Development of a Chip/Chip/SRM Platform Using Digital Chip Isoelectric Focusing and LC-Chip Mass Spectrometry for Enrichment and Quantitation of Low Abundance Protein Biomarkers in Human Plasma
| Autor: | William S. Hancock, Agnes Rafalko, Shujia Dai, Marina Hincapie, Barry L. Karger |
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| Rok vydání: | 2011 |
| Předmět: |
Male
Proteomics musculoskeletal diseases Analyte Molecular Sequence Data Protein Array Analysis Mass spectrometry Biochemistry Mass Spectrometry Article Limit of Detection Animals Humans Trypsin Sample preparation Amino Acid Sequence Biomarker discovery Immunosorbent Techniques Detection limit Chromatography Chemistry Isoelectric focusing Selected reaction monitoring Reproducibility of Results Blood Proteins General Chemistry Prostate-Specific Antigen Peptide Fragments Triple quadrupole mass spectrometer Linear Models Cattle Female Isoelectric Focusing human activities Biomarkers Chromatography Liquid |
| Zdroj: | Journal of Proteome Research. 11:808-817 |
| ISSN: | 1535-3907 1535-3893 |
| Popis: | Protein biomarkers are critical for diagnosis, prognosis, and treatment of disease. The transition from protein biomarker discovery to verification can be a rate limiting step in clinical development of new diagnostics. Liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM MS) is becoming an important tool for biomarker verification studies in highly complex biological samples. Analyte enrichment or sample fractionation is often necessary to reduce sample complexity and improve sensitivity of SRM for quantitation of clinically relevant biomarker candidates present at the low ng/mL range in blood. In this paper, we describe an alternative method for sample preparation for LC-SRM MS, which does not rely on availability of antibodies. This new platform is based on selective enrichment of proteotypic peptides from complex biological peptide mixtures via isoelectric focusing (IEF) on a digital ProteomeChip (dPC) for SRM quantitation using a triple quadrupole (QQQ) instrument with an LC-Chip (Chip/Chip/SRM). To demonstrate the value of this approach, the optimization of the Chip/Chip/SRM platform was performed using prostate specific antigen (PSA) added to female plasma as a model system. The combination of immunodepletion of albumin and IgG with peptide fractionation on the dPC, followed by SRM analysis, resulted in a limit of quantitation of PSA added to female plasma at the level of ∼1-2.5 ng/mL with a CV of ∼13%. The optimized platform was applied to measure levels of PSA in plasma of a small cohort of male patients with prostate cancer (PCa) and healthy matched controls with concentrations ranging from 1.5 to 25 ng/mL. A good correlation (r(2) = 0.9459) was observed between standard clinical ELISA tests and the SRM-based assay. Our data demonstrate that the combination of IEF on the dPC and SRM (Chip/Chip/SRM) can be successfully applied for verification of low abundance protein biomarkers in complex samples. |
| Databáze: | OpenAIRE |
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