Quantitative Proteomics

Quantitation AND Mass Spectrometry Condition A Condition B Identify and quantify differently expressed proteins resulting from a change in the environment (stimulus, disease) Lyse Improve understanding of biological function of a particular protein or posttranslational modification 5 4 3 2 1 Fractionate 5 4 3 2 1 Detect, & ID by MS

Available Methodology 1) Isotope-Coded Affinity Tag (ICAT) technology 2) itraq tagging technology 3) Absolute QUAntitation of proteins (AQUA) 4) Stable Isotope Labeling with Amino acids in Cell culture (SILAC)

ICAT (isotope coded affinity tagging) Label protein samples with heavy and light reagent. Reagent contains affinty tag and heavy or light isotope. Mix samples Digest protein sample mixture Affinity purify label peptides Analyze sample via LC/MS/MS

Isotope-coded affinity tag (ICAT) components A chemically reactive group Forms a covalent bond to the protein or peptide eg a thiol-reactive agent (will bind to Cys) An isotope-labeled linker heavy or light, depending on which isotope is used An affinity tag Enables the protein or peptide bearing an ICAT to be isolated by affinity chromatography in a single step

Example of an ICAT Reagent Biotin affinity tag binds tightly to streptavidinagarose resin O Thiol-reactive group note similarity to iodoacetamide HN NH S O H N * O O * O * * H N O I Linker Heavy version will have deuteriums at * Light version will have hydrogens at *

affinity isolation on streptavidin beads Lyse & Label mass spectrometry mix relative protein levels obtained from peak ratios proteolysis (eg trypsin) Target protein m/z Heavy ICAT reagent Light ICAT reagent

Protein Quantification & Identification w/icat Mixture 1 Quantitation 100 Light MS Heavy ICAT Reagent- labeled cysteines Optional fractionation Digest Optional fractionation Affinity separation 0 100 550 560 570 580 m/z NH 2 -EACDPLR-COOHCOOH MS/MS Mixture 2 2DLC or 2DLC with 1D gel Identification 0 200 400 600 800 m/z Quantitation and protein identification Ruedi Aebersold, 2000

Advantages of ICAT Estimates relative protein levels between samples with a reasonable level of accuracy (within 10%) Can be used on complex mixtures of proteins Cys-specific label reduces sample complexity Peptides can be sequenced directly if tandem MS-MS is used Disadvantage Yield and non specificity Slight chromatography differences Expensive Tag fragmentation Meaning of relative quantitation information (available Cys)

ICAT Improvements C13 labeling -resolves chromatography issue Solid state approach - improves yield and specificity Acid cleavage of Biotin tag - improves ionization and fragmentation interpretation Future Exploration of different ICAT chemistries Coupling to 2D separation strategies

itraq

http://www.proteome.soton.ac.uk/itraq.htm

AQUA Peptides are synthesized with incorporated stable isotopes (internal standards) Selected Reaction Monitoring (SRM) analysis in a tandem mass spectrometer is used for quantitation Internal peptides can be prepared with covalent modifications The sequence must be known Incomplete digestion not taken into account

Relative and Absolute Quantification of Postsynaptic Density Proteome Isolated from Rat Forebrain and Cerebellum MCP Papers in Press. Published on February 28, 2006 as Manuscript D500009-MCP200 Dongmei Cheng, Casper C. Hoogenraad, John Rush, Elizabeth Ramm, Max A. Schlager, Duc M. Duong, Ping Xu, Sameera Rukshan, John Hanfelt, Terunaga Nakagawa, Morgan Sheng, and Junmin Peng Used ICAT and AQUA to first identify proteins that are differently expressed in the forebrain vs cerebellum (indicating heterogeneity) Then used AQUA for molar quantitation to understand stoichiometric amounts of different types of proteins (scaffolds, kinases, receptors, GTPases.etc)

Stable Isotope Labeling with Amino acids in Cell culture (SILAC) 15 N purification One of the most accurate procedures for quantitation of proteins Each peptide produced by the cell and cleaved by enzymatic digestion will have an isotopically identical internal standard Works best with cell lines only Mix and digest Mass spectrometry

http://www.pil.sdu.dk/silac_intro.htm