Protein Expression and Standards

Stable Isotope-Labeled ApoA-1 as a Global Standard for Quantitative Proteomics Studies (Application Note 46)


  • Stable Isotope-Labeled ApoA-1 as a Global Standard for Quantitative Proteomics Studies (Application Note 46)
  • Full-Length Expressed Stable Isotope-Labeled Proteins for Quantification (Application Note 40)
  • Insect Cell Media
  • Mammalian Cell Media
 

Stable isotope labeled cellular biomass can be used in both proteomic and metabolomic investigations. In addition, quantitative, proteomic MS-based studies can benefit greatly from the use of purified, labeled intact protein as internal standards. The use of properly folded, labeled intact proteins are ideal internal standards because they will mimic, as close as possible, the physical and chemical properties of the target endogenous protein in a sample prior to, during and after digestion. In particular, they will undergo a similar degree of proteolytic cleavage as the unlabeled counterpart, thus improving the accuracy of the IDMS experimental result for both middle-down or bottom-up methodologies.

CIL is pleased to offer labeled cell growth media for E. coli, insect cells, yeast, and eukaryotic cells. Specific human proteins may be overexpressed in a variety of cell types using these media in conjunction with recombinant techniques so that one can obtain a relatively large amount of labeled purified protein for proteomic studies.

CIL is also please to offer reagents and kits for CellFree Sciences, which are used to produce uniform or selectively labeled proteins in yields ideal for MS-based proteomic applications.

Resources

 

Frequently Asked Questions 

What is a bottom-up quantitative proteomic workflow?

Bottom-up proteomics is a commonly used MS-based workflow used to identify and quantitate proteins in a given sample by analyzing unique peptides generated from enzymatic cleavage of the proteins. 

What protein-expression system is used to produce the heavy-labeled ApoA-1?

E.coli is used to produce the labeled protein. It is a robust method that has been optimized over the last few years.

Why does ApoA-1 (15N) contain a his-tag?

The his-tag is added to ease purification. It will not present a problem for most researchers. It can be removed if it is problematic, but it is a difficult process.

Can I use the heavy-labeled ApoA-1 in a top-down proteomic workflow?

The presence of the his-tag may limit the usefulness of this
standard in top-down approaches.

 

References 

Zhang, C.; Gao, S.; Molascon. A.J.; Liu, Y.; Andrews, P.C. 2014. Quantitative proteomics reveals histone modifications in crosstalk with H3 lysine 27 methylation. Mol Cell Proteomics, 13(3): 749-59 PMID: 24382802

Hessling, B.; Buttner, K.; Hecker, M. 2013. Global relative quantification with liquid chromatography-matrix-assisted laser desorption ionization time-of-flight (LC-Maldi-TOF)--cross-validation with LTQ-Orbitrap proves reliability and reveals complementary ionization preferences. Mol Cell Proteomic, 12(10):2911-20. PMID: 23788530

Zhang, C.; Liu, Y.; Andrews, P.C. 2013. Quantification of histone modifications using 15N metabolic labeling. Methods, 61(3):236-43. PMID: 23454290

Kevin Millis, PhD

Kevin Millis, PhD

Senior Scientist, Application Development Manager

Kevin Millis, PhD, is the Senior Scientist and Market Development Manager for all NMR and Mass Spectrometry product lines. Kevin is responsible for Technical Services both internally and externally for all CIL customers as well as being responsible for the application and market development for the CIL products.

More
Andrew Percy, PhD

Andrew Percy, PhD

Senior Applications Chemist - Mass Spectrometry

Dr. Andrew Percy is the Senior Applications Chemist for Mass Spectrometry. His responsibilities minimally involve overseeing product development, identifying new product market opportunities, assisting in the analysis of products for MS ‘omics applications, and providing technical support to customers.

More