Dawn Z Chen (Presenter)
Cedars-Sinai Medical Center
Bio: Dr. Dawn Chen, current an assistant professor at Cedar Sinai Medical Center at Los Angeles and also an Adjunct Assistant Professor at UCLA David Geffen School of Medicine. My primary interest is to translate the cutting edge mass spectrometry proteomics discoveries and validation technologies into high throughput medical and clinical researches and applications through the cross function collaborations with clinicians. . My research covers the development of quanlitative and quantitative mass spectrometry pipelines for small molecular metabolites and large biomolecules, such as protein and their diseases induced forms (PTMs) using high resolution and multiple reaction monitoring (MRM) mass spectrometers. I had Ph.D. in analytical chemistry from University of Alberta, Canada. Prior to Cedar Sinai, I worked as a Senior R & D Scientist in Quest Diagnostics Nichols Institute and also worked on proteomics researchers at Johns Hopkins.
Authorship: Dawn Z Chen, Weston Spivia, Shenyan Zhang, Jason Lee, Qin Fu, Jennifer E. Van Eyk
Advanced Clinical Biosystems Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA
| Short Abstract In the translation of building a clinical LC-MS/MS assay on MRM peptides in compliance with high throughput for patients, the initial small-scale experiments have to demonstrate the traceability along the workflow. Apolipoprotien L1 (ApoL1), an HDL lipoprotein with three genetic variants, is used as a model protein to assess the strategies of standards, matrix effects, and the quality controls for the enzymatic digestion. A simple, high-throughput, quantitative LC-MS/MS ApoL1 assay has been successfully developed, validated and then applied to 235 participants to examine the associations between chronic kidney disease with ApoL1 plasma levels and its genetic variants. |
Long Abstract
Introduction: Clinical applicability of a quantitative measurement of proteins based on their peptide mass spectrometry performance is critical for the success of the assay. Starting from small-scale laboratory developed experiments, the final stage method should be able to be in compliance with the high throughput capability and demonstrated the traceability along each important phase of the entire workflow to overcome the variety of human blood samples. However, the dynamic range of the human plasma and matrix effect greatly challenge the introduction of the standards and quality controls during the assay development. In this report, we use Apolipoprotien L1 (ApoL1) protein as a model protein to assess the strategies of the standards, effects of the matrix, and the most important, the quality control for the enzymatic digestion before, during and after the use of the trypsin.
ApoL1 is a lipoprotein component associated with HDL (High-density lipoprotein). Its secreted form circulating in the blood settles as HDL particle complex containing other proteins, such as Apolipoprotein A1 and haptoglobin-related protein. How to measure the ApoL1 reflects the true amount of endogenous level has always been challenging. Besides wildtype, two genetic coding variants G1 and G2 have been recently identified with relevance to human phenotypes and have been shown to associate with chronic kidney disease , especially among African populations (1). Many African Americans descendants consequently have a high prevalence of APOL1 risk alleles as well as APOL1 associated kidney diseases. In order to gain further understanding of the etiology of ApoL1 related kidney diseases, the quantification of ApoL1 protein total concentration and its allelic variants (isoforms) could provide more accurate epidemiological information to assess the risk of chronic kidney disease (CKD) among the American Populations.
Our discovery based on high-resolution tandem mass spectrometry from 36 patients found ApoL1 is among the top prognostics risk list for CKD. However, the commercial available immunoassay based experiments for total ApoL1 measurement completed in our laboratory did not show persuasive results on standards for us to continue our pursuit. Different cross-reactive antibodies had demonstrated variable assay results, either with limited linear range or un-reproducible data. On the other hand, there are no antibodies available to distinguish all the three variant forms. In contrast, liquid chromatography tandem Mass Spectrometry (LC-MS/MS) quantitation of ApoL1 based on multiple reaction monitoring (MRM) of tryptic digested peptides became our only choice and could be of significant use to provide a highly accurate alternative to immunoassays for measuring the total ApoL1 circulation concentration and highly specific assessment for allelic isoforms.
