultra high performance liquid chromatography (nanoUPLC) separation, to quantitate oxidation isomers with one oxidation at multiple adjacent sites in mixtures of peptides. Tandem mass spectrometry by ETD generates fragment ion ratios that accurately report on relative oxidative modification extent on specific sites, regardless of the charge state of the precursor ion. Conversely, CID was found to generate quantitative MS/MS product ions only at the higher precursor charge state. Oxidized isomers having multiple sites of oxidation in each of two peptide sequences in HRPF product of protein Robo-1 Ig1-2, a protein involved in nervous system axon guidance, were also identified and the oxidation extent at each residue was quantified by ETD without prior liquid chromatography (LC) separation. ETD has proven to be a reliable technique for simultaneous identification and relative quantification of a variety of functionally different oxidation isomers, and is a valuable tool for the study of oxidative stress, as well as for improving spatial resolution for HRPF studies.

Figure 1

Representative ETD spectrum of mixture of four oxidation isomers of the model peptide RPMFAIWK at (a) doubly-charged state and (b) triply-charged state by direct infusion. The mixture contains RP*MFAIWK, RPM*FAIWK, RPMF*AIWK, and RPMFA*IWK at a molar ratio 1:1:1:1. The asterisks indicate the product ions that are oxidized.

Figure 2

Representative CID spectrum of mixture of four oxidation isomers of the model peptide RPMFAIWK at (a) doubly-charged state and (b) triply-charged state by direct infusion. The mixture contains RP*MFAIWK, RPM*FAIWK, RPMF*AIWK, and RPMFA*IWK at a molar ratio 1:1:1:1. The asterisks indicate the product ions that are oxidized.

Figure 3

Comparison of theoretical and experimental quantification of mixtures of four oxidation isomers of the model peptide RPMFAIWK based on fragment c-type ions intensity by ETD at (a) doubly-charged state, and (b) triply-charged state, and on fragment b-type ions intensity by CID at (c) doubly-charged state and (d) triply-charged state, respectively. The mixtures contain: RP*MFAIWK, RPM*FAIWK, RPMF*AIWK, and RPMFA*IWK at five varying molar ratios of 1:1:1:1, 1:2:3:4, 4:3:2:1, 9:9:1:1, and 1:1:9:9 (n=3). Each replicate of the triplicate analysis is plotted as an individual data point.

Figure 4

Comparison of added molar ratio and experimental quantification calculated with RPM*FAIWK included (a) and excluded (b) of mixtures of four oxidation isomers of the model peptide RPMFAIWK based on SIM peak area after UPLC separation (n=3). Each replicate of the triplicate analysis is plotted as an individual data point.

Figure 5

The ETD spectrum of doubly-oxidized peptide LMITYTRK [M+2O+3H]³⁺. The single asterisks indicate the product ions that are singly oxidized. The double asterisks indicate the product ions that are doubly oxidized.

Figure 6

The ETD spectrum of singly oxidized peptide isomers IVEHPSDLIVSK [M+O+3H]³⁺. The asterisks indicate the product ions that are oxidized.