A dynamic range compression and three-dimensional peptide fractionation analysis platform expands proteome coverage and the diagnostic potential of whole saliva

Abstract

Comprehensive identification of proteins in whole human saliva is critical for appreciating its full diagnostic potential. However, this is challenged by the large dynamic range of protein abundance within the fluid. To address this problem, we used an analysis platform that coupled hexapeptide libraries for dynamic range compression (DRC) with three-dimensional (3D) peptide fractionation. Our approach identified 2340 proteins in whole saliva and represents the largest saliva proteomic dataset generated using a single analysis platform. Three-dimensional peptide fractionation involving sequential steps of preparative isoelectric focusing (IEF), strong cation exchange, and capillary reversed-phase liquid chromatography was essential for maximizing gains from DRC. Compared to saliva not treated with hexapeptide libraries, DRC substantially increased identified proteins across physicochemical and functional categories. Approximately 20% of total salivary proteins are also seen in plasma, and proteins in both fluids show comparable functional diversity and disease-linkage. However, for a subset of diseases, saliva has higher apparent diagnostic potential. These results expand the potential for whole saliva in health monitoring/diagnostics and provide a general platform for improving proteomic coverage of complex biological samples.

Figure 1. Optimization of proteominer treatment with whole saliva and results from 2D-peptide fractionation with/without dynamic range compression of saliva

(a) Increasing amounts of clarified whole saliva protein (25, 50 and 75 mg) were incubated with 100 µL of Proteominer beads overnight in the presence of protease inhibitors. Bound protein (eluate) and Flow through fractions from each treatment were collected. Untreated Saliva, flow through, and eluate fractions from samples were separated by SDS-PAGE and visualized by coomassie staining. (b) 100 µg peptides from Untreated Saliva, or Proteominer Eluate75 were trypsinized and fractionated by preparative IEF (OFFGEL) based on their isoelectric points (pH 3 to 10), and processed for µLC-MS/MS analysis. Total unique peptides identified in each OFFGEL fraction(s) are indicated for Untreated Saliva and Proteominer Eluate75. (c) Total proteins identified in Untreated Saliva versus Proteominer Eluate75.

Figure 2. 3D-peptide fractionation increased total proteins identified in Untreated and Proteominer-treated whole saliva

(a) Scheme for 3D-peptide fractionation. (b) Total proteins identified in Untreated Saliva and Proteominer Eluate75 by 2D- versus 3D-peptide fractionation. Results from combining both analyses are also shown. (c) Venn diagram illustrating total number of proteins those are specific to either Proteominer treatment or Untreated Saliva and those identified in both samples.

Figure 3. Additional identifications obtained after Library-2 treatment of whole saliva further expanded our total salivary dataset

(a) Library-2 eluates and flow through fractions were compared with those obtained after Proteominer treatment under identical conditions by SDS-PAGE analysis and coomassie staining. (b) Total unique peptides identified in each OFFGEL fraction/s after Library-2 treatment of saliva versus Proteominer treatment or Untreated Saliva. (c) Left panel, Venn diagram illustrating total proteins obtained from DRC of saliva and the individual contributions of Library-2 versus Proteominer hexapeptide libraries. Right panel, Venn diagram illustrating total proteins identified in this study and the relative contribution of DRC to these identifications.

Figure 4. DRC increased proteins across physicochemical and Gene Ontological categories without apparent selectivity

Proteins identified in Untreated Saliva, post-DRC saliva and total salivary identifications were grouped into individual categories as described in Results and Discussion section of the manuscript.

Figure 5. Comparison of total salivary protein identifications against those found in plasma for determining functional diversity and diagnostic potential of both fluids

Ingenuity pathway analysis (IPA) was done as described in Experimental Methods.