SABRe CVD

In total 338 pairs of CVD cases and controls were enrolled in this study. Atherosclerotic CVD events included myocardial infarction (MI, n=136), coronary heart disease (CHD) death (n=28), atherothrombotic brain infarction (ABI, n=70), coronary artery bypass grafting (CABG, n=57), and percutaneous transluminal coronary angioplasty (PTCA, n=47). For each case, one control was selected based on the following features: a) attended the same examination cycle as the case, b) matched for age (±5 years), sex, smoking status, and statin use, c) free of CVD on the date of event of the case. Clinical data and biological specimens for cases were selected from the FHS clinic examination visit immediately preceding CVD event occurrence, either examination cycle 5 (n pairs=85), 6 (n pairs=66), 7 (n pairs=150) or 8 (n pairs=37). Clinical data and biological specimens for each control were selected from the same examination cycles as its matched case.

33 markers that showed significant association with CVD either in our iTRAQ data or in literature or other resources, were measured by MRM on all subjects (targeted n=676). Data are available for 674 CVD cases and controls. (There were 675 observations in the data we received from BGM. One ID was not from the designing set, indicating wrong specimen was sent. Two IDs in the designing set were not measured for some reason, so we end up with 674 usable individuals for this study.)

MRM analysis was performed on 672 primary study samples. In this implementation primary samples were interleaved with reference sample replicates at regular intervals in the data acquisition queue for the purpose of signal normalization and trend correction. Advantages of such a workflow design without the need for a large number of isotope labeled peptide standards. Data acquisition was performed in measurement batches (36-44 hours long). Each batch consisted of 48 primary samples and 9 reference (QC) samples. Primary samples were randomized and distributed over 14 batches with cases and their matching controls analyzed in the same batch. Two injections of the “bulk plasma digest” preceded the first QC sample injection in every batch in order to carry the LC-MS instrumentation through the first couple of analyses that had tendency to produce outlying measurements. Normalization was performed by dividing the peak areas of individual transitions by the median of the same transition in the QC samples. In this manner, peptide quantities were reported ratios facilitating the conversion of peptide measurements into protein measurements through simple averaging.

Nine (9) μL of plasma was diluted five-fold with 8M freshly made urea and 50 mM ammonium bicarbonate containing 15 mM TCEP. Cysteine reduction was completed by incubation at room temperature for one hour. Alkylation was completed by adding five (5) µL 0.4M iodoacetamide and incubating in dark for 30 min. The reduced/alkylated protein solution is diluted with 250 µL 50 mM ammonium bicarbonate, 2% acetonitrile. Twenty (20) µL of this solution is carried forward to digestion and the remaining ~280 µL volume is stored at -80 ⁰C.

Digestion was completed by the addition of 4 μg trypsin, overnight, at room temperature. After 16h of digestion, it was stopped by adding 76 µL HPLC Solvent A (2% acetonitrile, 0.1% formic acid). The resulting peptide solution (of ~0.4 mg/mL concentration) was spiked with fibrinopeptide A standard to yield 5 μg/mL. This standard was used to monitor the steady level of instrument response during batches.

LC-MRM analysis was performed on 4000QTrap linear ion trap instrument (AB/SCIEX, Concord, ON) interfaced with a U3000 HPLC system (Dionex, Sunnyvale, CA). The system was plumbed to facilitate an in-line desalting step on a Poros R2 column with reverse flow. After desalting peptides were separated on a Targa C18 (5μm) 150x1.0 mm column (Higgins Analytical) utilizing a 200-μL/min flow rate. Peptide elution was carried out over a 21-min gradient from 2% B to 32%B (A: 5% acetonitrile, 0.1% formic acid, B: 95% acetonitrile, 0.1% formic acid). Following elution the HPLC columns were extensively washed with 95% B. The HPLC column compartment was kept at 50°C during analysis.

For each target protein, two peptides were selected with two transitions (fragments) per peptide. Selection of these fragments was preceded by screening larger numbers of peptides and transitions (typically five peptides and five fragments for each). Correct identity of peptides were confirmed by observing their correlation over a large number of individual samples.

• For unadjusted analysis, paired T test or Wilcoxon signed rank test depending on the distribution of a marker
• For adjusted analysis, if case status is the outcome, we recommend conditional logistic regression; if a marker as outcome, linear mixed model with a random effect for pairs is recommended.
• Interpretation of results from markers with majority values missing should be cautious.

The purpose of this section is to allow future users to be able to confirm correct alignment to covariates data. The covariates are not provided in this specific release.

For CVD_MRM set