Optimized sample preparation of endoscopic collected pancreatic fluid for SDS‐PAGE analysis

Abstract

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 (ePFT). Using SDS-PAGE protein profiling, we investigate (1) precipitation techniques to maximize protein extraction, (2) auto-digestion of pancreatic fluid following prolonged exposure to a range of temperatures, (3) effects of multiple freeze-thaw cycles on protein stability, and (4) the utility of protease inhibitors. Our experiments revealed that trichloroacetic acid (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°C and 37°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.

Analysis of Endoscopic Pancreatic Function Test (ePFT)-Collected Pancreatic Fluid Proteins Precipitated Via Ultracentrifugation

“…One aliquot of each specimen was subjected to TCA precipitation and the second aliquot to ultracentrifugation precipitation, for a total of 12 samples. The TCA-precipitated samples showed protein banding patterns similar to those seen in previous pancreatic fluid TCA precipitations [8, 10, 11, 35]. However, these protein banding patterns differed substantially from those observed in the ultracentrifugation-precipitated samples; the ultracentrifugation samples were far less concentrated and fewer stained bands were observed.…”

“…Protein concentration was determined using the BioRAD (Hercules, CA, USA) protein assay according to the manufacturer’s instructions. We have omitted protease inhibitors as we demonstrated previously that at 4°C, little proteolysis in pancreatic fluid occurs activity [11], without the caveats associated with the addition of protease inhibitors in mass spectrometry experiments [22, 23, 24]. …”

“…The proteins from 6 pancreatic fluid specimens (200 μL) were isolated by precipitation with the addition of 12.5% TCA, as described previously [10, 11]. This process limits protein degradation by instantaneously deactivating enzymes and removing salts that will interfere with the subsequent electrophoretic mobility-based fractionation by SDS-PAGE.…”

“…We have demonstrated previously that pancreatic fluid can be readily collected via the secretin-stimulated endoscopic pancreatic function test (ePFT) in volumes of 2 to 8 mL, and as such is a suitable specimen for mass spectrometry-based proteomic investigations [7, 8, 9, 10, 11]. We have screened a series of precipitation strategies to optimize protein extraction from pancreatic fluid [11], and we have demonstrated that pancreatic fluid can be maintained at 4°C for several hours with minimal proteolysis. We determined that trichloroacetic acid (TCA) precipitation was able to: 1) concentrate proteins; 2) eliminate non-protein contaminants; and 3) quickly inactivate pancreatic proteases (which are active at physiological pH).…”

“…Compared to TCA precipitation, ultracentrifugation requires fewer manual steps, thereby reducing the potential for sample loss. Furthermore, ultracentrifugation can be performed at 4°C, making it a potentially useful technique in the proteomic analysis of pancreatic fluid, as we have shown that a temperature at or below 4°C is an effective method of minimizing protease activity [11]. …”