Optimized sample preparation of endoscopic collected pancreatic fluid for SDS-PAGE analysis.
The standardization of methods for human body fluid protein isolation is a critical initial step for proteomic analyses aimed to discover clinically relevant biomarkers. Several caveats have hindered pancreatic fluid proteomics, including the heterogeneity of samples and protein degradation. We aim to optimize sample handling of pancreatic fluid that has been collected using a safe and effective endoscopic collection method (endoscopic pancreatic function test). Using SDS-PAGE protein profiling, we investigate (i) precipitation techniques to maximize protein extraction, (ii) auto-digestion of pancreatic fluid following prolonged exposure to a range of temperatures, (iii) effects of multiple freeze-thaw cycles on protein stability, and (iv) the utility of protease inhibitors. Our experiments revealed that TCA precipitation resulted in the most efficient extraction of protein from pancreatic fluid of the eight methods we investigated. In addition, our data reveal that although auto-digestion of proteins is prevalent at 23 and 37 degrees C, incubation on ice significantly slows such degradation. Similarly, when the sample is maintained on ice, proteolysis is minimal during multiple freeze-thaw cycles. We have also determined the addition of protease inhibitors to be assay-dependent. Our optimized sample preparation strategy can be applied to future proteomic analyses of pancreatic fluid.
Paulo
Joao A
JA
Department of Pathology, Children's Hospital Boston and Harvard Medical School, Boston, MA 02115, USA.
Lee
Linda S
LS
Wu
Bechien
B
Repas
Kathryn
K
Banks
Peter A
PA
Conwell
Darwin L
DL
Steen
Hanno
H
D000096:Acetone; D011232:Chemical Precipitation; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D016145:Endoscopy, Digestive System; D000431:Ethanol; D006801:Humans; D010189:Pancreatic Juice / Q000737:chemistry*; D055550:Protein Stability; D011506:Proteins / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods; D013048:Specimen Handling / Q000379:methods*
D011506:Proteins; D000096:Acetone; D000431:Ethanol