Protein Expression and Standards

Quantitative proteomic studies (through MS or NMR analysis) can benefit 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.

In addition to a few catalog stable isotope-labeled proteins, CIL is pleased to offer labeled cell growth media for E. coli, insect, 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. Also available are 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.

Stable Isotope-Labeled Peptides and Protein Reagents/Kits

➤ Stable Isotope Standards for Mass Spectrometry
➤ Insect Cell Media
➤ Mammalian Cell Media
➤ Yeast Media and Reagents
➤ Rich E. coli Media 
➤ CFS Products 

Application Notes
➤ Full-Length Expressed Stable Isotope-Labeled Proteins for Quantification
➤ Top Ten Tips for Producing ¹³C, ¹⁵N Protein in Abundance

Cell Growth Media and Protein Production view all

Cell-Free Protein Expression view all

❛❛We use CIL because, in addition to their labeled media, they carry an extensive line of reagents for biomolecular NMR, such as buffers, detergents, and reducing agents. They have also been quite helpful by adding new products in response to customer needs.❜❜

– Christopher Lepre, PhD
Research Fellow II, Structural Biology

❛❛We are very pleased with the quality of CIL’s BioExpress^® 6000 media. The mammalian cell growth media from CIL provides an excellent opportunity to obtain isotope-labeled samples of eukaryotic proteins for NMR studies. When comparing CIL’s BioExpress 6000 media with typical rich growth media no differences in growth or expression rates were found.❜❜

– Professor Dr. Harald Schwalbe
Institut für Organische Chemie

❛❛In our hands, CIL’s BioExpress^® 1000 worked like a charm. The cell growth rate and protein expression level essentially matched the results obtained with Luria broth, and the ¹⁵N labeling efficiency was excellent.❜❜

– Tero Pihlajamaa, PhD
Finnish Biological NMR Center, Institute of Biotechnology

Frequently Asked Questions

What is a bottom-up quantitative proteomic workflow? Bottom-up proteomics is a common MS-based workflow used to identify and/or quantify proteins in a given sample by analyzing unique peptides generated and from the enzymatic cleavage of its 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 Biosciences website for further information on their protein production services.

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