Ref 110?95) and gamma glutamyl transpeptidase 63 U/L and 60 U/L (ref ,35), respectively. In addition, plasma and urine samples were collected from 10 patients with suspected drug-induced acute liver injury that were admitted to the emergency room at Radboud University Nijmegen Medical Centre (Nijmegen, the Netherlands) and the Hagaziekenhuis (Den Haag, the Netherlands). The demographics of these patients are shown in Table 1.HistologyHematoxylin and eosin BIBS39 staining was performed on liver paraffin sections. Liver damage was evaluated by a qualified pathologist and scored blinded of 10 images taken from each liver section (106 magnification). For each image the degree of centrilobular necrosis was assessed by overlaying the images with a grid (Image J, 3700`2 Pentagastrin pixels) and counting the intersections in necrotic areas. Liver injury was reported as mean percentage of centrilobular necrosis. Kidney paraffin sections were stained using Periodic Acid Schiff staining.Urine protein profiling with MALDI-TOF MSUrine samples were normalized according to creatinine values to reduce sample protein variation [16]. Based on the method of Fiedler et al., urine samples were subsequently pretreated using affinity beads to isolate specific fractions of the urine proteome, before MALDI-TOF MS analysis [17]. We used weak cation exchange (WCX) Macro-PrepH carboxymethyl support beads (Bio-Rad Laboratories, Hercules, CA, USA) and Magnetic Beads based Hydrophobic Interaction Chromatography 8 beads (C8; Bruker Daltonics GmbH, Bremen, Germany), that bind positively charged proteins and hydrophobic proteins, respectively. Synthetic hepcidin-24 (Peptide International Inc., Louisville, KY, USA) was used as internal standard (IS) to enable comparison between samples. Of the prepared sample, 1 ml was applied to a MSP 96 polished steel MALDI target plate under nitrogen flow, followed by two times 0.5 ml of 5 mg/mL a-cyano-4-hydroxy-cinnamic acid in 50 ACN and 0.5 TFA. Mass-to-charge (m/z) spectra were generated using MALDI-TOF MS (Microflex LT with software flexControl Version 3.0, Bruker Daltonics) in positive, linear ion mode and 350 laser shots. Initial laser power; 50 for 1?0 kDa and 60 for 10?60 kDa measurements, Laser Attenuator; Offset 25 and Range 20 . Pulsed ion extraction was set to 250 24272870 ns. Samples prepared with the WCX support beads were measured in the 1?0 kDa mass range and those prepared with the C8 beads were measured in both the 1?0 kDa and 10?160 kDa mass range. Calibration was performed using protein calibration standard I for 1?0 kDa measurements and protein calibration standard II (both Bruker Daltonics) for 10?60 kDa measurements.described elsewhere [18,19]. Single C8 pretreated urine samples were used to identify specific protein masses smaller than 4 kDa directly with vMALDI LTQ. Proteins larger than 4 kDa were identified using 1D-gelelectrophoresis with a 15 SDS gel and silver-blue staining. Bands were excised and subjected to reduction, alkylation and trypsin digestion before being measured on the vMALDI-LTQ. For LC-MS/MS two pooled urine samples were used to identify differentially excreted proteins between control (n = 5) and APAP-induced liver injury (n = 5; plasma ALT.5000 U/L). Urine samples were in-solution digested, after reduction and alkylation. The digested samples were loaded on stagetips for desalting and concentrating, and eluted to a final volume of 20 mL, 8 mL of which was used for analysis. To avoid contamination with polymers.Ref 110?95) and gamma glutamyl transpeptidase 63 U/L and 60 U/L (ref ,35), respectively. In addition, plasma and urine samples were collected from 10 patients with suspected drug-induced acute liver injury that were admitted to the emergency room at Radboud University Nijmegen Medical Centre (Nijmegen, the Netherlands) and the Hagaziekenhuis (Den Haag, the Netherlands). The demographics of these patients are shown in Table 1.HistologyHematoxylin and eosin staining was performed on liver paraffin sections. Liver damage was evaluated by a qualified pathologist and scored blinded of 10 images taken from each liver section (106 magnification). For each image the degree of centrilobular necrosis was assessed by overlaying the images with a grid (Image J, 3700`2 pixels) and counting the intersections in necrotic areas. Liver injury was reported as mean percentage of centrilobular necrosis. Kidney paraffin sections were stained using Periodic Acid Schiff staining.Urine protein profiling with MALDI-TOF MSUrine samples were normalized according to creatinine values to reduce sample protein variation [16]. Based on the method of Fiedler et al., urine samples were subsequently pretreated using affinity beads to isolate specific fractions of the urine proteome, before MALDI-TOF MS analysis [17]. We used weak cation exchange (WCX) Macro-PrepH carboxymethyl support beads (Bio-Rad Laboratories, Hercules, CA, USA) and Magnetic Beads based Hydrophobic Interaction Chromatography 8 beads (C8; Bruker Daltonics GmbH, Bremen, Germany), that bind positively charged proteins and hydrophobic proteins, respectively. Synthetic hepcidin-24 (Peptide International Inc., Louisville, KY, USA) was used as internal standard (IS) to enable comparison between samples. Of the prepared sample, 1 ml was applied to a MSP 96 polished steel MALDI target plate under nitrogen flow, followed by two times 0.5 ml of 5 mg/mL a-cyano-4-hydroxy-cinnamic acid in 50 ACN and 0.5 TFA. Mass-to-charge (m/z) spectra were generated using MALDI-TOF MS (Microflex LT with software flexControl Version 3.0, Bruker Daltonics) in positive, linear ion mode and 350 laser shots. Initial laser power; 50 for 1?0 kDa and 60 for 10?60 kDa measurements, Laser Attenuator; Offset 25 and Range 20 . Pulsed ion extraction was set to 250 24272870 ns. Samples prepared with the WCX support beads were measured in the 1?0 kDa mass range and those prepared with the C8 beads were measured in both the 1?0 kDa and 10?160 kDa mass range. Calibration was performed using protein calibration standard I for 1?0 kDa measurements and protein calibration standard II (both Bruker Daltonics) for 10?60 kDa measurements.described elsewhere [18,19]. Single C8 pretreated urine samples were used to identify specific protein masses smaller than 4 kDa directly with vMALDI LTQ. Proteins larger than 4 kDa were identified using 1D-gelelectrophoresis with a 15 SDS gel and silver-blue staining. Bands were excised and subjected to reduction, alkylation and trypsin digestion before being measured on the vMALDI-LTQ. For LC-MS/MS two pooled urine samples were used to identify differentially excreted proteins between control (n = 5) and APAP-induced liver injury (n = 5; plasma ALT.5000 U/L). Urine samples were in-solution digested, after reduction and alkylation. The digested samples were loaded on stagetips for desalting and concentrating, and eluted to a final volume of 20 mL, 8 mL of which was used for analysis. To avoid contamination with polymers.