Methods: We aimed to develop a quantitative assay to determine the circulating level of ApoL1 and also discriminate its three variant isoforms. First, ApoL1 was pretreated, reduced and alkylated with other thousands proteins in plasma; and then the subsequent selection and resolution of the peptides was achieved by on line desalting LC columns coupled to a 6500 quadrupole ion trap MS (AB, USA). Based on our findings and the literatures(2), five proteolysis signature peptides (two common peptides represent the total apoL1 proteins and three peptides cover the wildtype, G1 and G2 isoforms) from ApoL1 and their stable isotopic labeled peptides and then the associated transitions have been chosen for the quantitation of ApoL1 total concentration and its potential CKD genetic variants in plasma by LC-MS/MS. Recombinant human wildtype apoL1 was used as a calibrator for total ApoL1 protein and wildtype while synthesized G1 and G2 peptides were spiked in non human plasma matrix for the calibrants of endogenous G1 and G2 measurements. Synthesized stable isotopic labeled (13C/15N) common peptides, wildtype, G1 and G2 peptides were used and compared as internal standards.
Results: During the development, ApoL1 recombinant protein, ApoL1 wing peptide and single tryptic digested non-cleavage peptides were evaluated against each other as the calibrants respectively according to the nature of the peptides and the need for quality control. Both external and internal calibration strategies were examined to investigate the effects of background matrix. In addition, the use of quality control peptides and proteins, and the introduction of internal standards at different time points and their effect on digestion efficiency and completion are also addressed. The assessment from different peptides associated to their sequence and spaces gives us better insight of the dynamic proteolysis process and guidelines for monitoring the assay quality. All the investigations and the recommendations provide necessary indications at different stages of the workflow and thus lead to a high quality assay to meet the clinical requirements, such as CLIA guidelines.
Conclusions: In conclusion, a simple, high-through, quantitative LC-MS/MS ApoL1 assay traceable with the quality controls indicators has been successfully developed and validated with linear range of 10-500 nM, correspond to the reported range(2) with coefficient of variation of 0.5-14.8% and 3.7-16.3% in intra-assay and interassay respectively. The assay has been successfully applied to 235 participants in the Atherosclerosis Risk in Communities study (ARIC) end-stage renal disease (ESRD) case-control study to examine the associations between chronic kidney disease with ApoL1 plasma levels and their ApoL1 genetic variants.
References & Acknowledgements:
1. Genovese G, Friedman DJ, Ross MD, Lecordier L, Uzureau P, Freedman BI, Bowden DW, Langefeld CD, Oleksyk TK, Uscinski Knob AL, Bernhardy AJ, Hicks PJ, Nelson GW, Vanhollebeke B, Winkler CA, Kopp JB, Pays E, Pollak MR. Association of trypanolytic ApoL1 variants with kidney disease in African Americans. Science 2010;329:841-5.
2. Zhou H, Hoek M, Yi P, Rohm RJ, Mahsut A, Brown P, Saunders J, Chmielowski RA, Ren N, Shuster D, Southwick K, Ayanoglu G, Gorman D, Laface D, Santino S, Conway J, Liu Z, Cully D, Cleary M, Roddy TP, Blom D. Rapid detection and quantification of apolipoprotein L1 genetic variants and total levels in plasma by ultra-performance liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom 2013;27:2639-47.
The authors would like to thank Dr. Josef Coresh, Adrienne Tin, Casey Rebholz at Department of Epidemiology, Johns Hopkins University for the study subjects and their participation, for the help provided accessing the ARIC-ESRD case-control samples. We also thank members of the Advanced Clinical Biosystems Research Institute and the heart institute at Cedar-Sinia Medical Center for excellent technical assistance and supports.
This work was supported by NIDDK grants U01 DK085689-0 “Proteomic Biomarkers of Chronic Kidney Disease”.
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