Robert English (Presenter)
Shimadzu Scientific Instruments
Bio: Scott Kuzdzal received his Ph.D. in Analytical Chemistry in 1997 from the University of California at Riverside. He served as a postdoctoral fellow at the Johns Hopkins University School of Medicine, where he co-founded the Johns Hopkins Center for Biomarker Discovery with Dr. Daniel Chan. He has a strong clinical chemistry background (former Director of Toxicology at Johns Hopkins Medical Institutions). He has published biomarkers for pancreatic cancer, Alzheimer's disease, Duchenne’s Muscular Dystrophy and ovarian cancer. Scott has lectured at continuing medical education courses at Johns Hopkins Hospital, George Mason University/INOVA Fairfax Hospital and the NIH, as well as conferences worldwide. He has co-authored several proteomics book chapters and has multiple patents. Scott currently serves as General Manager of Marketing at Shimadzu Scientific Instruments.
Authorship: Jeremy Post(1), Balamurugan Packialakshmi(2), Narayan Rath(3), Jackson Lay Jr.(2), Rohana Liyanage(2), Scott Kuzdzal (1), Chris Gilles(1) and Robert English (1)
(1) Shimadzu Scientific Instruments, (2) University of Arkansas, Fayetteville, AR, and (3) USDA, Fayetteville, AR.
| Short Abstract LC-MS/MS is a robust platform for quantifying targeted proteins in complex matrices. Typically, quantitative protein assays are developed using comparable synthetic or expressed protein standards to establish method conditions appropriate for measurement of clinical samples. Here a mixture of common proteins was used to develop a retention time library for a set UHPLC method, which Skyline utilized to estimate peptide retention times as well as MRM transitions and instrument parameters from known sequences effected in chicken plasma by Salmonella endotoxins. Utilizing key instrument and software capabilities, a targeted method was quickly developed without the use of comparable standards. |
Long Abstract
LC-MS/MS is a robust platform for quantifying targeted proteins in complex matrices. Typically, quantitative protein assays are developed using comparable synthetic or expressed protein standards to establish method conditions appropriate for measurement of clinical samples. Here a mixture of common proteins was used to develop a retention time library for a set UHPLC method in place of using protein standards.
Skyline software is a freely-available, open source Windows client application that aids in building selected reaction monitoring (SRM)/ multiple reaction monitoring (MRM) and targeted data dependent quantitative methods and analyzing the resulting mass spectrometer data [1]. Skyline was utilized to estimate peptide retention times as well as MRM transitions and instrument parameters from known sequences effected in chicken plasma by Salmonella endotoxins. The experimental workflow included building a retention time library from a known tryptic peptide mixture using UHPLC QQQ, optimizing the methods using Skyline software, refine the retention time (RT) curve to a 99% confidence level, and then use the RT curve to predict retention times for the target protein using the Skyline QQQ method.
To test protein expression differences in Chicken plasma, broiler chickens were treated with Salmonella endotoxins and after 24 hours, the plasma was prepared from the control and treated chickens. The method compares proteins quantified by QQQ protein data to data dependent analysis data, gel electrophoresis, and ELISA data. LC-MS/MS data dependent analysis of plasma protein extracts following abundant protein depletion showed four proteins to be differentially expressed, three up regulated and one down regulated. From other studies, it was observed that more proteins in plasma are expected to be regulated under stress conditions, such as ovotransferin and apolipoprotiens. Some of the plasma proteins that were monitored are heparanese, alpha 1-acid glycoprotiein and cathelidicin 2, to name a few. By utilizing key instrument and software capabilities, a targeted method was quickly developed without the use of comparable standards for a series of peptides.
References & Acknowledgements:
1. Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics (2010) 26 (7): 966-968. Brendan MacLean1, Daniela M. Tomazela1, Nicholas Shulman1, Matthew Chambers2, Gregory L. Finney1, Barbara Frewen1, Randall Kern3, David L. Tabb2, Daniel C. Liebler4 and Michael J. MacCoss1. 1 Department of Genome Sciences, University of Washington, Seattle, WA 98195, 2 Department of Biomedical Informatics, Vanderbilt University, Nashville, TN 37232, 3 Microsoft Corporation, Redmond, WA 98195 and 4 Department of Biochemistry, Vanderbilt University, Nashville, TN 37232, USA.
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Shimadzu |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |