Moving Away from the Reference Genome: Evaluating a Peptide Sequencing Tagging Approach for Single Amino Acid Polymorphism Identifications in the Genus <i>Populus</i>

The genetic diversity across natural
populations of the model organism, Populus, is extensive,
containing a single nucleotide polymorphism
roughly every 200 base pairs. When deviations from the reference genome
occur in coding regions, they can impact protein sequences. Rather
than relying on a static reference database to profile protein expression,
we employed a peptide sequence tagging (PST) approach capable of decoding
the plasticity of the Populus proteome. Using shotgun
proteomics data from two genotypes of P. trichocarpa, a tag-based approach enabled the detection of 6653 unexpected sequence
variants. Through manual validation, our study investigated how the
most abundant chemical modification (methionine oxidation) could masquerade
as a sequence variant (Ala→Ser) when few site-determining ions
existed. In fact, precise localization of an oxidation site for peptides
with more than one potential placement was indeterminate for 70% of
the MS/MS spectra. We demonstrate that additional fragment ions made
available by high energy collisional dissociation enhances the robustness
of the peptide sequence tagging approach (81% of oxidation events
could be exclusively localized to a methionine). We are confident
that augmenting fragmentation processes for a PST approach will further
improve the identification of single amino acid polymorphism in Populus and potentially other species as well.