Fig 1: Validation of identified proteins by western blotting. Urine samples were separated by 20% SDS-PAGE and analysed by western blotting using antibodies specific against CSTB, CTSB and albumin. Panels (A) and (C) (left) show the western blotting results of combined urine samples in the 1-100-kDa range. The samples were tested initially for the presence of fragments of CSTB and CTSB in the region of the measured m/z (open triangles), as well as full-length molecules (filled triangles) or other breakdown products. Reliable signals in the 35-kDa range for CSTB and 25-kDa for CTSB were then further analysed. Validation and confirmation of LC-MS/MS and Mascot results are shown in the strip-blots in panels (A) and (C), which show the results of (A) 8 or (C) 7 urine samples that were used in LC-MS/MS and subsequent Mascot searches, together with the cluster peak intensity matrices derived from the SELDI analysis (underneath the individual blots). Panels (B) and (D) depict the analysis of four random cancer and four control samples. The samples were selected based on the IMAC30 SELDI analysis for the presence of a peak cluster at m/z 2577 for CSTB, and on the CM10 SELDI peak pattern at m/z 2447 for CTSB (cancer samples) or the absence of these m/z peaks (healthy controls). ‘e’ corresponds to an estimated value (in brackets), where no peak at the m/z point could be detected above the S/N ratio. Qualitative loading control was performed by probing against the common urinary molecule serum albumin. Molecular weight indicators are depicted on the left, and the target proteins on the right of the blots. The cancer locus is the histologically assessed cancer site of duodenum (D), pancreas (P), oesophagogstric junction (OGJ), oesophagus (O), or non-cancerous control (C). CTSB, cathepsin-B; CSTB, cystatin-B; SELDI, surface-enhanced laser desorption/ionization; MS, mass spectrometry; S/N, signal-to-noise ratio.