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.
Authorship: Shenyan Zhang, Dawn Chen, Qin Fu, Mitra Mastali, Jennifer E. Van Eyk
Cedars Sinai Medical Center, Los Angeles, CA
| Short Abstract Chronic kidney disease (CKD) is a prevalent disorder that often remains undiagnosed until its most severe renal and cardiovascular complications arise. Although biomarkers for CKD are few, Apolipoprotein-L1 variants have been directly linked to an increased risk for this disease. In order to facilitate its routine detection, we built and evaluated a quantitative LC-MS/MS assay for Apo-L1 that is compatible with automated high-throughput upstream sample preparation capable of processing 96 samples in under 6 hours. |
Long Abstract
• Introduction
Chronic kidney disease (CKD) is a prevalent disorder that often remains undiagnosed until its most severe renal and cardiovascular complications arise. Although biomarkers for CKD are few, Aplipoproteino-L1 variants have been directly linked to an increased risk for this disease. In order to facilitate its routine detection, we built and evaluated a quantitative LC-MS/MS assay for Apo-L1 that is compatible with automated high-throughput upstream sample preparation.
• Methods & Results
Our MRM assay utilizes recombinant Apo-L1, spiked into chicken serum, to produce a standard curve based on area ratios relative to a heavy peptide. Our method was sensitive to remnant amounts of Apo-L1 contained in commercial apolipoprotein-depleted plasma. These endogenous interferences were eliminated with a chicken serum matrix. We did not detect any carryover between the highest standard injection and its adjacent blank, obviating the need for blanks between successive sample injections.
Our LC-MS method resulted in an LOQ of 0.281ug/mL (CV 7.1%, accuracy 116.7%), and a standard curve with a linear range spanning four orders of magnitude (R2=0.987). Method QC, performed by spiking three dilutions of recombinant Apo-L1 into chicken serum. The 128-fold dilution resulted in CV’s of 16% (intra-day) and 11.5% (inter-day); the 10-fold dilution resulted in CV’s of 6.1% (intra-day) and 5.7% (inter-day); the 4-fold dilution resulted in CV’s of 7.8% (intra-day) and 9.5% (inter-day). Study QC was performed in both lipoprotein-depleted serum and in pooled human serum, yielding intra-day CV’s from 4 replicates of 12.6% and 8.7%, and inter-day CV’s from 8 replicates of 11.7% and 9.8%, respectively.
In order to increase the throughput, sample preparation was performed on a Biomek Automated workstation from Beckman Coulter, which accomplished reduction, alkylation, and digestion of samples on a 96 well plate in less than 6 hrs. In addition to decreasing the preparative time, the automated workflow resulted in an assay reproducibility with a CV of 11% for an entire plate, where only 23% could be achieved by manually processing.
• Conclusions
In sum, we built and evaluated an LC-MS/MS based quantitative assay for Apo-L1, and an automated digestion protocol for clinical plasma samples was optimized and transferred to a robot workstation. Now we are expanding this work to include a multiplexed quantitative assay that encompasses the major Apo-L1 variants associated with CKD.
References & Acknowledgements:
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