Strategies in proteomics

Transcription

1 Strategies in proteomics Systems biology - understand cellpathways, network, and complex interacting (includes Genomics, Proteomics, Metabolomics..) Biological processes - characterize protein complexes, cellular machines, organelles, proteins induced by stimuli (e.g. chemokines) Biomarkers - proteins / peptides in urine, serum or tissues associated with a certain disease - for diagnosis and monitoring of therapy Drug targets discover biological processes involved in disease mechanism, which are targets for (rational) drug design (antigonists, inhibitors, antibodies etc.)

2 Strategies in proteomics Identification of proteins

3 Strategies in proteomics Identification of proteins 1.) From gel spots 2D-gels of 1D-gels of total proteins of cells / tissue prefractionated subset (solubility, pi etc.) mostly for immunoprecipitates Advantages: Disadvantages: quantitation on gel, pre-identification by size and pi reduced complexity of spots to be analyzed by MS blind at the extremes of size and pi problems with difficult to solubilize membrane proteins

4 Strategies in proteomics Identification of proteins 2) From total protein mixtures / tissues protease digestion performed on whole sample Advantages: Disadvantages: Solution: (one of a few) includes insoluble proteins (multispan-proteins*), very large or very basic etc. proteins extremely complex mixture of peptides mix of high and low abundance species two-dimensional LC before ESI-MS 1. - CIEX with salt pulses 2. - RP of CIEX fractions multi-dimensional protein identification = mudpit * special tricks for digestion of transmembrane region

5 Strategies in proteomics Functional protein complexes Immunoprecipitation Protein A Yeast two hybrid system on the DNA level Blue native PA-electrophoresis (BN-PAGE) for membran-proteins and protein-complexes mild detergent solubilizes + CB charges protein

6 Membrane proteins Membrane proteins in normal buffer solutions not soluble enrichment / fractionation Many membrane proteins can be solubilized with mild detergents e.g. Triton X-100, deoxycholate, octyl-ß-d-glucoside, u.v.a. but for many others these are inefficient. Radical solution: SDS, not suitable for later MS analysis.

7 Membrane proteins Blue Native polyacrylamide gel electrophoresis, BN-PAGE Protein-complexes solubilized with detergent (digitonin, dodecyl-maltoside) and Coomassie-blue G-250 CB confers charge to complexes, but does not denature them in contrast to SDS. BN-PAGE may be used as first dimension before SDS-PAGE

8 Comparing samples by 2D-PAGE Run normal 2D-PAGE, one for each sample Stain with silver, or with Sypro Ruby (more even staining of various proteins, better linearity of quantitation; less than 10 ng of protein; also Sypro Orange etc.; requires fluorimetric scanner) Compare the gels by eye or scan and compare with soft-ware Plus: Cheapest method Con: 2D-gels never identical, overlay problematic

9 Comparing samples by 2D-PAGE Example: liver cells (hepatocytes) control and drug treated from a work of Christopher Gerner, Wien 48 pi kda 20

10 Comparing samples by 2D-PAGE 2-D Fluorescence Difference Gel Electrophoresis (DIGE) 2 or even 3 samples can be compared proteins are reacted with a fluorescent dye (either Cy3, Cy5, or Cy6) mixed and separated by 2D-PAGE

11 Comparing samples by 2D-PAGE 2-D Fluorescence Difference Gel Electrophoresis Plus: Con: Samples run on same gel: no gel comparison guess work Sample pool can be used as quantitation standard: allows rather small relative differences in expression level to be measured (lod: ± 10 %) Expensive (only from GE healthcare) Only for 2D-PAGE accessible proteins

12 Cy3 Comparing samples by 2D-PAGE Cy5 T-plastin Dual ImageCy3/Cy5 Glutaminase C in breast cancer vs. control

13 Comparing samples by MS or MS/MS One pot using stable isotopes ICAT ITRAQ SILAC Sample by sample label free quantitation

14 Relative quantitation of protein expression by isotope-coded affinity tags (ICAT) Tryptic digest of sample 1 Tryptic digest of sample 2 -SH reaction with reagent containing only H -SH reaction with reagent containing only 8 D Mix MS Evaluate peak ratios for various peptides Identify peptides / proteins with significant difference

15 Relative quantitation of protein expression by ICAT isotope-coded affinity tag NH O NH Linker: Heavy version: D at * Light version : H at * S O H N * O O O * * * H N O I

16 Relative quantitation of protein expression by ICAT Two protein samples labeled with normal OR heavy versions of the isotope-coded affinity tag (ICAT) reagent, which binds cysteine residues and carries a biotin-tag. A B LC-MS-MS Quantification light heavy Samples are mixed, trypsin digested and ICAT-labeled peptides are recovered by affinity chromatography Identification MSMS on light peak m/z

17 Relative quantitation of protein expression by stable isotope labeling by amino acids in cell culture (SILAC) Mix MS Evaluate peak ratios for various peptides Identify peptides / proteins with significant difference

18 Relative quantitation of protein expression by ITRAC Tryptic digest of sample 1 Tryptic digest of sample 2 -NH 2 reaction with reagent containing only H -NH 2 reaction with reagent containing only 8 D Mix MS Evaluate peak ratios for various peptides Identify peptides / proteins with significant difference ITRAC is often performed on mixtures of proteins, often with 2D-HPLC

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21 Quantitative proteomics by shotgun principle 1-dimensional view of 182 MB file Options: a) TIC. total ion current b) BPI base peak intensity (bp is highest peak in scan) % Time Example spectrum 100 % AVSGLR LICDSR m/z Instrument program: 1.) survey scan select up to ten of the most promising peaks 2.) MSMS on selected peaks on after the other duration of cycle about 2 sec Problems: Re-selection of previously done peptide, often with isotope peaks ( lock time) Underrepresentation to total overlooking of less abundant peaks (dependent e.g. on instrument speed), somewhat stochastic!

22 Quantitative proteomics by shotgun principle Solutions: 1.) buy even faster operating instrument 2.) extend gradient time (up to 4 h with one-dimensional LC) 3.) enhance chromatographic separation (less overlap of peaks) using sub-2 µm LC (minimizes problem of hybrid spectra) 4.) pre-fractionate protein mixture e.g. by cation-exchange LC or RP-HPLC at different ph, e.g. ph 9 (use polymeric RP-phase!) 5.) fragment all ions without preselection parent and daughter ions will have the exactly same elution profile (one company going this way) MS E 6) Targeted quantitation E.g.in clinical diagnosis. protein-specific peptides for the biomarker, e.g. prostate specific antigen, are defined. Their elution time and their MRMs are determined. high dwell times, highest sensitivity.