Investigating key host, variant and microbial peptides for detection of oral cancer using a multi-omics approach

There is an unmet need for risk stratification biomarkers for precancerous oral leukoplakia patients. While host biomarkers are available for detection, there is a clear need to expand the biomarker panel to improve predictive performance. We have used the latest methods for enrichment of low-abundance proteins from non-invasively collected samples, sensitive mass spectrometry (MS) methods, and bioinformatic analysis to delve deeper into microbial proteome along with host and variant proteins from precancerous patients.

Oral rinse samples from six oral precancerous lesion patients (pre/post chemoprevention agent treatment) were processed using the PreOmics ENRICH kit to facilitate the detection of low abundance proteins. Digested proteins were analyzed by DIA-PASEF on a hybrid TIMS QTOF mass spectrometer (Bruker). RNASeq analysis was used to generate customized proteogenomics and metaproteomics databases. The MS data was searched against the human proteome along with variant proteins and microbial proteins using Spectronaut 19 (Biognosys). Bioinformatic and statistical analysis was performed to detect differentially expressed host proteins, microbial proteins and novel proteoforms.

Enrichment of low abundance proteins coupled with DIA-PASEF MS allowed for deeper quantitative analysis of oral host proteome and taxonomic and functional analysis of oral microbiome. Several human, microbial and variant proteins were detected to be differentially abundant in pretreatment and treated samples. Out of the 5894 human proteins quantified across the six replicates, 81 proteins were differentially abundant (51 in treated samples and 30 in pretreatment samples). Proteins associated with Gamma-carboxylation of protein precursors and complement cascade were upregulated and vesicle-mediated transport, apoptosis and inflammasome pathways were downregulated after treatment. Metaproteomics analysis detected 6830 microbial protein groups, out of which 38 microbial proteins were detected to be differentially abundant amongst treated and untreated samples. Notably, dextransucrase (glycotransferase involved in exopolysaccharide synthesis and promotes biofilm formation) from Streptococcus salivarius was abundant in treated samples. Additionally, D-Ala D-Ala carboxypeptidase (involved in peptidoglycan synthesis) from S. salivarius was also abundant in treated samples. Lastly, Alkyl hydroperoxide reductase subunit C (catalytic subunit responsible for the detoxification of reactive oxygen species) from Veillonella was found to be abundant in pretreated samples. Finally, variant proteins and peptides associated with PYCARD and GDI2 are upregulated after treatment. Taken together we have more than 300 peptides (127 host, 125 microbial and 80 variant peptides) that will prioritized to generate a biomarker panel which can be later assessed for a) risk stratification in oral leukoplakia and b) deciphering mechanisms for host-microorganism interaction in oral carcinogenesis.