The poor prognosis of head and neck cancer (HNC) is largely associated with the presence of metastasis in the lymph nodes (LN). Herein, the proteome of 140 multisite samples from a 59-HNC patient cohort, including primary and matched LN tissues, saliva, and blood cells, reveals insights into the biology and potential metastasis biomarkers that may assist in clinical decision making. The protein profiles are strictly associated with immune modulation across datasets, which provided the basis to investigate immune markers associated with metastasis. The proteome of LN metastasis cells quite recapitulates the proteome of primary sites. Conversely, the LN microenvironment proteome highlights candidate markers. By integrating selected markers at peptide, protein, and transcript levels with machine learning models, we indicate a nodal metastasis signature in blood and saliva. In summary, we present the deepest proteome characterization of multiple sampling sites in HNC, providing a promising basis to understand tumoral biology and indicating metastasis-associated biomarkers.

Peptides were injected into the LC system equipped with an Acclaim PepMap100 trap column (75µm x 2 cm, 3 µm, 100A) and PepMap RSLC analytical column (75 µm x 25 cm, 2 µm, 100A) for reversed-phase chromatography using (A) 0.1% formic acid in water, and (B) 0.1% formic acid in 80% acetonitrile / 20% water, as mobile phases. Peptides were resolved over a 120-min gradient (5-38% B) at 250 nl/min, 50°C. Eluting peptides were transferred to gas-phase using a EASY-Spray source operating a 1.6kV. Targeted ions were monitored within a 10-min window, with an isolation window set to 1.4 m/z, AGC Target set to 50%, Maximum Injection Time of 54 ms for CiRT and Pierce retention time calibration mixture and 120 ms for EMT-associated peptides. Precursor ions were activated by HCD with normalised collision energy (NCE) of 27, and product ions analysed in the Orbitrap at 30,000 resolution @200/mz.

In total, 22 FFPE samples previously analysed in the discovery phase were analysed via PRM to verify abudance changes of proteins associated with epithelial-mesenchymal transition. Microdissected samples were transferred to an 8M urea solution, then treated with 5 mM dithiothreitol for 25 min at 56°C and 14 mM iodoacetamide for 30 min at room temperature for cysteine reduction and alkylation, respectively. Urea was diluted to a final concentration of 1.6 M with 50 mM ammonium bicarbonate, and 1 mM calcium chloride was added to the samples. For protein digestion, a total of 2.5 μg of trypsin (Promega, USA) was added in three steps that included 1 μg for 16h, 1 μg for an additional 16h, and 0.5 μg for the last 16h at 37°C. The reactions were quenched with 0.4% formic acid, and the peptides were desalted with C18 stage tips (3M, USA) and dried in a speed-vac instrument. Tissue samples were reconstituted in 0.1% formic acid that was applied proportionally to the dissected area (10 μL of formic acid for 1,000,000 μm2), and iRT peptides were added to the digested tissue sample at a final concentration of 11.1 fmol/μL for LC- MS/MS quality control (Pierce™ Peptide Retention Time Calibration Mixture, Thermo Scientific, USA). A volume of 4.5 μL was used (50 fmol of iRT peptides).