A Practical Guide to the FLEXIQuant Method

A protein molecule exists as a heterogeneous population of posttranslationally modified forms, which are of potential interest to biologists. However, due to detection or methodology limitations, they remain uncharacterized. When a protein does become a prioritized interest in a laboratory, workflows aimed for its purification and characterization are implemented. Inherent in these workflows is the enrichment of the protein from the biological lysate, rendering it an ideal sample for mass spectrometry (MS), as detection of several peptides is greatly increased. In order to capitalize on this enhanced detection of the protein of interest, we have developed a full-length expressed protein quantification standard (FLEXIQuant standard) that is in vitro synthesized, devoid of posttranslational modifications (PTMs), and implemented into the purification workflow of the endogenous counterpart-as such it serves as an internal MS standard. FLEXIQuantification allows for the unbiased identification of peptides undergoing PTM as a function of a particular biological state. The extent of PTM is also quantified, providing further insight into the regulation of the protein.

[1]  William Stafford Noble,et al.  Assigning significance to peptides identified by tandem mass spectrometry using decoy databases. , 2008, Journal of proteome research.

[2]  Hanno Steen,et al.  FLEXIQuant: a novel tool for the absolute quantification of proteins, and the simultaneous identification and quantification of potentially modified peptides. , 2009, Journal of proteome research.

[3]  John Rush,et al.  Quantitative Proteomics Reveals the Function of Unconventional Ubiquitin Chains in Proteasomal Degradation , 2009, Cell.

[4]  Mu Wang,et al.  A multiple reaction monitoring method for absolute quantification of the human liver alcohol dehydrogenase ADH1C1 isoenzyme. , 2007, Analytical biochemistry.

[5]  S. Gygi,et al.  Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[6]  A. Schmidt,et al.  A novel strategy for quantitative proteomics using isotope‐coded protein labels , 2005, Proteomics.

[7]  S. Gygi,et al.  Quantitative analysis of complex protein mixtures using isotope-coded affinity tags , 1999, Nature Biotechnology.

[8]  F. Vandenesch,et al.  Isotope-labeled Protein Standards , 2007, Molecular & Cellular Proteomics.

[9]  M. Kirschner,et al.  Stable isotope-free relative and absolute quantitation of protein phosphorylation stoichiometry by MS. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[10]  T. Copeland,et al.  The P1' specificity of tobacco etch virus protease. , 2002, Biochemical and biophysical research communications.

[11]  D. Lauffenburger,et al.  Time-resolved Mass Spectrometry of Tyrosine Phosphorylation Sites in the Epidermal Growth Factor Receptor Signaling Network Reveals Dynamic Modules*S , 2005, Molecular & Cellular Proteomics.

[12]  Hiroko Yamada,et al.  Human protein factory for converting the transcriptome into an in vitro–expressed proteome , 2008, Nature Methods.

[13]  Hanno Steen,et al.  Different phosphorylation states of the anaphase promoting complex in response to antimitotic drugs: A quantitative proteomic analysis , 2008, Proceedings of the National Academy of Sciences.

[14]  M. Mann,et al.  Global, In Vivo, and Site-Specific Phosphorylation Dynamics in Signaling Networks , 2006, Cell.

[15]  M. Mann,et al.  Stable Isotope Labeling by Amino Acids in Cell Culture, SILAC, as a Simple and Accurate Approach to Expression Proteomics* , 2002, Molecular & Cellular Proteomics.