2.1. Subject Sample
The study was performed in accordance with the principles of the Declaration of Helsinki. A total of 174 patients and control subjects participated in the investigation after signing an informed consent form approved by the regional ethics committee. The patients and controls were of same ethnic origin, and had similar socio-economic backgrounds, but were stratified by sex, age, and smoking habit. None of the participants had co-existing malignant disease, uremia, diabetes, and neither statin nor anticoagulant therapy. Twenty participants were selected for an initial discovery study, while all participants were included in a subsequent validation study. For the discovery study, twelve male AAA patients and eight male controls without AAAs were carefully selected to minimize possible bias [13
]. Both patients and controls were former smokers. The average age (range) was 71 (59–83) years in the AAA group and 70 (62–78) years in the control group.
The 174 participants in the validation study were selected to give similar age, sex and current smoking habits in the controls and AAA patients (Table 1
). The validation study also included ten patients that were reinvestigated three years after they had undergone surgical treatment for non-ruptured AAA. These follow-up patients were treated by conventional open AAA repair through a standard midline laparotomy incision and transperitoneal approach with infrarenal polyester graft repair.
Both AAA patients and controls underwent ultrasonography investigation of the abdominal aorta. Peripheral venous blood samples were then taken from controls and patients. Samples were centrifuged within 30 min at 2000 g for 20 min and aliquots of citrated plasma were frozen and stored at −70 °C until analysis.
2.2. Discovery Study
The workflow of the discovery study included the following sequential steps as described in detail below: depletion, tryptic digestion, TMT labelling and sample pooling, fractionation, desalting, analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS), database search, TMT quantification, and statistical analysis.
2.2.1. Sample Preparation
Samples were depleted of high abundance plasma proteins (albumin and IgG) using the ProteaPrep Albumin and IgG Depletion kit (Protea Biosciences, Inc., Morgantown, WV, USA) according to the manufacturer’s instructions. For relative quantification (performed using 6-plex TMT-labeling, Pierce Biotechnology, Inc., Rockford, IL, USA), 10 µL aliquots from 20 randomly selected samples were pooled to create a standard protein mix. This standard pool was depleted in the same way as the samples and included as a reference in each prepared TMT-set (details on the TMT-sets are provided in Supplementary Table S1
The elution from the depletion step was evaporated to dryness (Savant SpeedVac™, Thermo Scientific, Basel, Switzerland) before proceeding with the Filter-Aided Sample Preparation (FASP) protocol for tryptic digestion [15
]. In brief, samples were solubilized in 50 µL of 4% SDS, 250 mM triehtylammonium bicarbonate (TEAB, Fluka, Sigma Aldrich, St. Louis, MO, USA) with 0.1 mM DTT. Protein concentrations were measured using the Pierce 660 nm protein kit (Thermo Scientific, Basel, Switzerland) according to the manufacturer’s guidelines. For each sample, 100 µg of total protein was digested using a 1:20 trypsin:protein ratio in a two-step digestion approach (sequencing grade modified porcine trypsin, Promega, Madison, WI, USA).
TMT labelling was performed according to the manufacturer’s recommendations with minor modifications. Specifically, reactions were quenched after 1 h by the addition of hydroxylamine to a final concentration of 0.25%. Labeled samples were combined into one set of samples prior to strong cation exchange (SCX) fractionation (100 × 2.1 mm PolySULFOETHYL A column, 5 µm particles, 300 Å pore size, PolyLc Inc., Columbia, MD, USA) using an ÄKTA purifier system (GE Healthcare Life Sciences, Uppsala, Sweden). At a flow rate of 0.25 mL/min the following gradient was applied: 100% A (25 mM ammonium formate, pH 2.8 in 25% acetonitrile) for 10 min; 0–20% B (500 mM ammonium formate, pH 2.8 in 25% acetonitrile) for 20 min; 20–40% B for 10 min and 40–100% B for 10 min and 100% B held for 10 min. UV absorbance at 280 nm was monitored while fractions were collected in tubes at 0.5 mL intervals. Per TMT-set, 18 fractions were collected for analysis by nano LC-MS/MS. Prior to this analysis, the SCX-fractions were desalted using Pierce C18 spin columns (Pierce Biotechnology, Inc., Rockford, IL, USA).
