Koen Raedschelders (Presenter)
Cedars Sinai Medical Center
Bio: I work in the Van Eyk lab in the Barbra Streisand Women's Heart Center at Cedars Sinai. Our focus is on proteomics, both in a basic discovery research sense and in terms of biomarker discovery and analysis for the clinical setting. My academic background focused on pharmacology and analytical chemistry during graduate school, which I integrated with projects that used proteomics to address biological problems during my postdoctoral work.
Authorship: Koen Raedschelders(1), Shenyang Zhang(1), Jennifer Van Eyk(1)
(1) Heart Institute, Cedars Sinai Medical Center, Los Angeles CA.
| Short Abstract B-type Natriuretic Peptide is a biologically active circulating factor whose concentration is routinely used in the diagnosis of heat failure. Although this analysis is routinely performed using ELISA platforms, these assays are incapable of differentiating between proteolytic forms of BNP. High-resolution mass spectrometry can easily resolve proteolytic variants with accurate quantitation, provided it is supplied with sufficiently resolved peaks. We are working on several different separation techniques that 1. Are compatible with mass spectrometry; 2. Can resolve intact BNP variants derived from a plasma matrix; 3. Can accommodate the large dynamic range with sufficient sensitivity. |
Long Abstract
B-type Natriuretic Peptide is a biologically active circulating factor whose concentration is routinely used in the diagnosis of heart failure. Although this analysis is routinely performed using ELISA platforms, these assays are incapable of differentiating between proteolytic forms of BNP. Because BNP variants may exhibit biologically distinct activities, and because N-terminal cleavage products of BNP can affect its binding properties, quantitative methods capable of differentiating between circulating BNP variants could reveal important clinical differences.
High-resolution mass spectrometry can easily resolve proteolytic variants with accurate quantitation, provided it is supplied with sufficiently resolved peaks. The subtle differences between BNP variants require intact protein analysis, which can be challenging given the complexity of the plasma matrix. The large concentration range of circulating BNP requires that analytical techniques display both high sensitivity (sub-pg/uL) and a large dynamic range.
Preliminary high-flow LC results using wide-pore columns, coupled to a Q-Exactive Plus MS, produced decently resolved peaks but with insufficient sensitivity (LOD 250pg/uL). Flow rate adjustment to 50uL/min with MRM acquisition improved the sensitivity by 3 orders of magnitude (~2pg/uL), but we were still unable to achieve sub-pg/uL sensitivity. Nanoflow LC-MS provided suffient sensitivity (fg/uL range), but this technique is incompatible with intact plasma.
Given the improved sensitivity resulting from decreasing flow rates, we sought an alternative low-flow separation strategy. Capillary electrophoresis offers a unique combination of picolitre sample injection, open tubular separations, and flow rates that can be tailored down to “No Flow”. Furthermore, the relatively high pKa of BNP is well suited to electrophoretic separations. Finally, recent advances in capillary coatings can help minimize protein adsorption to improve compatibility with plasma-derived samples. For these reasons, we are working towards a CE-MS based method for the analysis of BNP and its proteolytic variants.
Early results using a neutral-coated capillary show baseline electrophoretic resolution between BNP1-32 and BNP3-32, with sharp peak profiles. We are building on these successes by evaluating the sensitivity and reproducibility of this technique, and determining its suitability for plasma-derived samples.
References & Acknowledgements:
We would like to acknowledge and thank Sciex Separations for their helpful guidance with the CESI workflow
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