Salamanca, March 16th 2010 Participants: Laboratori de Proteomica-HUVH Servicio de Proteómica-CNB-CSIC Participants: Laboratori de Proteomica-HUVH Servicio de Proteómica-CNB-CSIC

STABLE ISOTOPIC LABELING (1) MAJOR POINTS: –Labeling one (or more) sample(s) with an stable isotope causes a mass shift that can be detected using mass spectrometry. –Samples can be labeled either metabolically (in vivo), chemically or enzymatically. –The samples (differentially labelled) are combined and digested at some point and analyzed. –The differences found in the peak intensity (calculated either using peak height or area) reflect the differences in abundance of the parental proteins between the samples. Salamanca, March 16th 2010

STABLE ISOTOPIC LABELING (2) 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). Salamanca, March 16th 2010

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

HPLC + MS instruments vs. Labeling strategy 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

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

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

ICPL Four-plex Labeling Salamanca, March 16th 2010

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

ICPL LABELING AT THE PEPTIDE LEVEL INCREASES THE NUMBER OF PROTEINS QUANTIFIED AND THE QUALITY OF THE QUANTIFICATION Sample A (50  g) Reduction and Alkylation Sample B (50  g) Reduction and Alkylation Label with ICPL-LIGHT (ReagentC 12-Nic ) LABEL WITH ICPL-HEAVY (Reagent C 13-Nic ) Combine both samples Proteolytic digestion (Trypsin, Endo Glu-C) Analyze the samples by LC ESI IT-MS for identification and Quantification Salamanca, March 16th 2010 Fractionation at the protein level (if necessary) Fractionation at the protein level (if necessary) Sample A (50  g) Reduction and Alkylation Sample B (50  g) Reduction and Alkylation Label with ICPL-LIGHT (ReagentC 12-Nic ) LABEL WITH ICPL-HEAVY (Reagent C 13-Nic ) Combine both samples Proteolytic digestion (Trypsin, Endo Glu-C) Analyze the samples by LC ESI IT-MS for identification and Quantification Proteolytic digestion (Trypsin, Endo Glu-C) Fractionation at the peptide level (if necessary) Fractionation at the peptide level (if necessary)

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

Salamanca, March 16th 2010

Peptide number CV% Peptide number Ratio n>2

sequence coverage % N / total % Salamanca, March 16th 2010

Peptide labelingProtein labeling

Salamanca, March 16th 2010 Peptide labelingProtein labeling

Salamanca, March 16th 2010 Peptide labelingProtein labeling

Salamanca, March 16th 2010 SUMMARY ICPL can be used for the quantitative proteomic analysis of complex samples (it would be desirable to check its true limits with state-of-the art instrumentation). Protein quantification should be avoided if # peptide < 2. ICPL-labeling at the peptide level increases dramatically the percentage of peptides suitable for quantification. ICPL-labeling protocol modifies drastically the population of peptides identified. ICPL-labeling at the peptide level increases 2-fold the complexity of the sample, hampering the identification of proteins. Complexity increase is “only” ~1.5-fold when labeling occurs at the protein level.

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