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Evaluation of iTRAQ and SWATH-MS for the quantification of proteins associated with insulin resistance in human duodenal biopsy samples
Insulin resistance (IR) is associated with increased production of triglyceride-rich lipoproteins of intestinal origin. In order to assess whether insulin resistance affects the proteins involved in lipid metabolism, we used two mass spectrometry based quantitative proteomics techniques to compare the intestinal proteome of 14 IR patients to that of 16 insulin sensitive (IS) control patients matched for age and waist circumference. A total of 3896 proteins were identified by the iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) mass spectrometry approach and 2311 by the SWATH-MS strategy (Serial Window Acquisition of Theoretical Spectra). Using these two methods, 208 common proteins were identified with a confidence corresponding to FDR < 1%, and quantified with p-value < 0.05. The quantification of those 208 proteins has a Pearson correlation coefficient (r2) of 0.728 across the two techniques. Gene Ontology analyses of the differentially expressed proteins revealed that annotations related to lipid metabolic process and oxidation reduction process are overly represented in the set of under-expressed proteins in IR subjects. Furthermore, both methods quantified proteins of relevance to IR. These data also showed that SWATH-MS is a promising and compelling alternative to iTRAQ for protein quantitation of complex mixtures.
Sample Processing Protocol
Frozen duodenal tissue biopsies (15 IS and 14 IR patients) were weighted (6 to 20mg), and disrupted using a mortar and pestle. Samples were kept frozen on dry ice, and grinded to fine powder. Then lysis buffer (50mM ammonium bicarbonate, 50mM dithiothreitol (DTT), 0.5% sodium deoxycholate (SDC)) containing protease inhibitors cocktail (Roche) was added, and the sample preparation was homogenized on ice by sonication with a Sonic Dismembrator (Fisher) with 1 sec. pulse (20 times). Samples were centrifuged 10 min at 16000g. The supernatants were mixed with 5 volumes of acetone (stored at -20°C) and incubated overnight at -20°C. Precipitated proteins were centrifuged 15min at 16000g. Protein pellets were air dried, and then resuspended in 0.5M Triethylammonium bicarbonate (TEAB) – 0.5% SDC. Finally, the protein concentration of each sample was determined by colorimetric Bradford assay. Equal amounts of protein (50µg) from each control (IS) or IR sample were combined to give a control group and a IR group, respectively. Experimentation was performed in duplicates. Protein concentrations in pooled control (IS) and IR groups were determined using the Bradford assay. Then a total of 100 µg of protein per group was used for iTRAQ labeling. TEAB and SDC were added to each sample to reach a final concentration of 0.5 M and 0.5 %, respectively. Proteins were then reduced and alkylated according to the iTRAQ kit manufacturer’s instructions (ABSciex). Samples were digested with trypsin (Sequence grade Modified, Promega) using 1:30 ratio overnight at 37°C. After digestion, peptides were acidified to precipitate SDC, and then purified with an oasis HLB cartridge (1cc, 10mg, Water Corp.) and lyophilized. Dried peptides were dissolved in 30µl 0.5M TEAB and labeled with iTRAQ label reagent (ABSciex). 4-plex labeling was performed for 2 h at room temperature in the dark. Labeled peptides were combined in one tube and dried with the SpeedVac. Samples were cleaned up using HLB cartridge (Water Corp.). Samples were dried and reconstituted in 200µl HPLC water and 1/100 ampholytes pH 3-10 (Biorad). Then peptides were fractionated with 7cm IPG strips pH 3-10 using IEF. IPG (immobilized pH gradient) strips were passively rehydrated 5 hours. Then they were conditioned 15min at 250V and voltage was increased to 4000V for 2 hours. Final focusing was performed at 4000V until 10000V.hours. Strips were cut in 14 fractions and peptides were extracted in 2% ACN - 1%FA solution followed by 50% ACN - 1% FA. Finally, fractions were dried with the SpeedVac. Fractions collected from the isoelectric focusing were resuspended in 25 ul 0.1% formic acid. Mass spectrometry analysis was performed on a TripleTOF 5600 mass spectrometer fitted with a nanospray III ion source (ABSciex, Concord, ON) and coupled to an Agilent 1200 HPLC (Agilent, California). Two ul samples were injected by the Agilent 1200 autosampler onto a trapping column (Zorbax 300SB-C18) 5 u, 5 x 0.3mm at ten ul/min for desalting then onto a 0.075 mm (internal diameter) self-packed PicoFrit column (New Objective) packed with a isopropanol slurry of 5um Jupiter C18 (Phenomenex) stationary phase using a pressure vessel (Proxeon) set at 700 p.s.i . The length of the column was 15 cm. Samples were run using a 90 min gradient from 5-35% solvent B (solvent A 0.1% formic acid in water; solvent B: 0.1% formic acid in acetonitrile) at a flow rate of 300 nl/min. Data were acquired using an ion spray voltage of 2.4kV, curtain gaz of 30 PSI, nebulizer gaz of 8 PSI an an interface heater temperature of 125C. A DDA method was set up with the MS survey range set between 400 amu and 1250 amu (250msec) followed by dependent MS/MS scans with a mass range set between 100 and 1800 amu (50m sec) of the 20 most intense ions in the high sensitivity mode with a 2+ to 5+ charge state. Dynamic exclusion was set for a period of 3 sec and a tolerance of 100 ppm. Rolling collision energy was used and iTRAQ reagent collision energy adjustment was on.
Data Processing Protocol
Data files were submitted for simultaneous searches using Protein Pilot version 4.5 software (ABSciex) utilizing the Paragon and Progroup algortihms and the integrated false discovery rate (FDR) analysis function. Protein Pilot was set up to search the uniprot ‘complete proteome’ human proteins database (84848 sequences) with MMTS as a fixed modification on cysteine. Variable peptide modifications included methionine (M) oxidation and iTRAQ labeling of the N-terminal, lysine (K) and tyrosine (Y). Automatic normalization of quantitative data (bias correction) was performed to correct any experimental or systematic bias.
Bourassa S, Fournier F, Nehmé B, Kelly I, Tremblay A, Lemelin V, Lamarche B, Couture P, Droit A. Evaluation of iTRAQ and SWATH-MS for the Quantification of Proteins Associated with Insulin Resistance in Human Duodenal Biopsy Samples. PLoS One. 2015 May 7;10(5):e0125934. eCollection 2015 PubMed: 25950531
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