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Serum peptidome for cancer detection: spinning biologic trash into diagnostic gold
Lance A. Liotta and Emanuel F. Petricoin
Center for Applied Proteomics and Molecular Medicine, College of Arts and Sciences, George Mason University, Manassas, Virginia, USA.
Address correspondence to: Lance A. Liotta or Emanuel F. Petricoin, Center for Applied Proteomics and Molecular Medicine, College of Arts and Sciences, George Mason University, 10900 University Boulevard, MS 4E3, Room 181A, Manassas, Virginia 20110, USA. Phone: (703) 993-9444; Fax: (703) 993-4288; E-mail: lliotta@gmu.edu (L.A. Liotta). Phone: (703) 993-8646; Fax: (703) 993-4288; E-mail: epetrico@gmu.edu (E.F. Petricoin).
Find articles by Liotta, L. in: JCI | PubMed | Google Scholar
Center for Applied Proteomics and Molecular Medicine, College of Arts and Sciences, George Mason University, Manassas, Virginia, USA.
Address correspondence to: Lance A. Liotta or Emanuel F. Petricoin, Center for Applied Proteomics and Molecular Medicine, College of Arts and Sciences, George Mason University, 10900 University Boulevard, MS 4E3, Room 181A, Manassas, Virginia 20110, USA. Phone: (703) 993-9444; Fax: (703) 993-4288; E-mail: lliotta@gmu.edu (L.A. Liotta). Phone: (703) 993-8646; Fax: (703) 993-4288; E-mail: epetrico@gmu.edu (E.F. Petricoin).
Find articles by Petricoin, E. in: JCI | PubMed | Google Scholar
Published January 4, 2006 - More info
J Clin Invest. 2006;116(1):26–30. https://doi.org/10.1172/JCI27467.
© 2006 The American Society for Clinical Investigation
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Abstract
Recent studies have established distinctive serum polypeptide patterns through mass spectrometry (MS) that reportedly correlate with clinically relevant outcomes. Wider acceptance of these signatures as valid biomarkers for disease may follow sequence characterization of the components and elucidation of the mechanisms by which they are generated. Using a highly optimized peptide extraction and matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF) MS–based approach, we now show that a limited subset of serum peptides (a signature) provides accurate class discrimination between patients with 3 types of solid tumors and controls without cancer. Targeted sequence identification of 61 signature peptides revealed that they fall into several tight clusters and that most are generated by exopeptidase activities that confer cancer type–specific differences superimposed on the proteolytic events of the ex vivo coagulation and complement degradation pathways. This small but robust set of marker peptides then enabled highly accurate class prediction for an external validation set of prostate cancer samples. In sum, this study provides a direct link between peptide marker profiles of disease and differential protease activity, and the patterns we describe may have clinical utility as surrogate markers for detection and classification of cancer. Our findings also have important implications for future peptide biomarker discovery efforts.
Authors
Josep Villanueva, David R. Shaffer, John Philip, Carlos A. Chaparro, Hediye Erdjument-Bromage, Adam B. Olshen, Martin Fleisher, Hans Lilja, Edi Brogi, Jeff Boyd, Marta Sanchez-Carbayo, Eric C. Holland, Carlos Cordon-Cardo, Howard I. Scher, Paul Tempst
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Carrier protein–bound peptidome
While Villanueva et al. (18) provide evidence to demonstrate one method by which the LMW peptidome is created ex vivo, there is a growing body of evidence that circulating blood already contains an abundance of protein fragments apparently derived from cells and tissues that are produced in vivo. Previous work by Tirumalai et al. (12) and most recently by Lowenthal et al. (16) has revealed a vast and diverse source of LMW and low-abundance fragments of cellular proteins that are not cleavage products of resident serum proteins. This view of the LMW peptidome is one in which low-abundance peptide biomarkers produced from specific and ongoing tumorigenic processes such as apoptosis, tumor-stromal interaction, vascularization, immune cell infiltration, and antigenic processing exist in a sequestered state, complexed to highly abundant resident blood proteins such as albumin (12, 16, 19) (Figure 1). The concentration of any LMW analyte in the circulation is completely dependant on its production and clearance rates. Given the ability of the glomeruli to efficiently and effectively remove molecules smaller than approximately 50 kDa, any LMW molecule generated in vivo would be cleared rather quickly, thereby reducing the concentration of the analyte at the time the blood is drawn to potentially undetectable levels. Sequestration of peptides by resident blood carrier proteins with long half-lives, such as albumin, protects the peptides from clearance. Two recent studies offer a first glimpse into the diagnostic potential of the LMW, carrier protein–bound biomarker pool. Lopez et al. report MALDI-TOF fingerprinting of serum for Alzheimer disease detection (20). They harvested albumin-bound LMW protein fragments as the direct input for their MS ion fingerprint analyses. Alzheimer disease–associated fingerprints found in a training set were validated using an independent test set. The approach of Lopez et al. (20) was distinct from that used by Villeneuva et al. (18). A second study investigated the identities of LMW, albumin-bound fragments obtained from the sera of ovarian cancer patients (16). The authors identified a large number of ovarian cancer–associated peptides that were different from the peptides found in high-risk, disease-free subjects. A subset of peptides was found only in the samples from patients with stage 1 ovarian cancer. Sequencing of these peptides indicated that they were fragments of low-abundance molecules such as BRCA2, tyrosine kinases, other signaling molecules, and intracellular scaffolding proteins. Thus, the endogenous circulating peptidome may be potentially redefined as a subset of the blood interactome — protein analytes that exist in the circulation as protein-protein complexes. Identification of the components of the LMW circulatory peptidome by MS sequencing provides a list of specific analytes that is independent of the measurement technology (e.g., MALDI-TOF). Fingerprints of MS ions can therefore be replaced with panels of named protein biomarkers. Regardless of whether the peptidome is derived from the tissue/microenvironment in vivo or ex vivo, the implications for the diagnostics arena are enormous.
