Conceived and designed the experiments: YS DGC. Performed the experiments: YS TL RZ BP MG RJM. Analyzed the data: YS NT SOP. Contributed reagents/materials/analysis tools: YS. Wrote the paper: YS NT TL DGC FE RDS. Provided breast cancer samples: FE.
The authors have declared that no competing interests exist.
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.
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.
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.
Breast cancer is the most common malignancy in Western women; in 2009, more than 192,000 women were estimated to be diagnosed and ∼40,000 died of this disease in the United States alone
Cancer progression, invasion, and metastasis require and/or bring about changes to tumor microenvironments that involve protein degradation by cancer degradome proteases
Herein, we report on degradomic behaviors based upon the analysis of the peptidomes of pooled breast cancer patients and control healthy persons. Our strategy involved comprehensive separations combined with tandem Fourier transform mass spectrometry and new data analysis approaches that facilitated top-down, global peptidomic analysis
Blood plasma samples for this work were collected under the identical lab protocol from15 breast cancer patients (BCP) (ER positive, Her2 negative; invasive ductal carcinoma; 5 are stage I, 7 are stage II and 3 are stage III) and 15 control healthy persons (HP) of matched age, race, and menopausal status. The blood plasma peptidomes of these samples were isolated without observable discrimination and analyzed in parallel under well-controlled conditions (see
The BCP and the control HP have very similar sets of plasma proteins (
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
Differences exist between the base peaks of the BCP and HP peptidome major components.
The cleavage positions P and P′ defined by Schechter I. and Berger A. (
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.
Plasmin (Plm) is an extracellular protease that physiologically and pathologically participates in the remolding of tissues and female reproductive organs
(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.
The prothrombin (FII)-antithrombin (AT) system is involved in angiogenesis, as well as in cancer invasion and metastasis
The matrix metalloproteinases (MMPs)
Tissue remodeling for cancer invasion and metastasis requires degradation of the ECM
(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).
The complement system is an essential part of the innate immune system
(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.
(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
The BCP degradome active substrates also encompass proteins that have been previously investigated individually for cancer therapy, although the roles of some of these proteins in tumor biology have not been well elucidated.
(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
The bottom-up proteomics results obtained in this study, generally with a detection limit of ∼ng/mL
However, it is recognized that this work was limited to 15 BCP and the matched control HP samples. The observations at the present time does not allow statistical evaluation of the differential degradomic-peptidomic signatures, and extensive investigations of the degradomes-peptidomes of a much larger BCP population need to be explored to validate whether every BCP displays all or part of the degradomic features described in this work. The BCP tested in this work are for stages I, II and III (maybe spread to nearby lymph nodes but not distant parts of the body), but not for late (advanced) stage IV breast cancer; the differences between the different stages of breast cancer were not studied in this work. Analysis of degradomic profiles for different stages of breast cancer, breast benign tumors and different molecular subgroups of breast cancers, and other types of cancers (e.g., uterine, liver, lung cancers, etc) and diseases (e.g., inflammation) are needed for statistical validation of which aberrances of the degradomic profile and/or which substrate sequence domains generating the peptidome peptides are specific to or unique for BCP before the BCP degradomic profile and/or individual peptidomic peptides reported in this work can be directly applied for the diagnostic purpose. We are now developing high-through measurement capabilities based on the accurate mass and time tags approach
We also note that degradomic-peptidomic analysis of blood plasma is highly sensitive to a variety of conditions such as specimen collection and storage, peptidomic sample processing, sample analysis processes etc. For extensive comparative studies, standard operation procedures need to be established and executed to minimize the unnecessary variation that could affect the analytical results. The present study, albeit with the relatively small cohort size, used carefully selected patient and healthy control samples that were collected using the same laboratory protocol and processed in parallel. The data generated with our global peptidomic strategy are highly suggestive and provide a foundation for future efforts in this area.
Human blood plasma samples were collected from the BCP and HP at the University of Texas M. D. Anderson Cancer Center (Houston, TX), following the same strict laboratory protocol for each sample. Approval for conducting this study was obtained from the Institutional Review Boards of the University of Texas M. D. Anderson Cancer Center and the Pacific Northwest National Laboratory in accordance with federal regulations.
The 15 breast cancer subjects selected for this study are patients with ER/PR positive and Her2 negative invasive ductal carcinoma; 5 are stages I, 7 are stage II and 3 are stage III; 11 Caucasians, 3 Hispanics and 1 Asian/Pacific Islander; 8 premenopausal and 7 postmenopausal; aged from 30–78 years old (median age is 48 years old). The 15 control healthy subjects were carefully selected to have matched age, race, and menopausal status as the BCP. Blood samples collected in EDTA tubes were first centrifuged at 1,500–1,600 g for 15 min at 4°C, and plasma (upper phase) was transferred to another EDTA tube and centrifuged at 14,000 g for 15 min at 4°C to remove any remaining cellular material. The top 90% of plasma was then transferred into cryovials with approximately 0.5 ml plasma in each and stored frozen at −80°C until further use.
