A Novel, High-Throughput Workflow for Discovery and Identification of Serum Carrier Protein-Bound Peptide Biomarker Candidates in Ovarian Cancer Samples

George Mason University, 페어팩스, Virginia, United States
Clinical Chemistry (Impact Factor: 7.91). 06/2007; 53(6):1067-74. DOI: 10.1373/clinchem.2006.080721
Source: PubMed


Most cases of ovarian cancer are detected at later stages when the 5-year survival is approximately 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 workflow is needed that provides an opportunity for rapid profiling along with direct identification of the underpinning ions.
We used carrier protein-bound affinity enrichment 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 subsequently directly subjected to tandem MS for sequence identification.
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 approach identified additional protein fragments, many from low-abundance proteins or proteins not previously seen in serum.
This workflow system using a highly reproducible, high-resolution MALDI-TOF platform enables 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.

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Available from: Kevin P Rosenblatt, Feb 18, 2014
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    • "It is important to highlight that higher peptide levels could be due either to an increasing level of circulating precursor protein, or to an increase of protein degradation pathways. In the case of complement C3, there are evidences of increased plasmatic level related in cancer (Liang et al., 2010; Lopez et al., 2007). Moreover, complement cascade is known to be involved in cancer development (Markiewski and Lambris, 2009; Rutkowski et al., 2010) and recently Dowling et al. (Dowling et al., 2012) demonstrated an increased complement activation in RC patients. "
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    ABSTRACT: Preoperative chemoradiotherapy is worldwide accepted as a standard treatment for locally advanced rectal cancer. Current standard of treatment includes administration of ionizing radiation for 45-50.4 Gy in 25-28 fractions associated with 5-fluorouracil administration during radiation therapy. Unfortunately, forty percent of patients have a poor or absent response and novel predictive biomarkers are demanding. For the first time, we apply a novel peptidomic methodology and analysis in rectal cancer patients treated with preoperative chemoradiotherapy. Circulating peptides (Molecular Weight <3 kDa) have been harvested from patients' plasma (n= 33) using nanoporous silica chip and analyzed by Matrix-Assisted Laser Desorption/Ionization-Time of Flight mass spectrometer. Peptides fingerprint has been compared between responders and non-responders. Random Forest classification selected three peptides at m/z 1082.552, 1098.537, and 1104.538 that were able to correctly discriminate between responders (n= 16) and non-responders (n= 17) before therapy (T0) providing an overall accuracy of 86% and an area under the receiver operating characteristic (ROC) curve of 0.92. In conclusion, the nanoporous silica chip coupled to mass spectrometry method was found to be a realistic method for plasma-based peptide analysis and we provide the first list of predictive circulating biomarker peptides in rectal cancer patients underwent preoperative chemoradiotherapy. This article is protected by copyright. All rights reserved
    Journal of Cellular Physiology 12/2014; 230(8). DOI:10.1002/jcp.24894 · 3.84 Impact Factor
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    • "It should be noted that the objective of our study was not to decipher the proteome of urine OvCa and thus, it is not surprising that more proteins were not overlapping with Fredolini et al. Another study, by Lopez et al., looked at the serum proteome of 110 healthy individuals and 453 patients with OvCa (including stages I-IV) [30]. The authors identified 160 proteins that were overexpressed in ovarian cancer compared to healthy controls. "
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    ABSTRACT: Background Ovarian cancer (OvCa) is the most lethal gynecological malignancy. The emergence of high-throughput technologies, such as mass spectrometry, has allowed for a paradigm shift in the way we search for novel biomarkers. Urine-based peptidomic profiling is a novel approach that may result in the discovery of noninvasive biomarkers for diagnosing patients with OvCa. In this study, the peptidome of urine from 6 ovarian cancer patients and 6 healthy controls was deciphered. Results Urine samples underwent ultrafiltration and the filtrate was subjected to solid phase extraction, followed by fractionation using strong cation exchange chromatography. These fractions were analyzed using an Orbitrap mass spectrometer. Over 4600 unique endogenous urine peptides arising from 713 proteins were catalogued, representing the largest urine peptidome reported to date. Each specimen was processed in triplicate and reproducibility at the protein (69-76%) and peptide (58-63%) levels were noted. More importantly, over 3100 unique peptides were detected solely in OvCa specimens. One such promising biomarker was leucine-rich alpha-2-glycoprotein (LRG1), where multiple peptides were found in all urines from OvCa patients, but only one peptide was found in one healthy control urine sample. Conclusions Mining the urine peptidome may yield highly promising novel OvCa biomarkers.
    Clinical Proteomics 06/2014; 11(1):23. DOI:10.1186/1559-0275-11-23
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    • "Differential expression of these proteins in sera was also confirmed by western blot and ELISA. Lopez et al. [47] set-up a workflow using carrier protein-bound affinity enrichment of serum samples directly coupled with MALDI/TOF. Subsequent tandem MS analysis defined the serum protein-bound peptides' sequence. "
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    01/2013; 2013(1):125858. DOI:10.1155/2013/125858
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