Shenyan Zhang (Presenter)
Cedars Sinai Medical Center
Bio: Dr. Shenyan Zhang is a postdoctoral scientist in Cedars Sinai Medical Center (CSMC), Los Angeles. She got her bachelor degree of Applied Chemistry in Tianjin University, China，then she joined Beijing Institute of Genomics, Chinese Academy of Science where she received her PhD in biochemistry and molecular biology. Now she worked at Heart insititute of CSMC, and main research area is clinical proteomics, including high throughput quantitation of protein biomarkers by MRM and SWATH and intact protein analysis in plasma.
Authorship: Shenyan Zhang, Koen Raedschelders, Jennifer Van Eyk
Advanced Clinical Biosystems Research Institute, Barbra Streisand Women’s Heart Center, Cedars-Sinai Heart Institute, Cedar-Sinai Medical Center, Los Angeles
| Short Abstract B-type Natriuretic Peptide (BNP) is a biologically active circulating hormone whose concentration is routinely used in the diagnosis of heart failure. Multiple enzymes cleave BNP before it binds its natriuretic peptide receptors. We coupled capillary electrophoresis to high-resolution mass spectrometry to profile BNP proteolysis in plasma with a view to assessing its potential relation to heart failure severity. Our method relies on electrokinetic introduction of minimally processed plasma samples to monitor the dynamic generation and breakdown of five BNP proteoforms. Combined with multisegment injection, this method can produce a multi-point BNP proteolysis profile of one patient within an hour. |
Long Abstract
Introduction
B-type Natriuretic Peptide (BNP) is one of the primary biomarkers whose quantitative analysis is used clinically in the diagnosis and risk stratification of heart failure. Plasma BNP levels reflect a dynamic equilibrium, in which it is synthesized and secreted by ventricular cells but also actively processed and degraded in plasma by at least 3 peptidases, neutral endopeptidase (NEP), dipeptydilpeptidase IV (DPPIV), and insulin degrading enzyme (IDE). Quantitative accuracy can be undermined by plasma storage conditions and storage time. Furthermore, routine clinical BNP analysis is performed by immunoassays that are by their nature incapable of distinguishing between cleaved or alternatively modified proteoforms. The overarching goal was to develop a method capable of reproducibly analyzing BNP and its cleavage products in plasma.
Challenges
In order to avoid skewing the BNP proteoform profiles by disfavoring or eliminating potential proteoforms during sample preparation, the method must involve intact protein analysis. Clinical BNP concentrations range between 100-400pg/uL; cleavage proteoforms require further sensitivity estimated in the sub-ng/uL range. Sensitivity is constrained by the complexity and broad dynamic range of inherent to plasma samples in the absence of enrichment and extensive sample processing that may overcome the complexity and broad dynamic range of plasma samples can introduce errors, can artificially skew profiles, and may be incompatible for routine clinical analysis.
Approach
Our sample preparation involves a 10-minute centrifugation of plasma samples through a 0.22um spin filter. Filtered plasma is spiked with 250ng/uL of BNP1-32 whereupon proteolysis is initiated. This solution is directly sampled for CESI-MS analysis at prescribed timepoints. The alkaline pI values of the five detected BNP cleavage proteoforms facilitates an electrokenetic injection strategy, which theoretically endows the sampling step with an embedded level of selectivity that favors BNP proteoforms and disfavors the cleavage enzymes.
Instrumentation
Capillary electrophoresis was performed using a CESI 8000 Plus system (SCIEX), with neutral coated cartridges (SCIEX) to minimize intact protein adsorption onto the inner silanol capillary surface. Mass Spectrometry was performed using a Q-Exactive Plus instrument (Thermo). Data analysis was performed using Biopharmafinder (Thermo) for identification and Tracefinder (Thermo) for quantitative analysis.
Results
Our method achieved an average intra-run CV of 7% (N=4) and an average of inter-run CV is 21.6% (N=5). We are able to produce a 5-point calibration curve in a single multi-segment injection run, from 50ng/uL to 250ng/uL, in with each concentration achieved a CV below 15% (N=3) and accuracy ranging from 89% to 130%.
When using an artificial plasma control matrix, BNP1-32 was stable with very little BNP 3-32 detected up to 900min of incubation. When spiked in human heparin plasma, BNP1-32 was rapidly turned into BNP3-32 with a slope 2e4 times higher than in artificial plasma and 28 times higher than in the presence of protease inhibitor (Pierce). Early qualitative assessment of cleavage profiles shows clear differences between freeze-thawed plasma, plasma stored for 24hr at room temperature, plasma stored at 4 degrees, and directly analyzed plasma.
Applying MSI to the same sample can provide a rapid profile with five successive timepoints. In this mode, sample preparation steps require 15 minutes. Sample incubation with 250ng/ul BNP1-32 requires another 15 mins but can be automated and temperature controlled in the CE autosampler. Finally, samples can be electrokinetically injected every 3 mins for a total 5 injections, requiring another 15. Finally, CE-MS analysis followed by another 15 minutes of CE time. This one-hour assay was used to compare profiles from plasma sampled by heparin and EDTA storage tubes. BNP3-29 was only detected within an hour in plasma from heparin tubes. Detection of BNP3-29 in plasma from EDTA tubes requires longer incubation times, since the activity of IDE was partially inhibited by the chelation of EDTA and its cofactor Zn2+.
For clinical cohort study, our goal is a more comprehensive profile of proteoforms by extending analysis time. In order to increase throughput, multiple samples need to be analyzed in one run. This approach requires both external QC and internal QC for longer experiment period. We ran five injections of 250ng/uL BNP1-32 in water as a daily external QC. Preliminary AUC data showed that the intra-run CV of 3-day external QC are all below 4% and average of inter-run CV is about 37%. Internal QC analyses can be performed using the same internal standard solution introduced as the first MSI segment then followed by several sample injections in the same run. The CV for this internal standard QC, in the absence of labeled standard, is 26.7%(N=12).
Conclusions
We present a new method capable of dynamically profiling five BNP cleavage proteoforms as they are generated in minimally processed plasma samples. When combined with multi-segment injection (MSI), this method can be used to produce a multi-point BNP proteoform profile from a single plasma sample across the protracted timeframe of a single CESI-MS run. Similarly, MSI also allows for the parallel analysis of multiple plasma samples, with successive CESI-MS runs providing a time course for BNP proteoform profiling.
References & Acknowledgements:
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