Product List for "Isotope-coded Affinity Tagging of Proteins" Protocol

The following Cold Spring Harbor Laboratory Press protocols are brought to you by BioSupplyNet in a partnership with Sigma-Aldrich. A list of products is available to use in each protocol.

Selected Protocol from "Proteins and Proteomics: A Laboratory Manual", Richard J. Simpson.

Isotope-coded Affinity Tagging of Proteins

Richard J Simpson

ABSTRACT
Quantitative proteomics has traditionally been performed using 2D gel electrophoresis where quantitation is accomplished by recreating differences in the staining patterns of proteins derived from two states of cell populations or tissues from a similar biological system. More recently, Mass Spec (MS) methods based on stable isotope quantitation have been developed that show significant potential for differential expression proteomic studies. One such in vitro method, described in this protocol, involves the use of isotope-coded affinity tags (ICATs). This approach uses a protein tag with three functional moieties: a cysteine reactive moiety, a linker with either eight hydrogens (the light form of the reagent) or eight deuteriums (the heavy form of the reagent, having an isotope code or mass tag of 8 daltons), and a biotin moiety (the affinity tag). Using this technique, the cysteine side chains in complex mixtures of proteins from two different states of a cell population (e.g., normal vs. disease) are reduced and alkylated using the light form of the reagent (d0-labeled tag) in one cell state and the heavy form of the reagent (d8-labeled tag) for proteins in the second cell state. The two mixtures (d0- and d8-labeled) are then combined and subjected to proteolytic digestion (typically, with trypsin and/or Lys-C). Generated cysteine-containing peptides are affinity-purified using an avidin column resulting in a “simplified” mixture of peptides that contains about tenfold fewer peptides than the original mixture. These peptides are analyzed by MS, and quantitation information based on the relative abundance of the d0 and d8 isotopes is obtained. The identification of proteins is obtained from the peptide molecular mass and MS/MS-derived amino acid sequence. This protocol was kindly provided by Ruedi Aebersold, Timothy J. Griffin, and Sam Donohoe (The Institute for Systems Biology). Four stages comprise this protocol:

• Stage 1: Labeling Proteins with ICAT
• Stage 2: Cation Exchange Cleanup of ICAT Samples
• Stage 3: Selection of Tagged Proteins Using an Avidin Column
• Stage 4: Analysis of ICAT-labeled Peptides by MS

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