15289-s-bos

8 123 | Novel protein biomarker predictors of CAD in FH then used; (i) the diseased segment score (DSS), granting 1 point for each narrowing >20%; (ii) the CAD severity score, granting 1, 2 or 3 points per segment narrowing of 21-50%, 51-70% and 70%, respectively; and (iii) the CAC extent score, granting 1, 2, 3 or 4 points per segment narrowing of 1-20%, 21-50%, 51-70% and >70%, respectively. iTRAQ Proteomics Isobaric tag for relative and absolute quantification (iTRAQ) was performed by Proteomics International (PI) on fasting EDTA plasma that had previously been stored at -80 o C. The process involved an initial discovery phase followed by a validation phase. In the discovery phase the samples were depleted of the top 14 high abundance proteins, diafiltrated, reduced, alkylated and trypsin digested. The samples from each group were then labelled with iTRAQ reagents and combined to make a pooled sample (100 μL) for each individual group (FH, FH + Ca and FH + CAD) and an overall pooled sample (all 60 samples). Samples were then desalted on a Strata-X 33 μm polymeric reversed phase column (Phenomenex) and dissolved in buffer (10 mM KH 2 PO 4 , pH3 in 10% acetonitrile) before separation by strong cation exchange liquid chromatography (SCX, Agilent 1100 HPLC System) using a PolySulfoethyl column (4.6 x 100 mm, 5 μm, 300A). Peptides were eluted with a linear gradient of 0 – 400 mM KCl. Eight fractions containing the peptides were collected and desalted on Strata-X columns. The fractions were then analysed using electrospray ionisation mass spectrometry (Agilent 1260 Infinity HPLC system) coupled to an Agilent 1260 Chipcube Nanospray interface on an Agilent 6540 mass spectrometer, before being loaded onto a ProtlD-Chip-150 C18 column (Agilent) and separated with a linear gradient (water/acetonitrile/0.1% formic acid v/v). In the validation phase, samples (20 μL) were again depleted of the top 14 high abundance proteins, diafiltrated, reduced, alkylated and trypsin digested. Samples were then desalted on a Strata-X 33 μmpolymeric reversed phase column (Phenomenex) and analysed by electrospray ionisation mass spectrometry (LC/MS) using a Dionex UltiMate 3000 nanoflow HPLC system coupled to a 4000 Q-TRAP mass spectrometer (AB Sciex). Duplicate runs were performed for all samples. A 1 μL volume containing 1:1 (v/v) ratio of tryptic unlabelled and 18 O-labelled reference standard plasma peptides was then loaded onto an Agilent Zorbax 300SB-C18, 3.5 μm column and separated with a linear gradient of water/acetonitrile/0.1% formic acid (v/v) over 90 mins. A reference plasma sample was used as a control to determine the representative peptides of the new proteins and as an 18 O-labelled reference standard for relative peptide quantification. MRM transitions for unlabelled and 18 O-labelled peptides were created and searched for