{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,6,3]],"date-time":"2026-06-03T05:07:12Z","timestamp":1780463232113,"version":"3.54.1"},"posted":{"date-parts":[[2026,6,2]]},"group-title":"Bioengineering","reference-count":0,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,6,2]],"date-time":"2026-06-02T00:00:00Z","timestamp":1780358400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"name":"National Institute of Allergy and Infectious Disease (NIAD) of the National Institutes of Health","award":["R21AI178442"],"award-info":[{"award-number":["R21AI178442"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,6,2]]},"abstract":"<jats:p>Background:\nDetecting multiple viruses in biofluids such as saliva remains challenging because of rapid mutation and low abundance presenting in the complex matrix. Proteomics analysis provides a complementary method by directly measuring viral peptides and resolving protein-level sequence variation in rapidly evolving viruses such as influenza and SARS-CoV-2. However, the ongoing viral evolution challenges search sensitivity by introducing numerous mutations. Here, we develop a saliva viral proteomics strategy that integrates mutation profiling of clinically relevant viral sequences with single amino acid substitution mutation discovery to enable sensitive detection of both known and emerging variants.\nResults:\nIn silico analysis of SARS-CoV-2 and influenza viral sequences reveals bimodal prevalence distributions of mutated peptides. For mutation profiling of clinically relevant viral sequences, we prioritize high-prevalence peptides, which presumably have higher clinical significance, within a 3-month timespan. Applying a 10% prevalence threshold reduces database size by more than 82.1% for SARS-CoV-2 and 94.5% for influenza. Optimized mutated peptide databases cause &lt;0.8% sensitivity loss compared with searches using viral reference sequences alone, while still covering 97.8% of SARS-CoV-2 and 98.4% of influenza viral populations in the subsequent month. G-PTM-D-based mutation discovery further mitigates the loss of database coverage and enables detection of new variants. In peptide-spiked saliva samples, G-PTM-D detected single-amino-acid variant peptides with femtogram-level sensitivity, outperforming conventional PTM searches and de novo sequencing.\nConclusions:\nThis study provides a scalable framework for mutation profiling and discovery proteomics analysis that enables sensitive detection of both known and emerging viral variants in complex clinical samples.<\/jats:p>","DOI":"10.64898\/2026.05.30.729008","type":"posted-content","created":{"date-parts":[[2026,6,3]],"date-time":"2026-06-03T04:30:13Z","timestamp":1780461013000},"source":"Crossref","is-referenced-by-count":0,"title":["A Viral Mutation Profiling and Discovery Strategy for Sensitive Multiplex Detection of Viruses and Variants in Saliva by Proteomics"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0009-0005-8972-2273","authenticated-orcid":false,"given":"Yanjia","family":"Zhang","sequence":"first","affiliation":[{"name":"University of Pennsylvania;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael","family":"Shortreed","sequence":"additional","affiliation":[{"name":"UW-Madison"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aaron T.","family":"Timperman","sequence":"additional","affiliation":[{"name":"University of Pennsylvania;"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.64898\/2026.05.30.729008","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,3]],"date-time":"2026-06-03T04:30:13Z","timestamp":1780461013000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.64898\/2026.05.30.729008"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,6,2]]},"references-count":0,"URL":"https:\/\/doi.org\/10.64898\/2026.05.30.729008","relation":{},"subject":[],"published":{"date-parts":[[2026,6,2]]},"subtype":"preprint"}}