Abstract B4: Depletion of antibodies and serum-derived proteins to facilitate LC-MS detection of pancreas-specific proteins in pancreatic ductal secretions.

Abstract

Pancreatic ductal secretions are an abundant source of proteins that may serve as biomarkers for the early detection of pancreatic adenocarcinoma. Most pancreatic ductal secretion proteomes published in the literature are dominated by serum proteins. Due to the invasive nature of procedures used to sample pancreatic secretions, it remains uncertain whether the serum proteins present in the samples are artifacts of blood introduced during sampling or true indicators of disease. In addition, serum proteins are not generally useful as specific disease markers and mask other less abundant pancreas-specific proteins of interest in the sample. The enrichment of pancreas-specific proteins in pancreatic ductal secretions increases the opportunity for uncovering specific biomarkers of pancreatic cancer. Pancreatic ductal secretions were collected from ten patients with pancreatic adenocarcinoma undergoing pancreatectomy during surgery according to IRB-approved protocols. Protease inhibitors were added and samples were stored at -80°C. Samples were extracted 3x with chloroform to remove hydrophobic contaminants followed by IgA depletion using SSL-7 agarose (Invivogen) and depletion with a Vivapure Anti-HSA human albumin depletion (Sartorius) or Seppro IgY 14 Spin Column (Sigma) serum protein depletion column. Depleted samples were precipitated with methanol/chloroform/water, dissolved into 2M urea with 50mM ammonium bicarbonate/ 10mM TCEP and digested with trypsin. The digest was processed on a C-18 column on an HPLC, dried, resuspended in 0.1% TFA and characterized by ESI-LC-MS/MS on an LTQ-Orbitrap. Data were searched using Mascot against the NCBI RefSeq database. The top 20 proteins in pancreatic secretions taken from pancreatic adenocarcinoma patients in order of abundance without depletion were albumin, transferrin, beta globin, alpha 2 globin, IgA-1 chain constant region, alpha-1 antitrypsin, complement component 3, carboxyl ester lipase, keratin 1, apolipoprotein A-1, haptoglobin, delta globin, Predicted protein similar to complement component C3, alpha-2 macroglobulin, pancreatic carboxypeptidase B1, IgA-2 chain constant region, apolipoprotein E, polymeric immunoglobulin receptor, IgG-1 constant region, and Ig lambda chains. Mascot scores for albumin were about 20 times higher than scores for the pancreas-specific proteins and about three times higher than scores for the other serum proteins. Twelve of the fourteen proteins which are depleted by the Seppro IgY 14 depletion columns are present as dominant proteins in most pancreatic secretion samples. Of the remaining top proteins, alpha-2, beta and delta globin are blood proteins which are not depleted. These are most likely the result of hemolyzed red blood cells during freezing. Carboxyl ester lipase, pancreatic carboxypeptidases A1, B1, elastase 3A and 3B, trypsin, regenerating islet-derived 1 alpha and beta, pancreatic lipase, chymotrypsin B1 and B2, pancreatitis-associated protein, pancreatic amylase alpha 2A and B, mesotrypsin, and phospholipase A2 are pancreas specific proteins which are detected with increased sensitivity once the interfering serum proteins are depleted. Data will be shown comparing the performance of the two columns in depletion efficiency as well as the effect of the addition of an additional depletion step of secretory IgA from the samples, which depleted IgA-1, IgA-2, polymeric immunoglobulin receptor, Ig-lambda and Ig-kappa chain proteins. In conclusion, IgA, albumin, and IgY 14 serum protein depletion improve the LC-MS analysis of pancreas-derived proteins in pancreatic secretions by reducing interference from abundant serum proteins. Citation Format: Jana D. Rocker, Carlo Contreras, Lee W. Thompson, Russell E. Brown, Jack A. DiPalma, Lewis K. Pannell. Depletion of antibodies and serum-derived proteins to facilitate LC-MS detection of pancreas-specific proteins in pancreatic ductal secretions. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr B4.