Candice Johnson (Presenter)
National Institute for Standards and Technology
Authorship: Candice Johnson, Ashley Beasley-Green, Karen Phinney
National Institute of Standards and Technology, Biomolecular Measurement Division, 100 Bureau Drive, MS 8314, Gaithersburg, MD 20899
| Short Abstract The emergence of mass spectrometry (MS) based proteomic platforms utilized in biochemical and biomedical research has increased the need for high quality MS measurements. In an attempt to standardize MS metrics for the proteomics community, Saccharomyces cerevisiae yeast protein extract has been used in several successful inter-laboratory studies that demonstrated the yeast proteome as a suitable proteomic reference material (RM). To further improve on MS performance materials, we are developing a tryptic digest of yeast protein extract (RM 8313: Digested Yeast Protein Extract) to function as a quality control material to benchmark the performance of MS-based instrumentation. RM 8313 will facilitate the intra- and inter-laboratory assessment of MS-based instrument performance and measurement quality by eliminating the variability in laboratory protein digestion protocols. |
Long Abstract
The emergence of mass spectrometry (MS) based proteomic platforms as a prominent technology utilized in biochemical and biomedical research has increased the need for high quality MS measurements. The growing interest in the application of proteomics in clinical laboratory assays has also increased the need for insuring reproducibility of MS measurements and instrument performance. Reference Material (RM) 8323: Yeast Protein Extract, an intact yeast protein material, was previously designed to assess the pre-analytical and analytical variables that influence measurement quality in proteomics-based workflows. However, due to the overwhelming interest in a highly complex uniformly digested peptide material, we are developing RM 8313: Digested Yeast Protein Extract as a performance quality control material to benchmark the performance of MS-based instrumentation. This peptide reference material will further facilitate the intra- and inter-laboratory assessment of MS-based instrument performance, measurement quality, and reproducibility by eliminating the variability in laboratory protein digestion protocols.
Saccharomyces cerevisiae is an attractive organism for a MS-based performance reference material because of the proteome complexity, extensive dynamic range of the proteome, and reduced production costs. The extracted yeast protein will be digested with trypsin to generate tryptic yeast peptides. To optimize the digestion reaction, several grades of trypsin (i.e. sequencing grade and non-sequencing grade trypsin) will be evaluated to determine the most cost effective enzymatic material for the large scale digestion of the extract yeast protein. In addition to the trypsin material, the following parameters of the conventional tryptic digestion protocol will be evaluated to determine the optimal conditions: digestion reaction temperature and duration; and enzyme-to-substrate ratio. To determine the optimal digestion conditions, thirty yeast candidate proteins will be selected on the basis of abundance, molecular weight, charge (pI) and hydrophobicity and the digestion efficiency will be determined by the degree of sequence coverage of the selected protein metrics. The homogeneity of the digested yeast protein extract will be evaluated using various LC-MS platforms and the MS measurements will be quantitatively assessed via the NIST MS Quality Control (MSQC) performance metrics.
RM 8313 will support MS measurement quality for protein characterization in complex protein mixtures, such as those used in clinical proteomics. Once available publicly, RM 8313 will provide research and clinical laboratories with a standard complex digested protein mixture to benchmark and improve their MS protocols and assess instrument performance.
References & Acknowledgements:
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