Bile samples were directly frozen at −80°C and were thawed only once just before proteomic analysis. Bile samples were diluted in H2O to a final protein concentration of 1 mg/mL, as verified with the bicinchoninic acid assay (Interchim, Montlucon, France). For CE-MS analysis, 0.7 mL diluted bile was added
to 0.7 mL n-butanol/iso-proyl ether 4:6 (v/v) and centrifuged for 10 minutes at 14,000 rpm and 4°C. The lower aqueous phase was extracted and diluted with 0.5 mL of 8 M urea, followed by 1 mL H2O, and passed over a 10 kDa MWCO Centrisart ultrafilter (Sartorius, Goettingen, PLX-4720 datasheet Germany) at 3,000 rpm until 1.4 mL filtrate was obtained. The filtrate was desalted on a PD-10 column (GE Healthcare, München, Germany) preequilibrated in 0.01% aqueous NH4OH (Roth, Karlsruhe, Germany). After elution with ammonium buffer, the sample was lyophilized, stored at 4°C, and resuspended
in CE-MS running buffer containing 20% acetonitrile and 1% formic acid before analysis. CE-MS analysis was performed as described using a P/ACE MDQ capillary electrophoresis system (Beckman Coulter, Fullerton, CA) on-line coupled to a Micro-TOF MS (Bruker Daltonic, Bremen, Germany).19, PLX3397 supplier 22 The ESI sprayer (Agilent Technologies, Palo Alto, CA) was grounded, and the ion spray interface potential was set between −4.0 and −4.5 kV. Data acquisition and MS acquisition methods were automatically controlled by the CE via contact-close-relays. Spectra medchemexpress were accumulated every 3 seconds over a range of m/z 350 to 3,000. Details regarding accuracy, precision,
selectivity, sensitivity, reproducibility, and stability of the CE-MS method have been described.19 Mass spectral ion peaks representing identical molecules at different charge states were deconvoluted into single masses using MosaiquesVisu software.23 Only signals were included with a charge >1 observed in a minimum of three consecutive spectra and with signal-to-noise ratios >4.24 The software employs probabilistic clustering and uses isotopic distribution and conjugated masses for charge-state determination of peptides/proteins. The resulting peak list characterizes each peptide by its molecular mass, CE-migration time, and ion signal intensity (amplitude). Because these parameters are influenced by the amount of salt and peptides in the sample, comparison of peptide spectra requires normalization. CE migration time and MS-detected mass were normalized by the definition of 339 clusters of peptides covering a range of 19.39 to 37.93 minutes in CE-migration time and 0.830 to 6.456 kDa in molecular mass. Amplitude calibration was based on 38 peptides with >60% abundance, >100 counts ion signal intensity above baseline, and <130% amplitude deviation. Detected peptides were deposited, matched, and annotated in a Microsoft SQL database, allowing comparison of multiple samples (patient groups).