Global analysis of the rat and human platelet proteome - the molecular blueprint for illustrating multi-functional platelets and cross-species function evolution

Abstract

Emerging evidences indicate that blood platelets function in multiple biological processes including immune response, bone metastasis and liver regeneration in addition to their known roles in hemostasis and thrombosis. Global elucidation of platelet proteome will provide the molecular base of these platelet functions. Here, we set up a high-throughput platform for maximum exploration of the rat/human platelet proteome using integrated proteomic technologies, and then applied to identify the largest number of the proteins expressed in both rat and human platelets. After stringent statistical filtration, a total of 837 unique proteins matched with at least two unique peptides were precisely identified, making it the first comprehensive protein database so far for rat platelets. Meanwhile, quantitative analyses of the thrombin-stimulated platelets offered great insights into the biological functions of platelet proteins and therefore confirmed our global profiling data. A comparative proteomic analysis between rat and human platelets was also conducted, which revealed not only a significant similarity, but also an across-species evolutionary link that the orthologous proteins representing "core proteome", and the "evolutionary proteome" is actually a relatively static proteome.

Figure 1. Schematic illustration of rat and human platelet proteome analysis

A two-step extraction method with 8M urea and the lysis buffer was applied to extract platelet proteins for either SDS gel-based separation and LC-MS/MS or in-solution digestion respectively.

Figure 2

(A & B) Radar plots illustrating the distribution of protein subcellular localization from rat and human platelets. Proteins belonging to Other locations were not shown in the radar plots (rat, 20.3%; human, 29.6%). ER, endoplasmic reticulum. (C & D) Radar plots showing the categories of molecular functions and biological processes of the proteins identified in the rat and human platelets.

Figure 3. The distribution of quantified proteins versus the number of matched approaches used

Low, emPAI<0.1 and without emPAI annotated; medium, 0.1≤emPAI<1; high, emPAI≥1.

Figure 4. Results of the database search using dual engines including Mascot and Sequest

The Venn plot demonstrates the overlap and exclusive matched proteins or peptides of MASCOT and SEQUEST. The number of peptides or proteins was marked in the corresponding areas. (B) The histogram shows the matched ratios of search engines in each abundance category. All of the peptides and proteins were confirmed through bioinformatics analysis.

Figure 5. Biological process (A) and pathway (B) analyses of the differentially expressed proteins after thrombin stimulation

Metabolism group (in A) and other pathways group (in B) were further classified into 7 and 11 groups, respectively, according to the bioinformatics analysis results.

Figure 5. Biological process (A) and pathway (B) analyses of the differentially expressed proteins after thrombin stimulation

Metabolism group (in A) and other pathways group (in B) were further classified into 7 and 11 groups, respectively, according to the bioinformatics analysis results.

Figure 6. Bioinformatics analysis of orthologous proteins

(A) The Venn plot (left) demonstrates the orthologous proteins between human and rat platelets. (B, C) All orthologous proteins in rat platelets are classified according to their subcellular localization and molecular functions. (D, E) The histogram demonstrates the ratios of the orthologous proteins in each category versus the overall proteins in the same category. The dotted lines show the average value of the distributions.