Comprehensive and quantitative proteomic analyses of zebrafish plasma reveals conserved protein profiles between genders and between zebrafish and human

Abstract

Omic approaches have been increasingly used in the zebrafish model for holistic understanding of molecular events and mechanisms of tissue functions. However, plasma is rarely used for omic profiling because of the technical challenges in collecting sufficient blood. In this study, we employed two mass spectrometric (MS) approaches for a comprehensive characterization of zebrafish plasma proteome, i.e. conventional shotgun liquid chromatography-tandem mass spectrometry (LC-MS/MS) for an overview study and quantitative SWATH (Sequential Window Acquisition of all THeoretical fragment-ion spectra) for comparison between genders. 959 proteins were identified in the shotgun profiling with estimated concentrations spanning almost five orders of magnitudes. Other than the presence of a few highly abundant female egg yolk precursor proteins (vitellogenins), the proteomic profiles of male and female plasmas were very similar in both number and abundance and there were basically no other highly gender-biased proteins. The types of plasma proteins based on IPA (Ingenuity Pathway Analysis) classification and tissue sources of production were also very similar. Furthermore, the zebrafish plasma proteome shares significant similarities with human plasma proteome, in particular in top abundant proteins including apolipoproteins and complements. Thus, the current study provided a valuable dataset for future evaluation of plasma proteins in zebrafish.

Figure 1. Overview of plasma protein profiles based on shotgun MS.

(A) Cumulative percentage of plasma proteome ranked according to protein abundance. (B) Cumulative plasma protein numbers (shown as % of total identified proteins, 642 in female and 595 in male) ranked based on estimated concentrations of proteins detected. Most proteins (~70%) were in the range of 2–10 μg/mL. (C,D) Abundance distribution of plasma proteins in females (C) and in males (D). Top 10 proteins account for about 65% of plasma proteins in female and about 50% in male.

Figure 2. Characteristics of plasma protein composition.

(A–C) GO distribution of plasma proteins based on number of protein entries (left) and protein abundance (right) in both genders as indicated. GO annotation was retrieved for a total of 791 unique UniProt entries of zebrafish plasma proteins. Categories of GO are: Biological Process (A), Cellular Component (B) and Molecular Function (C). (D) Comparison of types of plasma proteins between the two genders as classified by IPA.

Figure 3. Tissue expression of plasma proteins.

(A) Distribution of plasma proteins based on tissues of expression. (B) Comparison of tissue expression of plasma proteins between females and males. Only those proteins expressed in a single tissue are used for comparison.

Figure 4. Overlaps in ranks of plasma proteins between human and zebrafish based on the study of Liuet al..

(A) Rank of top 30 abundant human plasma proteins in zebrafish. (B) Comparison in ranks of all mapped overlapping plasma proteins between human and zebrafish female (left, 255 proteins) and male (right, 250 proteins). Proteins are presented based on ranks in human plasma. Two dashed lines in (B) mark approximately the rank of 500 and 5000 in human plasma proteins. Hs: human; Zf: zebrafish. Black arrows indicate Alpha-1-antitrypsin precursor (human) and Serpina1 protein (Zebrafish), and green arrows indicate Ig mu chain C region.

Figure 5. Comparison of zebrafish and human plasma proteomes based on Plasma Proteme Database (PPD).

(A–C) GO distribution of human plasma proteins in the categories of Biological Process (A), Cellular Component (B), and Molecular Function (C). Both the ranks of terms and the corresponding percentages are similar to those in zebrafish plasma (Fig. 2A–C). (D) Pearson correlation between zebrafish female and male plasma proteins based on concentration of overlapping proteins. (E,F) Pearson correlation between human plasma proteins and zebrafish female (E) and male (F) plasma proteins, based on corresponding concentrations of homologous proteins.

Figure 6. Overview of SWATH profile of female and male plasma proteins depicting abundance of detected proteins.

Median total ion current (TIC) of proteins identified in male (x-axis) and female (y-axis) plasma were compared. Majority of plasma protein have similar abundance in both sexes (Pearson correlation = 0.88), with representative sex-biased plasma proteins as outliers.