Qin Fu (Presenter)
Cedars Sinai Medical Center
Bio: Qin Fu obtained her PhD degree in Genetics from University of Minnesota and post-doctoral training from University of California, San Francisco. She has broad research experiences in autoimmune, drug resistance, cardiovascular diseases and biomarker development and validation. Her interests are multiplex analysis (both antibody-based and mass spectrometry-based technologies) for biomarker targeted discovery and validation. Dr. Fu, was the biomarker development director at the Johns Hopkins University Bayview Proteomic Center. At Cedar Sinai Medical Center, Dr. Fu is the director of the high throughput protein quantification applying the immuno-assay and LC MSMS platforms
Authorship: Qin Fu, and Jennifer E. Van Eyk
Advanced Clinical Biosystems Institute, Heart institute, Cedars Sinai Medical Center, Los Angeles, CA, USA
| Short Abstract Precise protein quantification is essential for both discovery and targeted protein workflows. It requires reproducible protein proteolysis that consistently generates the same peptide in each sample type. Here, we have developed a robotic MS samples preparation workflow by using a Biomek NXP workstation. The workflow is completely hands free with flexible denaturation and proteolysis time for sample specific conditions (e.g. plasma, urine, IPSC, and tissue). It improved reproducibility and throughput by approximately 4 and 7 fold compared to manual processing. We will present expanded automation workflows, to assist in quality control (QC), digestion optimization, and digestion reproducibility testing for large scale LC-MS based analysis. |
Long Abstract
Mass spectrometry (MS) based protein and peptide quantification has become an increasingly applied bio-analytical tool in basic and in pre-clinical research. For new biomarker candidate validation, a large number of samples are needed and reproducible sample preparation is a critical element of a robust and quantitative assay. Currently, it is the bottleneck that limits throughput. Manual sample processing is highly time-consuming, labor intensive and low throughput. We developed a workflow using the Biomek NXP automation workstation to carry out the basic steps of protein denaturation, reduction, alkylation and specific enzymatic (usually trypsin) digestion of samples in 96 well format.
There are many advantages in using robotic sample preparation for proteomic analysis. The first advantage is to improve the throughput and save time in comparison to the manual operation. The second advantage is to reduce analytic variation across large sample batches by reducing the imprecision introduced when performing multistep manual operations (we see 2-4 fold of reduction in %CV). Third advantage is that highly trained staff is freed to work on other aspects of projects. Generation of tryptic peptides in a digestion reaction is complicated by matrix and stoichiometry of a protein. We often see different tryptic fragments releasing with different kinetics from the same protein. Therefore, to quantitate specific tryptic fragments using MS, it is important to have good precision in the digestion process (such as denaturation time, temperature and enzymatic incubation time) for a large number of samples. The robotic sample process provides consistency in digestion, and hence increases the reaction precision. The last advantage is that the 96 well plate format allows for the establishment of a pipeline for digestion and quality control for a large scale proteomic analysis.
The workflow is completely hands free with flexible denaturation and proteolysis time for sample specific condition (e.g. plasma, urine, IPSC, and tissue). We present expanded automation workflows, to assist in quality control (QC), digestion optimization, and digestion reproducibility testing for large scale LC-MS based analysis.
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