2.2.2. Nano LC-MS Analysis
Samples were analyzed on a Q Exactive mass spectrometer interfaced with an Easy-nLC 1000 nano LC system (both Thermo Fisher Scientific, Inc., Waltham, MA, USA). Peptides (3 µL injection volume) were separated using an in-house constructed pre-column and analytical column set up (45 mm × 0.075 mm I.D. and 200 mm × 0.750 mm I.D., respectively) packed with 3 μm Reprosil-Pur C18-AQ particles (Dr. Maisch GmbH, Ammerbuch, Germany). The following gradient was run at 150 nL/min; 7–27% B-solvent (acetonitrile in 0.2% formic acid) over 60 min, 27–40% B over 10 min, 40–80% B over 5 min with a final hold at 80% B for 10 min.
The mass spectrometer was operated in a so-called Top10 data acquisition approach. Full scan MS spectra were taken at a resolution of 35,000 (full width at half maximum, FWHM, at 200) over an m/z range of 400–1800 with an automatic gain control (AGC) target value of 3 × 106 and a maximal injection time of 100 ms. An ion from ambient air at m/z 445.12003 was used for internal calibration.
Based on each full scan spectrum, the 10 most abundant ions above an intensity of 1.0 × 105 (“underfill ratio”: 0.7%) were selected for fragmentation using a stepped higher energy collisional dissociation regime (stepped HCD, NCE 35 +/− 25%) and an isolation width of 2 Da. Unassigned, singly charged as well as ≥5 charge states were excluded from fragmentation and fragmented precursor masses were excluded dynamically for 30 s. Fragment spectra were recorded at a resolution of 17,500 for a target AGC-value of 1 × 106 and a maximal injection time of 64 ms was used for MS/MS scans.
For increased coverage and sensitivity using an exclusion list strategy, samples were injected a second time using a longer gradient and with exclusion lists based on the identifications made in the first round of analysis. The following gradient was run at 150 nL/min: 7–25% B-solvent (acetonitrile in 0.2% formic acid) over 90 min, 25–40% B over 10 min, 40–80% B over 10 min with a final hold at 80% B for 15 min. A smaller mass range (m/z 300–1400) was used compared to the first analysis. For the exclusion list runs, the 10 most abundant ions above an intensity of 1.0 × 104 (“underfill ratio”: 0.1%) were selected for fragmentation using a stepped higher energy collisional dissociation regime (stepped HCD, NCE 30 +/− 25%) and an isolation width of 1.2 Da. Unassigned, singly charged as well as ≥8 charge states were excluded from fragmentation and fragmented precursor masses were excluded dynamically for 30 s. Fragment spectra were recorded at a resolution of 35,000 for a target AGC value of 1 × 106 and a maximal injection time of 120 ms.
2.2.3. Database Search and TMT Quantification
MS raw data files from all 18 SCX fractions (including re-injection/exclusion list runs) for each TMT set were merged for relative quantification and identification using Proteome Discoverer version 1.3 (Thermo Fisher Scientific, Inc., Waltham, MA, USA). A database search for each set was performed with the Mascot search engine version 2.3 (Matrix Science LTD., London, UK) using the UniProtKB/Trembl database (downloaded 2012-11-19, Swiss Institute of Bioinformatics, Lausanne, Switzerland) with MS peptide tolerance of 10 ppm and MS/MS tolerance of 100 millimass units (mmu). Tryptic peptides were accepted with one missed cleavage and variable modifications of methionine oxidation, cysteine methylthiolation and fixed modifications of N-terminal TMT6plex and lysine TMT6plex were selected.
The detected peptide threshold in the software was set to 1% False Discovery Rate by searching against a reversed database and the corresponding result was filtered by selecting proteins with at least one unique peptide per protein. Identified proteins were grouped by sharing the same sequences to minimize redundancy. For TMT quantification, the ratios of the TMT reporter ion intensities in MS/MS spectra ([M + H]+ m/z 126–131) from raw data sets were used to calculate fold changes between samples. The ratios were derived by Proteome Discoverer version 1.3 using the following criteria: fragment ion tolerance as 100 mmu for the most confident centroid peak, TMT reagent purity corrections factors were used and missing values were replaced with minimum intensity. Only peptides unique for a given protein were considered for relative quantitation, excluding those common to other isoforms or proteins of the same family. The quantification was normalized using the protein median.
2.2.4. Statistical Analysis
The results were exported into MS Excel (Microsoft, Redmond, WA, USA) for manual data interpretation and statistical analysis. Welch’s unequal variances t-tests were performed after log-transformation of the data.