Blood peptidome-degradome profile of breast cancer

Abstract

Background: Cancer invasion and metastasis are closely associated with activities within the degradome; however, little is known about whether these activities can be detected in the blood of cancer patients.

Methodology and principal findings: The peptidome-degradome profiles of pooled blood plasma sampled from 15 breast cancer patients (BCP) and age, race, and menopausal status matched control healthy persons (HP) were globally characterized using advanced comprehensive separations combined with tandem Fourier transform mass spectrometry and new data analysis approaches that facilitated top-down peptidomic analysis. The BCP pool displayed 71 degradome protein substrates that encompassed 839 distinct peptidome peptides. In contrast, the HP 50 degradome substrates found encompassed 425 peptides. We find that the ratios of the peptidome peptide relative abundances can vary as much as >4000 fold between BCP and HP. The experimental results also show differential degradation of substrates in the BCP sample in their functional domains, including the proteolytic and inhibitory sites of the plasmin-antiplasmin and thrombin-antithrombin systems, the main chains of the extracellular matrix protection proteins, the excessive degradation of innate immune system key convertases and membrane attack complex components, as well as several other cancer suppressor proteins.

Conclusions: Degradomics-peptidomics profiling of blood plasma is highly sensitive to changes not evidenced by conventional bottom-up proteomics and potentially provides unique signatures of possible diagnostic utility.

Figure 1. The proteome and degradome profiles for the 15 BCP and control HP samples.

The degradome is a sub-proteome that participates in the proteolytic activities and produces peptidome (top). The proteomic and degradomic measurements are represented by tryptic peptides and peptidome peptides, respectively (middle). The BCP degradome is compared to the HP degradome using the peptidome peptide abundances (bottom). Details for each degradome substrate, peptidome peptide and its quantification, proteome protein, and tryptic peptide are given in Tables S1 and S2.

Figure 2. The comparative base peak chromatograms of the BCP and HP peptidome components.

Differences exist between the base peaks of the BCP and HP peptidome major components.

Figure 3. The net cleavage specificity of BCP and HP proteases for the degradome-wide substrates.

The cleavage positions P and P′ defined by Schechter I. and Berger A. (Papain Biochem Biophys Res Commun 1967, 27: 157–162) are adopted herein to present the specificity measured from ∼1000 peptidome peptides listed in Table S2.

Figure 4. The net cleavage specificity of BCP and HP proteases for the degradome individual substrate.

Protein substrate apoA-IV is used for this examination. The counts and frequencies (percentages) for amino acids at P1 in the BCP and HP are used to present the specificity. The BCP degradome proteases are more active than the HP ones to cleave more sites at P1-Ala, Lys, Leu, Arg, and Tyr. For the BCP, the P1-Leu, Tyr, and Ala are relatively preferred for cleavage in comparison with other P1-amino acids.

Figure 5. Selective degradation of function domains of protease systems observed for the tested BCP.

(A) For Plm-α₂-AP system, Plg degrades on its preactivation peptide; while α₂-AP on the inhibitory function bonds. (B) For FII-AT system, FII degrades on its heavy chain, not on its proteolytic function domain; while AT degrades on its inhibitory function bonds to prevent formation of thrombin-antithrombin (TAT) complex. The red lines along the sequences represent the peptidome peptides solely observed from the BCP; the differential degradation is represented by the numbers (red for the BCP and blue for the HP) of different peptides observed.

Figure 6. Selective degradation of ITI HC1-3 observed for the tested BCP.

(A) The peptidome peptides were observed solely from the HC1 main chain, not from the two propeptides (blank-filled ovals). (B) The peptidome peptides were observed dominantly from the HC2 main chain, except for one (not labeled) from the propeptide (black color-filled oval). (C) The peptidome peptides were observed solely from the HC3 main chain, not from the two propeptides (blank-filled ovals).

Figure 7. Degradation of complement system convertases observed for the tested BCP.

(A) For C3, the degradation was solely on the C3d, C3g, C3α′1 and C3β (black color-filled ovals), not on the other fragments (blank-filled ovals); the C3 sequence domains are colored for comparison of the domains observed with degradation. (B) For C4, the degradation was solely on the C4b and C4β (black color-filled ovals), not on the other fragments (blank-filled ovals); the degradation of the front portion of the C4b fragment was observed solely for the tested BCP. (C) For complement factor B, the degradation was solely on the complement factor Ba and Bb (black color-filled ovals), not on the other fragments (blank-filled ovals). The red, blue, and black lines along the amino acid sequences represent the peptidome peptides identified solely from the tested BCP, HP, and both the BCP and HP, respectively.

Figure 8. Degradation of complement system MAC key components observed for the tested BCP.

(A) For C8, the degradation was solely on the C8α (black color-filled ovals), not on the other fragments (blank-filled ovals). (B) For C9, the degradation was solely on the C9α (black color-filled ovals), not on the other fragments (blank-filled ovals). The red, blue, and black lines along the amino acid sequences have the same significances as for Figure 7.

Figure 9. The differential degradation of other protein substrates observed for the tested BCP.

(A) Pigment epithelium-derived factor; specific truncates from the N-terminal direction exist solely to produce the multiple BCP peptidome peptides. (B) Gelsolin; the sequence zone in the middle of the substrate sequences specifically produce the multiple BCP peptidome peptides. (C) Ceruloplasmin; the sequence zone toward the substrate C terminus specifically generated the multiple BCP peptidome peptides. (D) ApoA-IV; the cleavage P1-Leu, P1-Tyr, and Lys (labeled in the figure) specifically for the BCP exist to produce the multiple BCP peptidome peptides. The red, blue, and black lines along the amino acid sequences have the same significances as for Figure 7.