All in one peptide

Serum samples were collected and cryopreserved from each patient at baseline, 6 months, 12 months and 24 months. Samples were not available from all patients at each time-point; a total of 475 samples were available for SELDI-TOF mass spectrometry. Samples were randomized, and staff were blinded to the treatment arms. CM10 ProteinChip arrays (Bio-Rad Laboratories) were primed with binding buffer (50 mM ammonium acetate, 0.01% Triton X-100, pH 4·0) and incubated at room temperature (RT) for 5 min. A 1:10 dilution of serum in binding buffer was then applied to the array and incubated at RT for 1 hr. The arrays were washed twice with binding buffer and deionized water. Saturated sinapinic acid (0.7 µL) was applied twice to each spot on the arrays. Time-of-flight spectra were generated using a PCS-4000 mass spectrometer (Bio-Rad). Low-range spectra (mass/charge (m/z) ratio 0 – 20,000) were obtained at a laser energy of 3000 nJ, with a focus mass of 6000 and the matrix attenuated to 1000. High-range spectra (m/z 10,000 – 75,000) were obtained at a laser energy of 3900 nJ, with a focus mass of 30,000 and the matrix attenuated to 10,000. Mass accuracy was calibrated externally using All-in-One Peptide or Protein molecular mass standards (Bio-Rad).