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J Proteome Res. 2010 Jan;9(1):333-40. doi: 10.1021/pr900658d.

Development of high-throughput chemical extraction techniques and quantitative HPLC-MS/MS (SRM) assays for clinically relevant plasma proteins.

Author information

1
Quotient Bioresearch Ltd, Newmarket Road, Fordham, Cambridgeshire CB7 5WW, United Kingdom.

Abstract

The clinical application of targeted plasma protein analysis by selective reaction monitoring of peptides using LC-MS/MS requires the development of robust, inexpensive protein extraction techniques with the potential for high-throughput applications. We present the development of a novel mixed-mode solid phase extraction (SPE) technique for the removal of high abundance and high molecular weight proteins from plasma. This technique, coupled with fused-core HPLC-MS/MS analysis is compared to a previously developed extraction method to study a range of proteins in plasma, including routinely measured biomarkers of growth hormone action. To further validate this technique, it was used for the quantification of insulin-like growth factor I (IGF-I) levels and compared to a state-of-the-art immunoassay on a fully automated analyzer. Clinical reference materials were applied for method development to allow for further interlaboratory comparisons. The LC-MS/MS approach quantified IGF-I in plasma with an accuracy that is within the guidelines for macromolecular assays in a regulated laboratory environment. Furthermore, IGF-I levels determined using the SPE and ACN methods with LC-MS/MS analysis correlated well with the immunoassay results. This demonstrates the applicability of mixed-mode SPE coupled with fused-core HPLC-MS/MS to quantify plasma proteins with results suitable for clinical applications.

PMID:
19921931
DOI:
10.1021/pr900658d
[Indexed for MEDLINE]

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