We used 2D-LC-MS/MS methods to examine the impact of the biologically important PTM citrullination and its analogue carbamylation on peptide retention. Three first-dimension separation systems were evaluated: high-pH, HILIC and SCX, with the instrument interface separation happening at low-pH RP for all three. As we have well developed models for all of these separation systems, our goal was to enhance peptide assignment using retention prediction.
We induced the PTMs citrullination and carbamylation on a mixture of integrins using PAD2 and urea respectively. As deamidation often confounds the assignment of citrullination we also examined its retention properties. Identified peptides were organized into pairwise modified and unmodified counterparts to assign shifts in both separation dimensions.
Under both HILIC and SCX separations peptide retention times were significantly shifted by both PTMs, with SCX exhibiting shift magnitudes greater than the prediction error of our current computational model. Conversely deamidation's retention impact across all separation models was small. These findings gave us the capacity to assign the target PTMs without needing to identify their unmodified counterparts.
We applied this approach to an un-depleted sample of human synovial fluid from an RA patient, identifying 14 citrullinated peptides which were supported by prior RA literature.
[doi:10.25345/C5C49Q]
[dataset license: CC0 1.0 Universal (CC0 1.0)]
Keywords: citrullination ; carbamylation ; PTMS ; 2D LC-MS/MS ; retention prediction ; peptide assignment ; arthritis
Principal Investigators: (in alphabetical order) |
Oleg Krokhin, University of Manitoba, Canada |
Submitting User: | vspicer1 |
Neale Q, Prefontaine A, Battellino T, Mizero B, Yeung D, Spicer V, Budisa N, Perreault H, Zahedi RP, Krokhin OV.
Compendium of Chromatographic Behavior of Post-translationally and Chemically Modified Peptides in Bottom-Up Proteomic Experiments.
Anal Chem. 2023 Oct 3;95(39):14634-14642. Epub 2023 Sep 22.
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