MSACL 2017 US Abstract

Evaluation of Quantitative Proteomic Methods

Clark Henderson (Presenter)
University of Washington Medical Center

Bio: I am currently a postdoctoral fellow in Dr. Andrew Hoofnagle’s laboratory at the University of Washington Medical Center in Seattle. Prior to joining Dr. Hoofnagle’s laboratory, I received my PhD in Biophysics from the University of California Davis in 2013. While at UC Davis, I developed lipidomic methodologies using LC-MS/MS to elucidate the critical membrane bilayer components in yeast that allow them to grow and thrive in a high ethanol environment. While in Dr. Hoofnagle’s laboratory, I have developed a number of discovery and targeted proteomic methods using nano-flow chromatography coupled on-line to a Q Exactive Plus mass spectrometer. For several of these assays, I assisted in the development of a work flow to transition these methods to normal flow LC-MS/MS to allow for high-throughput analysis of large sample sets from epidemiological studies, as well as patient samples.

Authorship: Clark M. Henderson
University of Washington Medical Center

Short Abstract

Along with peptide selection and proper development of calibration curves, other experiments should be performed to evaluate the analytical performance of a quantitative proteomic method. This session will discuss some of the fundamental experiments that should be used to evaluate an LC-MS/MS method for reliable and robust quantification of proteins.

Long Abstract

The hallmarks of any reliable quantitative biomarker assay include imprecision, linearity, specificity and stability. In bottom-up proteomics, quantification of proteins relies upon judicious selection of peptides that act as surrogates for the protein of interest. These peptides can be used to quantify proteins using properly constructed calibration curves using proteomic methods that have been rigorously evaluated to determine if they have the potential to reliably and specifically detect these peptides. These experiments include determining assay reproducibility, potential interferences and sample collection and stability. While method evaluation may seem a daunting task, the number of experiments and samples required for elucidation a method’s performance and potential for clinical use are relatively few.

By the end of the session users should be able to:

1. Describe a general proteomic method workflow.

2. Define intra-day, inter-day and total variability.

3. Describe how matrix effects can affect protein quantification.

4. Describe sources of bias in an assay and how they can affect protein quantification.

5. Describe the minimum number of experiments that should be performed to demonstrate a robust and reliable method for quantification of proteins using LC-MS/MS.


References & Acknowledgements:


Financial Disclosure

DescriptionY/NSource
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SalaryyesUniversity of Washington
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IP Royalty: no

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