LC7 Proteomic Analysis of Matched Control and NSCLC Lung Adenocarcinoma Tissue.
Non-small cell lung adenocarcinoma is the most frequently diagnosed lung cancer type and remains the leading cause of cancer mortality for men and women in the United States. Management of lung cancer is hindered by high false-positive rates due to the inability to resolve benign versus malignant tumors. Therefore, better molecular analysis comparing malignant and non-malignant tissues will provide additional evidence of the underlying biology contributing to tumorigenesis. In the current study, we utilized a proteomics approach to analyze 38 malignant and non-malignant paired tissue samples obtained from current or former smokers with early stage (Stage IA/IB) lung adenocarcinoma. Statistical mixed effects modeling and orthogonal partial least squares discriminant analysis were used to identify key cancer-associated perturbations in the malignant tissue proteome. Identified proteins were subsequently assessed against clinicopathological variables.
Sample Processing Protocol
Sample Digestion Protein pellets were solubilized in 100µL of 6M urea in 50mM ammonium bicarbonate (AMBIC). 200mM of dithiothreitol (DTT) was added to a final concentration of 5mM and samples were incubated for 30min at 37°C. Next, 20mM iodoacetamide (IAA) was added to a final concentration of 15mM and incubated for 30min at room temperature, followed by the addition of 20 µL DTT to quench the IAA reaction. Lys-C/trypsin (Promega) was next added in a 1:25 ratio (enzyme:protein) and incubated at 37°C for four hours. Samples were then diluted to <1M urea by the addition of 50mM AMBIC and digested overnight at 37°C. The following day, samples were desalted using C18 Macro Spin columns (Nest Group) and dried down by vacuum centrifugation. LC-MS/MS Analysis LC separation was done on a Waters Nano Acquity UHPLC (Waters Corporation) with a Proxeon nanospray source. The digested peptides were reconstituted in 2% acetonitrile /0.1% trifluoroacetic acid and roughly 3µg of each sample was loaded onto a 100 micron x 25 mm Magic C18 100Å 5U reverse phase trap where they were desalted online before being separated on a 75 micron x 150 mm Magic C18 200Å 3U reverse phase column. Peptides were eluted using a gradient of 0.1% formic acid (A) and 100% acetonitrile (B) with a flow rate of 300nL/min. A 120 minute gradient was ran with 5% to 35% B over 100 minutes, 35% to 80% B over 8 minutes, 80% B for 1 minute, 80% to 5% B over 1 minute, and finally held at 5% B for 10 minutes. Each of the gradients was followed by a 1h column wash. Mass spectra was collected on an Orbitrap Q Exactive Plus mass spectrometer (Thermo Fisher Scientific) in a data-dependent mode with one MS precursor scan followed by 15 MS/MS scans. A dynamic exclusion of 15 seconds was used. MS spectra were acquired with a resolution of 70,000 and a target of 1 × 106 ions or a maximum injection time of 30ms. MS/MS spectra were acquired with a resolution of 17,500 and a target of 5 × 104 ions or a maximum injection time of 50ms. Peptide fragmentation was performed using higher-energy collision dissociation (HCD) with a normalized collision energy (NCE) value of 27. Unassigned charge states as well as +1 and ions >+5 were excluded from MS/MS fragmentation.
Data Processing Protocol
Raw files were converted to mzXML using MSConvert and peptide spectrum matching was perfromed using Myrimatch and MSGF+ .PSM's were loaded into IdPicker 3 for analysis and filtered on a spectra FDR of 0.29% which equated to a protein FDR of 6.66%. At this FDR 4.5M spectra resulted in 79K distint peptide matches and 10,713 protein Groups identified. Spectral counts and MS1 precursor intensity was analyzed by IdPicker 3
Fahrmann JF, Grapov D, Phinney BS, Stroble C, DeFelice BC, Rom W, Gandara DR, Zhang Y, Fiehn O, Pass H, Miyamoto S. Proteomic profiling of lung adenocarcinoma indicates heightened DNA repair, antioxidant mechanisms and identifies LASP1 as a potential negative predictor of survival. Clin Proteomics. 2016 Oct 27;13:31 PubMed: 27799870