Characterization of the Human Gastric Fluid Proteome Reveals Distinct pH-Dependent Protein Profiles: Implications for Biomarker Studies

Abstract

Gastric fluid is a source of gastric cancer biomarkers. However, very little is known about the normal gastric fluid proteome and its biological variations. In this study, we performed a comprehensive analysis of the human gastric fluid proteome using samples obtained from individuals with benign gastric conditions. Gastric fluid proteins were prefractionated using ultracentrifuge filters (3 kDa cutoff) and analyzed by two-dimensional gel electrophoresis (2-DE) and multidimensional LC-MS/MS. Our 2-DE analysis of 170 gastric fluid samples revealed distinct protein profiles for acidic and neutral samples, highlighting pH effects on protein composition. By 2D LC-MS/MS analysis of pooled samples, we identified 284 and 347 proteins in acidic and neutral samples respectively (FDR ≤1%), of which 265 proteins (72.4%) overlapped. However, unlike neutral samples, most proteins in acidic samples were identified from peptides in the filtrate (i.e., <3 kDa). Consistent with this finding, immunoblot analysis of six potential gastric cancer biomarkers rarely detected full-length proteins in acidic samples. These findings have important implications for biomarker studies because a majority of gastric cancer patients have neutral gastric fluid compared to noncancer controls. Consequently, sample stratification, choice of proteomic approaches, and validation strategy can profoundly affect the interpretation of biomarker findings. These observations should help to refine gastric fluid biomarker studies.

A simple biomarker scoring matrix for early gastric cancer detection

“…In a critical discussion of the impact of proteolytic degradation on GC biomarker validity , it was proposed that either a peptide‐centric or protein‐centric biomarker identification approach should be adopted depending on the extent of possible degradation, and that up‐regulated protein biomarkers could simply be protected from degradation due to hypoacidity of a cancerous stomach. This is however not a concern in this study, since four out of five of the proposed GC biomarkers are down‐regulated in GC, and not the other way round.…”

Discovery of biomarkers for gastric cancer: A proteomics approach

“…A higher number of proteins (<3 kDa) were identified in the acidic component than in the neutral component of the specimens. Furthermore, full-length potential biomarkers were rarely discovered in the acidic component derived from normal, healthy individuals [37].…”

“…However, the presence of specific gastric fluid biomarkers can further aid in the evaluation of symptomatic patients, thus leading to a definitive diagnosis. Of particular importance are pepsin-related proteins such as pepsinogen C [35], pepsin A [35], α1-antitrypsin [36], and pepsinogen II [37] that have been shown to have altered expressions in gastric cancer. These findings suggest that the activation of proteolytic enzymes in gastric fluid may play an important factor in gastric carcinogenesis.…”

Dynamic Monitoring of Systemic Biomarkers with Gastric Sensors

“…[ 8 ] This fluid contains a cocktail of digestive compounds, such as pepsin and hydrochloric acid. It also contains many analytes present in serum [ 9 ] including urea, [ 10 ] ammonia, [ 11 ] or cortisol [ 12 ] as well as intravenously administered antibiotics like metronidazole [ 13 ] and clarithromycin. [ 14 ] To better understand the coincidence of markers in serum and GF we first analyzed a broad array of metabolic biomarkers in serum and time‐matched porcine GF.…”