Computational protocol: Label-Free Quantitative Mass Spectrometry Reveals a Panel of Differentially Expressed Proteins in Colorectal Cancer

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[…] After desalting, the peptides were dried using a vacuum centrifuge and then resuspended with loading buffer (5 mM ammonium formate containing 5% acetonitrile, pH 3.0), separated, and analyzed by two-dimensional (2D) strong cation-exchange (SCX)/reversed-phase (RP) nanoscale liquid chromatography/mass spectrometry (2D nano-LC/MS) []. The experiments were performed on a nanoACAUITY UPLC system (Waters Corporation, Milford, USA) connected online to an LTQ Orbitrap XL mass spectrometer (Thermo Electron Corp., Bremen, Germany) equipped with a nanoelectrospray ion source (Michrom Bioresources, Auburn, USA). A 180 μm × 2.4 cm SCX column (Waters Corporation, Milford, USA), packed with 5 μm PolySULFOETHYL Aspartamide (PolyLC, Columbia, MD, USA), was used for the first dimension. A 20 μL peptide sample was loaded onto the SCX column to form the first salt gradient before the other gradient plugs were injected. After the first RP analysis was completed, 20 μL of salt solutions of differing concentrations was injected each time to form eight-step gradients followed by eight-RP analyses. The salt concentrations (ammonium formate) of the gradient were set as follows: 10, 20, 50, 100, 150, 200, 300, and 500 mM. The plugs were loaded onto the SCX column with a loading buffer for 4 min at a flow rate of 4 μL/min. The eluted peptides were captured by a trap column (Waters), while salts were diverted to waste. The trap column (2 cm × 180 μm) was packed with 5 μm Symmetry C18 packing material (Waters). The RP analytical column (20 cm × 75 μm) was packed with 1.7 μm Bridged Ethyl Hybrid (BEH) C18 packing material (Waters) and was used for the second-dimension separation. The peptides were eluted at a flow rate of 0.500 μL/min using a linear gradient of acetonitrile with 0.1% formic acid from 5% to 50% over 120 min. The eluted peptides were ionized at 1.9 kV, and the ions were analyzed on an LTQ-Orbitrap XL mass spectrometer. The spectrometer was operated in data-dependent mode and was set to switch automatically between MS and MS/MS acquisition. Survey full-scan MS spectra with two micro scans (m/z 300 to m/z 1800) were acquired in the Obitrap with a mass resolution of 70,000 at m/z 200, followed by 10 sequential LTQ-MS/MS scans. Dynamic exclusion was used with two repeat parameters: a 10 s repeat duration and a 60 s exclusion duration. For MS/MS, precursor ions were activated using 35% normalized collision energy at the default activation q of 0.25.All MS/MS spectra were identified by searching with SEQUEST [v.28 (revision 12), Thermo Electron Corp.] against the human UniProtKB/Swiss-Prot database (Release 2011_12_14, with 20, 249 entries). A decoy database, in which the sequences had been reversed, was appended to reduce false positive identification. The searching parameters were as follows: full tryptic cleavage with two missed cleavage sites was considered; variable modifications were oxidation (M) and acetyl (protein N-term); the peptide mass tolerance was 20 ppm; and the fragment ion tolerance was 1 Da.Trans Proteomic Pipeline software 4.0 (Systems Biology, WA) was utilized to identify proteins based on corresponding peptide sequences with ≥95% confidence. PeptideProphet [] with a P value of >0.95 was used for the peptide results, and ProteinProphet [] with a probability of 0.95 was used for the protein identification results. In label-free proteomics, the quantification of peptides is done by using spectral characteristics, such as retention time, m/z ratio, and peak intensity, and by comparing the direct mass spectrometric signal intensity for any given peptide (Perseus. In this study, the peak intensity for each individual spectrum was determined, and the comparison of spectra between multiple LC-MS runs provided quantitative measurements for thousands of peptides. From this large amount of data, a selected list of differentially expressed peptides was produced for subsequent fragmentation by LC-MS/MS for sequence determination and protein identification. Perseus software was used to match the large amount of spectra data according to retention time and precursor m/z characteristics. Once matched, the expression ratio in peak intensity was calculated based on peak areas.Cellular localization of identified proteins was further analyzed based on information available from Gene Ontology (GO) ( Biological function classifications and signaling pathway analysis were performed with the tools available in DAVID Bioinformatics Resources 2011 ( The association between the most-changed proteins was analyzed by the STRING 9.0 software-Known and Predicted Protein-Protein Interactions. […]

Pipeline specifications

Software tools Comet, PeptideProphet, ProteinProphet, Perseus, DAVID
Databases UniProt UniProtKB
Application MS-based untargeted proteomics
Diseases Colorectal Neoplasms