A Novel, High-Throughput Workflow for Discovery and Identification of Serum Carrier Protein-Bound Peptide Biomarker Candidates in Ovarian Cancer Samples Mary F. Lopez, 1* Alvydas Mikulskis, 1 Scott Kuzdzal, 1 Eva Golenko, 1 Emanuel F. Petricoin III, 2 Lance A. Liotta, 2 Wayne F. Patton, 1 Gordon R. Whiteley, 3 Kevin Rosenblatt, 4 Prem Gurnani, 4 Animesh Nandi, 4 Samuel Neill, 5 Stuart Cullen, 5 Martin O’Gorman, 5 David Sarracino, 6 Christopher Lynch, 1 Andrew Johnson, 1 William Mckenzie, 1 and David Fishman 6 Background: Most cases of ovarian cancer are detected at later stages when the 5-year survival is 15%, but 5-year survival approaches 90% when the cancer is detected early (stage I). To use mass spectrometry (MS) of serum proteins for early detection, a seamless work- flow is needed that provides an opportunity for rapid profiling along with direct identification of the under- pinning ions. Methods: We used carrier protein– bound affinity en- richment of serum samples directly coupled with MALDI orthagonal TOF MS profiling to rapidly search for potential ion signatures that contained discriminatory power. These ions were subsequent- ly directly subjected to tandem MS for sequence identification. Results: We discovered several biomarker panels that enabled differentiation of stage I ovarian cancer from unaffected (age-matched) patients with no evidence of ovarian cancer, with positive results in >93% of samples from patients with disease-negative results and in 97% of disease-free controls. The carrier protein– based ap- proach identified additional protein fragments, many from low-abundance proteins or proteins not previously seen in serum. Conclusions: This workflow system using a highly reproducible, high-resolution MALDI-TOF platform en- ables rapid enrichment and profiling of large numbers of clinical samples for discovery of ion signatures and integration of direct sequencing and identification of the ions without need for additional offline, time- consuming purification strategies. © 2007 American Association for Clinical Chemistry The need for development of early screening methods for epithelial ovarian carcinoma is particularly urgent. Ovar- ian cancer is the 4th leading cause of cancer-related deaths among women in the United States (1, 2). Regrettably, 70%–75% of new cases are diagnosed in stage III or IV, with a predicted 5-year survival of 15% (3). The 5-year survival approaches 90%, however, if cancer is detected when confined to the ovary (stage I) (3). More sensitive and specific tests for earlier detection may improve pa- tient survival rates by facilitating early treatments such as surgical intervention (4–8). Recently, new methods of disease detection based on discriminant mass spectral analysis (serum pattern profil- ing) have been proposed (9 –14 ). The power of this approach is 4-fold: (a) it is unbiased and does not presup- pose any particular disease mechanism, (b) multiple dif- ferences, i.e., multiple putative disease markers, are often discovered and combinations of markers are likely to be 1 PerkinElmer Life and Analytical Sciences, Wellesley, MA. 2 Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, VA. 3 Clinical Proteomics Reference Laboratory, Gaithersburg, MD. 4 Department of Pathology, Division of Translational Pathology, Univer- sity of Texas Southwestern Medical Center, Dallas, TX. 5 Nonlinear Dynamics, Cuthbert House, All Saints, Newcastle upon Tyne, United Kingdom. 6 Harvard Partners, Cambridge, MA. 7 New York University Medical School, Division of Obstetrics and Gyne- cology, New York, NY. * Address correspondence to this author at: PerkinElmer Life and Analyt- ical Sciences, 45 Williams St., Wellesley, MA 02481-4008. Fax 617-574-9864; e-mail mary.lopez@perkinelmer.com. Received September 29, 2006; accepted March 27, 2007. Previously published online at DOI: 10.1373/clinchem.2006.080721 Clinical Chemistry 53:6 1067–1074 (2007) Proteomics and Protein Markers 1067 Downloaded from https://academic.oup.com/clinchem/article/53/6/1067/5627461 by guest on 22 August 2022