Protein Expression and Standards
Share
Full-Length Expressed Stable Isotope-Labeled Proteins for Quantification (Application Note 40)
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 isotope dilution mass spectrometry (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 pleased to offer reagents and kits from CellFree Sciences (CFS), which are used to produce uniform or selectively labeled proteins in yields ideal for MS-based proteomic applications.
Resources
Stable Isotope-Labeled Peptide and Protein Reagents/Kits
Frequently Asked Questions
What is a bottom-up quantitative proteomic workflow?
Bottom-up proteomics is a common MS-based workflow used to identify and quantitate proteins in a given sample by analyzing unique peptides generated from enzymatic cleavage of the proteins.
Does CIL have the ability to express other proteins?
The primary and secondary structures will need to be evaluated. Please contact Nexomics Biosciences (info@nexomics.com) with full request details (e.g., UniProt ID, labeling type, characterization requirements, quantity). Also, please refer to Nexomics website for further information on their protein production services.
References
Fogeron, M.L.; Lecoq, L.; Cole, L.; et al. 2021. Easy synthesis of complex biomolecular assemblies: Wheat germ cell-free protein expression in structural biology. Front Mol Biosci, 8, 639587. PMID: 33842544
Minikel, E.V.; Kuhn, E.; Cocco, A.R.; et al. 2019. Domain-specific quantification of prion protein in cerebrospinal fluid by targeted mass spectrometry. Mol Cell Proteomics, 18(12), 2388-2400. PMID: 31558565
Lacabanne, D.; Fogeron, M.L.; Wiegand, T.; et al. 2019. Protein sample preparation for solid-state NMR investigations. Prog Nucl Magn Reson Spectrosc, 110, 20-33. PMID: 30803692
Zhong, F.; Xu, M.; Metz, P.; et al. 2018. A quantitative metabolomics study of bacterial metabolites in different domains. Anal Chim Acta, 1037, 237-244. PMID: 30292298
Reddy, P.T.; Brinson, R.G.; Hoopes, J.T.; et al. 2018. Platform development for expression and purification of stable isotope labeled monoclonal antibodies in Esherichia coli. MAbs, 10(7), 992-1002. PMID: 30060704
Zhang, C.; Gao, S.; Molascon, A.J.; et al. 2014. Quantitative proteomics reveals histone modifications in crosstalk with H3 lysine 27 methylation. Mol Cell Proteomics, 13(3), 749-759. 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), 2911-2920. PMID: 23788530
Zhang, C.; Liu, Y.; Andrews, P.C. 2013. Quantification of histone modifications using 15N metabolic labeling. Methods, 61(3), 236-243. PMID: 23454290
Singh, S.; Springer, M.; Steen, J.; et al. 2009. FLEXIQuant: a novel tool for the absolute quantification of proteins, and the simultaneous identification and quantification of potentially modified peptides. J Proteome Res, 8(5), 2201-2210. PMID: 19344176
Andrew Percy, PhD
Senior Applications Chemist – Mass Spectrometry
Dr. Andrew Percy is the Senior Applications Chemist for Mass Spectrometry and the MS ‘Omics Product Manager at CIL. His responsibilities minimally involve providing technical support, overseeing product development, identifying new product market opportunities, assisting in the analysis of product-related applications, and writing/reviewing marketing literature.
More
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
