The composition of buffer A was water with 0.1% formic acid and buffer B was 80% acetonitrile with 0,08% formic acid. Each LC run was preceded by a blank run ensuring lack of carryover ABC294640 clinical trial of the material from the previous run. MS analysis was performed in positive ion mode, with a mass range of 250–600 m/z. MS/MS analyses were performed on the reporter peptide fragment CP-AP for sequence confirmation. Reproducibility of reporter peptide spiking To monitor the reproducibility of reporter peptide spiking, two distinct quality control samples were generated comprising serum specimens

from five colorectal tumor patients (QCT) and five healthy control individuals (QCH), respectively. Both samples were aliquoted and stored at −80°C until further use. The QCT and QCH-samples were spiked with the reporter peptide and internal

standard and incubated for 3 h, 6 h and 22 h at 37°C as described above. The proteolytic processing of the reporter peptide CP-RP resulted in the accumulation of CP-AP and the respective peak areas were used for quantification using LCQuan that is part of the Xcalibur software package (Thermo Fisher Scientific). Each QC-specimen was processed 5 times and median, standard deviation (SD) and coefficient of variation (CV) of the m/z 515.795 peak was calculated with Microsoft Excel software. Statistics The D’Agostino-Pearson Epigenetics inhibitor test, Mann–Whitney test and the receiver operating characteristics (ROC) calculations were performed with MedCalc (MedCalc Software). Results for continuous variables were expressed with the medians and standard deviations. Calculated P ADAMTS5 values of less than 0.05 were considered to indicate statistical significance. Correlation analyses were performed with Microsoft Excel 2002 SP-2 using the ‘add trendline’ functionality.

Acknowledgement We gratefully acknowledge that the costs of publication were supported by the LESSER-LOEWE Foundation e.V. Electronic supplementary material Additional file 1: Figure S1: Amino acid sequence confirmation of the anchor peptide Ahx-ateeqlkv (see Table 1). Print screen of the MS/MS spectra decoding of m/z 515.795 that was performed with PEAKS software (Bioinformatics Solutions). The unusual amino acid Ahx cannot be handled by the software and instead is displayed as Lysine (L) that is an isomer of Ahx and thus produces a fragment with the same mass. (PPT 72 KB) Additional file 2: Figure S2: Inhibition of protease-activity with iodoacetamide. The protease inhibitor iodoacetamide together with CP-RP and IS was added to a serum specimen from one tumor patient and incubated for 22 h prior to LC-MS analyis. Iodoacetamide concentrations ranged from 5 to 25 and 100 mmol/L. The CP-AP concentration of the serum specimen without iodoacetamide was set to 100%.