trypsinogen and Carcinoma--Ductal--Breast

This work explores the use of 20-microm-i.d. polymeric polystyrene-divinylbenzene monolithic nanocapillary columns for the LC-ESI-MS analysis of tryptic digest peptide mixtures. In contrast to the packing of microparticles, capillary columns were prepared, without the need of high pressure, in fused-silica capillaries, by thermally induced in situ copolymerization of styrene and divinylbenzene. The polymerization conditions and mobile-phase composition were optimized for chromatographic performance leading to efficiencies over 100000 plates/m for peptide separations. High mass sensitivity (approximately 10 amol of peptides) in the MS and MS/MS modes using an ion trap MS was found, a factor of up to 20-fold improvement over 75-microm-i.d. nanocolumns. A wide linear dynamic range (approximately 4 orders of magnitude) was achieved, and good run-to-run and column-to-column reproducibility of isocratic and gradient elution separations were found. As samples, both model proteins and tissue extracts were employed. Gradient nano-LC-MS analysis of a proteolytic digest of a tissue extract, equivalent to a sample size of approximately 1000 cells injected, is presented.

Topics: Amino Acid Sequence; Animals; Carcinoma, Ductal, Breast; Caseins; Catalase; Cattle; Chickens; Chromatography, Liquid; Cytochromes c; Databases, Protein; Female; Horses; Humans; Lactoglobulins; Microscopy, Electron, Scanning; Molecular Sequence Data; Myoglobin; Neuropeptides; Ovalbumin; Peptide Fragments; Polystyrenes; Proteins; Reproducibility of Results; Spectrometry, Mass, Electrospray Ionization; Tissue Extracts; Trypsin; Trypsinogen

Expression of pancreatic trypsinogen and cathepsin B in 23 surgically resected pancreatic ductal adenocarcinomas was evaluated immunohistochemically, using a monoclonal antibody against human pancreatic trypsinogen and a polyclonal antibody against human cathepsin B. Fifteen of 20 invasive tubular adenocarcinomas (75%) expressed pancreatic trypsinogen in a coarse granular pattern located in the supranuclear cytoplasm of the carcinoma cells. In addition, metastatic lesions, including those in peripancreatic lymph nodes and neural plexuses, expressed pancreatic trypsinogen. In contrast, three intraductal (non-invasive) papillary adenocarcinomas did not express pancreatic trypsinogen. Cathepsin B expression was recognised in 14 of 20 invasive tubular adenocarcinomas (70%) in a fine granular pattern located diffusely in the cytoplasm of the carcinoma cells, while none of the three intraductal papillary adenocarcinomas had detectable cathepsin B. These findings suggest that pancreatic invasive ductal adenocarcinomas express pancreatic trypsinogen and cathepsin B immunoreactive peptides, raising the possibility that pancreatic trypsinogen and cathepsin B may act independently of each other in the process of carcinoma invasion and metastasis, like other different classes of proteases involved in the proteolytic modification of the matrix barrier.

Topics: Adult; Aged; Antibodies, Monoclonal; Antibody Specificity; Carcinoma, Ductal, Breast; Cathepsin B; Female; Humans; Immunohistochemistry; Lymphatic Metastasis; Male; Middle Aged; Pancreatic Neoplasms; Trypsinogen