PROTEORED MULTICENTRIC EXPERIMENT 5
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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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PROTEORED MULTICENTRIC EXPERIMENT 5

ICPL RESULTS

Participants:

Laboratori de Proteomica-HUVH

Servicio de Proteómica-CNB-CSIC

Salamanca, March 16th 2010


Stable isotopic labeling 1
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


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).

STABLE ISOTOPIC LABELING (2)

Salamanca, March 16th 2010


When to use the different strategies
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
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


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


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


ICPL Four-plex Labeling

Salamanca, March 16th 2010


Isotopic labeling schema
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


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


Experimental PROTEINS QUANTIFIED AND THE QUALITY OF THE QUANTIFICATIONWorkflow

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


RESULTS: SUMMARY PROTEINS QUANTIFIED AND THE QUALITY OF THE QUANTIFICATION

Salamanca, March 16th 2010


QUANTITATION VALUES: SPIKED PROTEINS PROTEINS QUANTIFIED AND THE QUALITY OF THE QUANTIFICATION

Salamanca, March 16th 2010


QUANTITATION VALUES: E. PROTEINS QUANTIFIED AND THE QUALITY OF THE QUANTIFICATIONcoli PROTEINS

Ratio

n>2

Peptide number

CV%

CV%

Peptide number

Salamanca, March 16th 2010


Differences PROTEINS QUANTIFIED AND THE QUALITY OF THE QUANTIFICATION in proteinsequencecoverages

N / total %

sequence coverage %

Salamanca, March 16th 2010


Comparison PROTEINS QUANTIFIED AND THE QUALITY OF THE QUANTIFICATIONbetweenlabeling at the

protein and peptidelevel

Salamanca, March 16th 2010


Peptide PROTEINS QUANTIFIED AND THE QUALITY OF THE QUANTIFICATIONprofileisdramaticallyaffected

bythe experimental workflow (1)

Peptide labeling

Protein labeling

Salamanca, March 16th 2010


Peptide PROTEINS QUANTIFIED AND THE QUALITY OF THE QUANTIFICATIONprofileisdramaticallyaffected

bythe experimental workflow (2)

Peptide labeling

Protein labeling

Salamanca, March 16th 2010


Peptide PROTEINS QUANTIFIED AND THE QUALITY OF THE QUANTIFICATIONprofileisdramaticallyaffected

bythe experimental workflow (3)

Peptide labeling

Protein labeling

Salamanca, March 16th 2010


Summary
SUMMARY PROTEINS QUANTIFIED AND THE QUALITY OF THE QUANTIFICATION

  • 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


Thank you for your attention PROTEINS QUANTIFIED AND THE QUALITY OF THE QUANTIFICATION

Questions?

Salamanca, March 16th 2010


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