Posters Enhancement of Biomarker
نویسندگان
چکیده
Nadeem Ali-Khan, Hsin-Yao Tang, Lynn Echan and David W. Speicher The wide range of protein concentrations found in human serum poses a considerable challenge with regard to proteomic analysis, as it restricts the detection and quantitation of low abundance proteins, including cancer biomarkers. A critical component of several alternative protein profiling strategies designed to detect lower abundance proteins in serum is a high resolution fractionation method developed in our laboratory. This micro-scale solution isoelectrofocusing (MicroSol-IEF) method is now available from Invitrogen Corp. as the Zoom-IEF Fractionator. This simple, inexpensive and efficient device is used to separate proteins according to their isoelectric points (pI) into up to seven fractions, thereby reducing sample complexity and consequently simplifying and enhancing the detection capacities of downstream analytical methods, including top-down approaches such as 2-D gels and bottom-up LC/LC-MS/MS approaches. We previously reported the advantages of this approach for analysis of human breast cancer cell extracts. In addition we have used the device to fractionate human and mouse serum either with or without major protein depletion. We are currently optimizing fractionation of human serum for biomarker discovery using Micro-Sol IEF by systematically evaluating the effects of different experimental conditions on serum proteome fractionation. Parameters examined include maximum sample loads with and without major protein depletion, optimal focusing conditions, and effects of various buffer formulations. Recent results show that either MicroSol IEF prefractionation or major protein depletion can substantially enhance our ability to detect lower level proteins but the combination of these two steps in tandem provide limited synergy. In contrast, a new 4-D separation method developed in our laboratory shows great promise for detection of low abundance serum proteins. This protein array-pixelation method utilizes depletion of the six most abundant serum proteins, MicroSol IEF fractionation, 1-D gel electrophoresis and LC-MS/MS. Based upon initial results, it may be advantageous to fractionate serum proteomes into more than 5 to 7 pI range fractions.
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