Figure 1Proteinases generate biomarker fragments. Circulating protein fragments generated in the diseased tissue microenviroment may serve as diagnostic protein markers. Proteolytic cascades within the tissue (a product of the interacting cellular ecology such as stromal-epithelial interactions), immune cell MHC presentation, or apoptosis generate protein fragments that passively diffuse into the circulation. Shed LMW peptides are protected from kidney-mediated clearance by sequestration on abundant resident blood proteins such as albumin. According to the results presented by Villanueva et al. (18) in this issue of the JCI, diagnostic protein fragments can also be generated ex vivo by circulating enzymes derived from the diseased tissue microenvironment acting on exogenously derived peptides produced by serum collection methodology (see Figure 10 in ref. 18).
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Immuno-MS: the diagnostic platform of the future?
Conventional immunoassay platforms cannot measure panels of analyte fragments. This is because immunoassays, by their very definition, rely on antibody-based capture and detection methods. An antibody-based assay cannot distinguish the parent molecule from its cleaved fragments (the latter of which could possess the greatest diagnostic potential) since the antibody recognizes its cognate epitope in both the parent and fragment molecules. Thus, the future of fragment-based diagnostics will require the invention and adoption of wholly new technologies that read out both the identity and the exact size of the molecule. Immuno-MS is 1 example of how this could be achieved (Figure 2). With this approach, a microaffinity antibody column, perhaps in a multiplexed microwell format, would first be used to capture the parent protein along with any of its fragment isoforms that contain the antibody recognition site. Next, the captured fragments would be eluted from the antibody column directly into a mass spectrometer (such as a MALDI-TOF MS). MS analysis of the eluted peptides would provide an extremely accurate mass determination of the entire population of captured peptides. Thus, in only 2 steps, a panel of peptide fragments derived from a known parent molecule could be rapidly tabulated. Future rounds of investigation into the nature and diagnostic potential of the serum peptidome will likely uncover many more surprises. Nevertheless, based on results to date, we now expect that the diagnostic potential of the LMW serum peptidome will likely be dependent on the examination of the following: (a) low-abundance, tissue-derived peptides, many of which avoid clearance by binding to carrier proteins; (b) size- and cleavage-specific fingerprints of fragments derived from cellular parent molecules; and (c) now, based on the findings of Villanueva et al. (18), additional signatures of cleavage products produced ex vivo after blood clotting during serum collection. Far from drying up, the LMW blood-borne biomarker pipeline is surging with potential. Nevertheless, this potential will never be realized unless commonly adopted and specific blood collection protocols are implemented across clinical and research laboratories, serum and plasma reference sets are developed and standardized, instrumentation for measuring panels of specific fragments are proven to be both sensitive and reproducible, and extensive validation of the diagnostic utility of these biomarkers is examined in clinical trials conducted in accordance with regulatory guidelines developed by the College of American Pathologists and Clinical Laboratory Improvement Amendments.
Figure 2Immuno-MS provides a means for rapidly determining the specific size and identity of each member of a panel of peptide marker fragments present within patient sera. As part of a high-throughput assay performed in clinical diagnostic laboratories, patient serum would be applied to a multiplexed plate of microcolumns filled with antibody-immobilized beads. Each microcolumn captures both the parental and fragment isoforms of the candidate marker since they both contain the antibody recognition site. The captured population of analytes, including the fragment(s) with potential for disease detection and/or discrimination, are eluted and analyzed directly by MALDI-TOF MS. The presence of the specific peptide biomarker at its precise mass/charge ratio (m/z) would be used as a diagnostic test result. This analysis could be performed rapidly by simple software that determines if the ion peak is present or absent at the predefined m/z location.
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