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Salamanca, March 16th 2010

PROTEORED MULTICENTRIC EXPERIMENT 5 ICPL RESULTS. Participants : Laboratori de Proteomica -HUVH Servicio de Proteómica -CNB-CSIC. Salamanca, March 16th 2010. STABLE ISOTOPIC LABELING (1). MAJOR POINTS:

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Salamanca, March 16th 2010

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  1. PROTEORED MULTICENTRIC EXPERIMENT 5 ICPL RESULTS Participants: Laboratori de Proteomica-HUVH Servicio de Proteómica-CNB-CSIC Salamanca, March 16th 2010

  2. STABLE ISOTOPIC LABELING (1) • MAJOR POINTS: • Labelingone (or more) sample(s) withanstableisotope causes a massshiftthat can bedetectedusingmassspectrometry. • Samples can belabeledeithermetabolically (in vivo), chemicallyor enzymatically. • Thesamples (differentiallylabelled) are combined and digested at somepoint and analyzed. • Thedifferencesfound in thepeakintensity (calculatedeitherusingpeakheightorarea) reflectthedifferences in abundance of the parental proteinsbetweenthesamples. Salamanca, March 16th 2010

  3. Disadvantages Reagents are expensive. Analytical instruments (i.e., liquid chromatographers and mass spectrometers) are not easily accesible. Protein isoforms are difficult to detect and quantify. In most cases, specific software for the analysis of data is not reliable enough. Some labeling techniques (SILAC, ICPL, ICAT) increase the complexity of the sample (which are already complex). Advantages Methodologically is a relatively simple technique. Each labeled peptide is an independent data point in the quantification of the protein. Some approaches allow the simultaneous analysis of 4 (ICPL and iTRAQ), 6(TMT) and 8 samples (iTRAQ). STABLE ISOTOPIC LABELING (2) Salamanca, March 16th 2010

  4. When to use the different strategies? • METABOLIC (SILAC): • less experimental error. • Theoretically, any aminoacid could be used for labeling. • Quantitation is performed at the MS level. • Only for living organisms (not “higher organisms”). • CHEMICAL: • ICPL, ICAT, Lys-tag, etc: • Applicable to all kind of biological samples. • Quantitation is performed at the MS level. • Reagents are expensive. • iTRAQ,TMT: • Applicable to all kind of biological samples. • Quantitation is performed at the MS/MS level. • Reagents are expensive. • Only a limited set of MS instruments are adequate for the analysis of iTRAQ labelled samples. • Enzymatic (O16/O18): • Applicable to all kind of biological samples. • Quantitation is performed at the MS level. • High resolution mass spectrometers required to perform quantitation. • Label-free: • Applicable to all kind of biological samples. • Superb chromatographic reproducibility. • High accuracy (ideally in the range of 10-5 ppm). Salamanca, March 16th 2010

  5. HPLC + MS instruments vs. Labelingstrategy Bruker HCT ultra PTM (with ETD) ion trap iTRAQ reporter ions (114, 115, 116 and 117) can not be detected Discard iTRAQ-based approaches Salamanca, March 16th 2010

  6. Quantitative information is stored in the MS spectra (Metabolic, chemical, and enzymatic labeling) Detail Q 6 Da MS Peak intensities (or areas) are related to protein relative abundances. MSMS spectra help us to identify the peptide Salamanca, March 16th 2010

  7. Q 6 Da MS Same peptide, from 2 different samples NH2 NH2 25°C NH2 2h NH2 ICPL-BASED CHEMICAL LABELLING (ICPL: Isotope Coded Protein Labeling) Schmidt, Kellermann, Lottspeich, Proteomics 2005, 5, 4–15 Proteins in sample A carry a light label Proteins in sample B carry a heavy label 12C/13C-Nicotinoyloxy-succinimide Nicotinoyloxy-succinimide Light label: X = 12C Heavy label: X = 13C Dm = 6 Da Salamanca, March 16th 2010

  8. ICPL Four-plex Labeling Salamanca, March 16th 2010

  9. Isotopic labeling: schema Less risk of experimental error Sample SILAC Protein extraction ICPL , ICAT, etc 16/18O iTRAQ, TMT Digestion ICPL LC-MS/MS “Label Free” More risk of Experimental error Salamanca, March 16th 2010

  10. ICPL LABELING AT THE PEPTIDE LEVEL INCREASES THE NUMBER OF PROTEINS QUANTIFIED AND THE QUALITY OF THE QUANTIFICATION Sample A (50g) Sample B (50g) Sample A (50g) Sample B (50g) Reduction and Alkylation Reduction and Alkylation Reduction and Alkylation Reduction and Alkylation Labelwith ICPL-LIGHT (ReagentC12-Nic) LABEL WITH ICPL-HEAVY (Reagent C13-Nic) Proteolyticdigestion (Trypsin, EndoGlu-C) Proteolyticdigestion (Trypsin, EndoGlu-C) Labelwith ICPL-LIGHT (ReagentC12-Nic) LABEL WITH ICPL-HEAVY (Reagent C13-Nic) Combine bothsamples Fractionation at theproteinlevel (ifnecessary) Combine bothsamples Proteolyticdigestion (Trypsin, EndoGlu-C) Fractionation at thepeptidelevel (ifnecessary) Analyzethesamplesby LC ESI IT-MS foridentification and Quantification Analyzethesamplesby LC ESI IT-MS foridentification and Quantification Salamanca, March 16th 2010

  11. Experimental Workflow 20-25 individual fractions (slices) ICPL-labeling at the protein level ICPL-labeling at the Peptide level nanoRP-LC ESI-IT ( Bruker HCT Ultra) MS ICPL Quantification WarpLC (ProteinScape) Identification (Mascot) MS2 MS2 Salamanca, March 16th 2010

  12. RESULTS: SUMMARY Salamanca, March 16th 2010

  13. QUANTITATION VALUES: SPIKED PROTEINS Salamanca, March 16th 2010

  14. QUANTITATION VALUES: E. coli PROTEINS Ratio n>2 Peptide number CV% CV% Peptide number Salamanca, March 16th 2010

  15. Differences in proteinsequencecoverages N / total % sequence coverage % Salamanca, March 16th 2010

  16. Comparisonbetweenlabeling at the protein and peptidelevel Salamanca, March 16th 2010

  17. Peptideprofileisdramaticallyaffected bythe experimental workflow (1) Peptide labeling Protein labeling Salamanca, March 16th 2010

  18. Peptideprofileisdramaticallyaffected bythe experimental workflow (2) Peptide labeling Protein labeling Salamanca, March 16th 2010

  19. Peptideprofileisdramaticallyaffected bythe experimental workflow (3) Peptide labeling Protein labeling Salamanca, March 16th 2010

  20. SUMMARY • ICPL can beusedforthequantitativeproteomicanalysis of complexsamples (itwouldbedesirabletocheckits true limitswithstate-of-the art instrumentation). • Proteinquantificationshouldbeavoidedif # peptide < 2. • ICPL-labeling at thepeptidelevelincreasesdramaticallythepercentage of peptidessuitableforquantification. • ICPL-labelingprotocolmodifiesdrasticallythepopulation of peptidesidentified. • ICPL-labeling at thepeptidelevelincreases 2-fold thecomplexity of thesample, hamperingtheidentification of proteins. • Complexityincreaseis “only” ~1.5-fold whenlabelingoccurs at theproteinlevel. Salamanca, March 16th 2010

  21. Thank you for your attention Questions? Salamanca, March 16th 2010

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