LNBio Mas Lab - Projeto1_BuffyCoat-SalivaPellet_2019

Multisite proteome provides insights into the biology and prognostic markers for head and neck cancer
Data License: CC BY 4.0 | ProteomeXchange: PXD027984 | doi: https://doi.org/10.6069/7795-a573
  • Organism: Homo sapiens
  • Instrument: Xevo TQ-XS,ACQUITY UPLC
  • SpikeIn: Yes
  • Keywords: Head and Neck Squamous Cell Carcinoma, Mouth Neoplasms, Proteomics, Lymph Node Metastasis, Buffy coat, Saliva, Biomarkers, Prognosis, Immune System, Machine Learning
  • Lab head: Adriana Paes Leme Submitter: Adriana Paes Leme
Abstract
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.
Experiment Description
1.6 µg of buffy coat or saliva digests were resolved over a 60-min gradient using an Acquity UPLC-Class M equipped with a trap column (Acquity UPLC BEH C18 130A, 5 μm, 300 μm × 50 mm, Waters, USA) and a BEH Shield C18 IonKey column (10-cm × 150-μm ID packed with 1.7-μm C18 particles, Waters, USA) at 1.2 μl/min flow rate and temperature set to 40 °C. MeCN gradient started at 2% B (MeCN, 0.1% formic acid), following a linear ramp to 40% B over 45 min, followed by a step increase to 85% B until 47 min and conditioning at 2% B until 60 min. Mass spectrometry analysis of eluting peptides was performed via SRM-MS mode, with quadrupoles Q1 and Q3 operating as unit mass resolution (0.7 Th full width at half maximum). The optimal collision energy was determined for each peptide by Skyline 19.1 (MacLean et al. 2010). Scheduled SRM-MS acquisition was adjusted to a 3-min elution window, with dwell times automatically set in MassLynx v4.2 to achieve at least ten points per peak over a 16-s elution profile.
Sample Description
Proteins were isolated from buffy coat and saliva cells using TRizol reagent (Invitrogen, USA) and resuspended in 200 μL of urea buffer (100 mM Tris-HCl pH 7.5, 8 M urea, and 2 M thiourea). An ultrasound treatment was carried out for 10 min on ice for homogenization. Samples were reduced with 5 mM dithiothreitol for 25 min at 56°C and alkylated with 14 mM iodoacetamide for 30 min at room temperature. Urea was diluted to a final concentration of 1.6 M with 50 mM ammonium bicarbonate, and 1 mM of calcium chloride was added to the samples. For protein digestion, 2.5 μg of trypsin (Promega, USA) were added for 48 h at 37°C. The reactions were quenched with 0.4% formic acid, peptides were desalted with C18 stage tips (3M, USA) (Rappsilber, Mann, and Ishihama 2007), and dried in a speed-vac instrument. Samples were reconstituted in 0.1% formic acid , and quantified using the Pierce™ Quantitative Colorimetric Peptide Assay (Thermo Scientific, USA) and 1.6 μg were submitted to subsequent analysis.
Created on 8/17/21, 10:46 AM
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FINAL_Buffycoat_SIL_labelfree_19samples_4_2021-08-17_10-49-18.sky.zip2021-08-17 10:45:5515283716119
FINAL_Pelletsaliva_SIL_labelfree_25samples_1_2021-08-04_09-37-03.sky.zip2021-08-17 10:45:5515283716625