Computational protocol: Glycoprotein Biomarker Panelfor Pancreatic CancerDiscovered by Quantitative Proteomics Analysis

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Protocol publication

[…] TMT-labeled peptide mixtures were dissolved in 0.1% formic acid (FA) and loaded onto an Eksigent Nano 2D System (ABsciex) equipped with a commercial New Objective ProteoPepID trap column (150 um × 25 mm) and an analytical column (75 um × 100 mm, C18, 5 μm, 300A) coupled to an Orbitrap Velos mass spectrometer (Thermo Fisher Scientific). Peptides were separated with 0.1% FA in water (solvent A) and 0.1% FA in acetonitrile (solvent B) using a 100 min linear gradient from 2 to 32% solvent B at a flow rate of 300 nL/min. The mass spectrometer was operated by taking one full MS scan followed by ten HCD MS/MS scans on the ten most intense ions from the MS spectrum. Other mass spectrometer operating conditions included: 45% NCE; ± 1.5 Da isolation window; and dynamic exclusion enabled with a 10 ppm exclusion window. Exclusion settings were set with a repeat count of 2 using a repeat duration of 20 s and exclusion duration of 20 s. The resolution of full scans (m/z 400.0–1800.0) and HCD scans (fixed start from m/z 100.00) was set to 30 000 and 7 500, respectively. Ions with +1 or unassigned charge states were rejected for MS/MS analysis. The maximum injection time was 250 ms for the FTMS full scan and 200 ms for the FTMS MSn scan. The AGC target value was set as 100 000 for the FTMS scan and 40 000 for the FTMS MSn scan.Acquired MS/MS spectra were searched against a forward-reverse database generated from the UniProt human database (released Nov. 2010) using SEQUEST in Proteome Discoverer 1.1 (Thermo). Searches were performed using the following settings: precursor ion m/z tolerance, ± 10 ppm; fragment ion m/z tolerance, ± 0.03 Da; two missed cleavages allowed; static modification, carbamidomethylation (+57.02146 Da, C) and TMT 6-plex (+219.163 Da) of lysines and protein N-termini; dynamic modifications: oxidation (+15.99492 Da, M) and deamidation (+0.98402 Da, N). Identified peptides were filtered using a 1% peptide-level false discovery rate (FDR) and quantification was performed using reporter ions. For quantification, reporter ion intensities were extracted using Proteome Discoverer with the following parameters: (1) reject all quantitative values if not all quantitative channels are present; (2) do not replace missing quantitative values with the minimum intensity; (3) consider only proteins from different protein groups for peptide uniqueness; (4) the tolerance for reporter ion extraction is 0.01 Da. […]

Pipeline specifications

Software tools Comet, Proteome Discoverer
Application MS-based untargeted proteomics
Organisms Homo sapiens
Diseases Diabetes Mellitus, Pancreatic Neoplasms, Pancreatitis, Chronic