Affinity Tagging of Proteins

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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		Buy this book, Z700428

Products Available for this Protocol
Protocol Material Description	Product #	Product Name	Add to Cart
Buffers, Solutions, and Reagents
Acetonitrile	34967	Acetonitrile
Ammonium bicarbonate	A6141	Ammonium bicarbonate
Trypsin	T6567	Trypsin from porcine pancreas, Proteomics Grade, Dimethylated
Methanol	M1775	Methanol, Absolute - Acetone free
Acetic Acid	A6283	Acetic acid, >99%
TFA	299537	Trifluoroacetic acid, >99%, purified by redistillation, for protein sequencing
Dithiothreitol (DTT)	D9779	DL-Dithiothreitol, for molecular biology
EDTA	E7889	Ethylenediaminetetraacetic acid disodium salt solution
Tributylphosphine (TBP)	90827	Tributylphosphine
SDS	L4390	Sodium dodecyl sulfate
Urea	U5378	Urea, for molecular biology
Tris	T5941	Trizma® hydrochloride, for molecular biology, Biotechnology Performance Certified
Silver stain	HT101A	Silver stain, modified steiner-steiner
KH₂PO4	P2222	Potassium phosphate dibasic, meets USP testing specifications
KCl	P9541	Potassium chloride, for molecular biology
d-Biotin	47868	D-Biotin
Phosphate-buffered saline (PBS)	P5368	Phosphate buffered saline, pH 7.4, Biotechnology Performance Certified
Glycine	50046	Glycine
Heptafluorobutyric acid	77247	Heptafluorobutyric acid

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