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Monitoring Mass Spec Instrument Performance and Method Development with Whole Cell Human and Yeast Protein Reference Extracts

Prior to joining industry Dr. Saveliev worked on various academic projects including epigenomics, DNA repair and recombination, mechanisms of radioresistance and circadian rhythm. Dr. Saveliev’s research efforts were supported by US government funding, and the results were published in the leading academic journals such as the EMBO Journal, PNAS, PLOS, and Genes & Development.

After joining Promega Corporation in 2004, Dr. Saveliev utilized his expertise to develop tools for life sciences including antibodies, tools for bioassays and affinity protein purification. Now, his product development efforts are focused on protein mass spectrometry sample preparation, and he has played the key role in building mass spectrometry portfolio at Promega including a comprehensive set of proteases and additional mass spec reagents. His current efforts are directed toward the development of solutions that help overcome the major challenges in protein mass spec sample analysis such as reproducibility and standardization.

  • Sergei Saveliev, Ph.D.

  • Sr. Research Scientist

  • Original Webinar Date: Tuesday, July 8, 2014

To address the need for a complex protein reference material suitable for mass spectrometry analysis, we developed intact and pre-digested whole cell yeast and human protein extracts. This webinar will outline the development and testing of these reference extracts with emphasis on LC/MS compatibility, material reproducibility and amenability to efficient mass spec analysis as measured by high numbers of peptide and protein identifications. Read full abstract below.

Abstract

The protein complexity of biological samples such as tissue, cells, and plasma places a high demand on mass spec analytical capability. Adequate monitoring of instrument performance for proteomics studies requires equally complex reference material, and whole cell protein extracts provide the necessary sample complexity. Preparation of such reference extracts is, however, a challenging task because the extracts must meet a number of design requirements:

  • compatibility with LC/MS (lack of detergents, salts, etc.)
  • high protein integrity (minimal level of protein degradation and non-biological PTMs)
  • compatibility with common sample preparation methods such as proteolysis, PTM enrichment and mass-tag labeling
  • reproducible lot-to-lot protein composition and quantity

To meet these requirements, we developed yeast and human whole cell protein extracts in two formats. Each extract is available in a pre-digested, tryptic peptide form and used primarily for instrument performance monitoring. Intact extracts are also available for use in optimizing mass spec sample preparation methods. The yeast extract is designed for researchers who appreciate the convenience of a relatively small and well-annotated proteome, whereas the human extract provides a complex proteome with a large dynamic range. This webinar will primarily focus on the utility of these extracts for monitoring mass spec instrument performance, and we will also demonstrate their use as model systems for testing phosphopeptide enrichment resign, isobaric tag labeling and other applications.