Proteomic analysis of endoscopically (endoscopic pancreatic function test) collected gastroduodenal fluid using in-gel tryptic digestion followed by LC-MS/MS

Abstract

Purpose: Proteomic analysis of gastroduodenal fluid offers an alternative strategy to study diseases, such as peptic ulcer disease and gastric cancer. We use in-gel tryptic digestion followed by LC-MS/MS (GeLC-MS/MS) to profile the proteome of gastroduodenal fluid collected during the endoscopic pancreatic function test (ePFT).

Experimental design: Gastroduodenal fluid specimens collected during ePFT from six patients with upper abdominal pain were subjected to proteomic analysis. We extracted proteins using three chemical precipitation reagents (acetone, ethanol, and trichloroacetic acid) and analyzed each sample by SDS-PAGE and GeLC-MS/MS for protein identification. Cellular origin and molecular function of the identified proteins were determined via gene ontology analysis.

Results: All three precipitation techniques successfully extracted protein from gastroduodenal fluid, with acetone resulting in excellent resolution and minimal protein degradation compared with the other methods. A total of 134 unique proteins were found in our GeLC-MS/MS analysis of ePFT-collected gastroduodenal fluid samples. Sixty-seven proteins were identified in at least two of the three samples. Gene ontology analysis classified these proteins mainly as being peptidases and localized extracellularly.

Conclusions and clinical relevance: ePFT, followed by acetone precipitation, and coupled with LC-MS/MS, can be used to safely collect gastroduodenal fluid from the upper gastrointestinal tract for MS-based proteomic analysis.

Figure 1

Experimental workflow. Gastroduodenal fluid is collected from six individuals, particulates are pelleted. Samples from the six individuals are pooled and subsequently divided into 3 aliquots to be precipitated by three different chemical (acetone, ethanol, and TCA) precipitation techniques. Samples are analyzed by GeLC-MS/MS methodology.

Figure 2

SDS-PAGE analysis on gastroduodenal fluid of each of the six individuals. Patient-to-patient variability is observed among the six samples. Subsequent proteomic analyses use a pooled sample of gastroduodenal fluid from these six individuals.

Figure 3

Protein extraction from gastroduodenal fluid. A) SDS-PAGE analysis of gastroduodenal fluid precipitated by (1) acetone, (2) ethanol, and (3) trichloroacetic acid (TCA). B) Quantitative gel densitometry measurements of gel lanes (98kDa to 7kDa molecular weight marker) as determined using ImageJ software. Data points were normalized with respect to the maximum intensity value.

Figure 4

Venn diagram depicting the number of proteins identified by GeLC-MS/MS of gel lanes in gastroduodenal fluid precipitated by acetone, ethanol, and TCA. Tallied proteins were identified with two or more peptides that were of 95% or greater confidence as determined by the Paragon Algorithm [19].

Figure 5

Gene ontology analysis of A) subcellular localization and B) molecular function. Gene ontology characterization using the categories listed of the proteins identified in 2 of the 3 samples was performed manually with the UniProt [21] database or using the GoFact online tool [22, 23].