Multiple Reaction Monitoring Cubed for Protein Quantification at the Low Nanogram/Milliliter Level in Nondepleted Human Serum
Mass spectrometry-based strategies for the quantification of low-abundance putative protein biomarkers in human blood currently require extensive sample fractionation steps which hamper their implementation in a routine and robust way across clinical laboratories. We demonstrate that a technique usi...
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Published in | Analytical chemistry (Washington) Vol. 81; no. 22; pp. 9343 - 9352 |
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Main Authors | , , , , , , , |
Format | Journal Article |
Language | English |
Published |
Washington, DC
American Chemical Society
15.11.2009
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Subjects | |
Online Access | Get full text |
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Summary: | Mass spectrometry-based strategies for the quantification of low-abundance putative protein biomarkers in human blood currently require extensive sample fractionation steps which hamper their implementation in a routine and robust way across clinical laboratories. We demonstrate that a technique using MS3 reconstructed chromatograms on a signature of secondary ions issued from a trapped primary product ion, termed multiple reaction monitoring cubed (MRM3), enables targeting protein biomarkers in the low nanogram/milliliter range in nondepleted human serum. The simple two-step workflow is based on a trypsin proteolysis of whole serum (100 μL) followed by enrichment of targeted proteotypic peptides on a solid phase extraction column using mixed-cation exchange resin. MRM3’s fidelity of peak detection extends the dynamic range and limit of quantitation (LOQ) of protein biomarkers to the low nanogram/milliliter range, corresponding to a concentration that is 106-fold lower than the concentration of the most abundant proteins in serum. The power of the MRM3 method is illustrated by the assay of prostate specific antigen in nondepleted human sera of patients. The results correlate well with the established method for determining PSA levels in serum, i.e., enzyme-linked immunosorbent assay (ELISA) tests. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0003-2700 1520-6882 |
DOI: | 10.1021/ac901447h |