The method reported
Quantitation of peptidome peptides was achieved through extraction of the FT- measured precursors of the FT-MS/MS Spectra. ICR2LS was used to deisotope high-resolution spectra and produce lists of neutral masses for both precursor MS and MS/MS spectra. Functions were built specifically for this study to isolate MS spectra only and merge re-enumerated spectra to form a single continuous dataset. ICR2LS was also used to assign absolute intensity (abundance) values for the deisotoped masses using the area under the curve (peak area) as a measurement. To compile quantitative measurement of the identified peptides, the datasets for the two samples were processed for finding features (unique mass classes) based on the algorithm described previously
Conventional bottom-up approach was used for proteomic analysis of the BCP and HP plasma samples. The samples were depleted using the same procedure as used for preparation of the peptidomic samples. The IgY12 flow-through proteins were denatured and reduced in 50 mM ammonium bicarbonate buffer (pH 8.2), 8 M urea, 10 mM dithiothreitol for 1 h at 37°C, followed by alkylation in 40 mM iodoacetamide for 1 h at room temperature in the dark. The resultant protein mixtures were diluted 10 fold with 50 mM ammonium bicarbonate buffer (pH 8.2), and then sequencing grade modified porcine trypsin (Promega, Madison, WI) was added at a trypsin:protein ratio of 1∶50 (w/w). The samples were incubated overnight at 37°C for digestion. The tryptic digest samples were loaded on a 1-mL SPE C18 column (Sigma, St. Louis, MO) and cleaned with 4 mL of 0.1% trifluoroacetic acid/5% acetonitrile. The peptides were eluted from the SPE column with 1 mL of 0.1% trifluoroacetic acid/80% acetonitrile and lyophilized. The peptide samples were stored at −80°C for analysis.
The 300 µg of the prepared two tryptic digestion samples were individually reconstituted with 300 µL of 10 mM ammonium formate (pH 3.0)/25% acetonitrile and fractionated by strong cation exchange (SCX) chromatography on a Polysulfoethyl A 200 mm×2.1 mm column (PolyLC, Columbia, MD) that was preceded by a 10 mm×2.1 mm guard column. The separations were performed on an Agilent 1100 series HPLC system (Agilent, Palo Alto, CA) at a flow rate of 200 µL/min, and with mobile phases that consisted of 10 mM ammonium formate (pH 3.0)/25% acetonitrile (A) and 500 mM ammonium formate (pH 6.8)/25% acetonitrile (B). After loading 300 µL of each sample separately onto the column, the gradient was maintained at 100% A for 10 min. The peptides were separated using a gradient from 0 to 50% B over 40 min, 50–100% B over 10 min, and then held at 100% B for 10 min. A total of 30 fractions were collected for each sample, and each fraction was dried under vacuum. The fractions for each sample were dissolved in 30 µL of 25 mM NH4HCO3, and 5 µL was used for the capillary LC-MS/MS analyses.
Peptides LC-MS/MS analyses were carried out using a custom-built automated capillary LC system coupled online to an LTQ mass spectrometer (Thermo Scientific)
The LC-MS/MS raw data were converted into .dta files using Extract_MSn (version 3.0) in Bioworks Cluster 3.2 (Thermo Scientific), and the SEQUEST algorithm (version 27, revision 12) was used to independently search all the MS/MS spectra against the IPI database (version 3.39, released February 2008) with dynamic oxidation on methionine residues and static alkylation on cysteine residues. The false discovery rate (FDR) was estimated based on the reported decoy-database searching methodology
The identification details for each proteomic and peptidomic peptides were given in
Supplementary materials are included with this manuscript.
We have obtained the written informed consent from all patients involved in this study.
The proteome tryptic peptides identified from the pooled breast cancer patients (BCP) and control healthy persons (HP) blood plasma samples.
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The peptidome peptides identified from the pooled breast cancer patients (BCP) and control healthy persons (HP) blood plasma samples.
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The MMPs identified from the pooled breast cancer patients (BCP) and control healthy persons (HP) blood plasma proteomic samples.
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The HC1-3 composed of protein complexes IαI, PαI and IαIL that function to stabilize the ECM. The red and blue numbers represent the different peptidome peptides observed for the BCP and the control HP, respectively.
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The selective degradation of the BCP complement system convertases and MAC components would lead to the system dysfunction. The key convertases C3b, C4b, and factor B and MAC components C8 and C9 in the complement system that limits tumor growth are differentially degraded on their function domains for the BCP; the grey arrows represent the activation routes to be limited due to the excessive degradation
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The BCP and the control HP peptidome peptide molecular weight distributions. The BCP and HP peptidomes were isolated with use of size exclusion chromatography.
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Work was performed in the Environmental Molecular Science Laboratory, a DOE/BER national scientific user facility located on the campus of Pacific Northwest National Laboratory (PNNL) in Richland, Washington. PNNL is a multi-program national laboratory operated by Battelle Memorial Institute for the DOE under contract DE-AC05-76RLO-1830.