lecture 11 some q uantitation methods with lc-ms icat itraq p roteolytic 18 o labelling silac

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Lecture 11Some quantitation methods with LC-MSa.ICATb.iTRAQc.Proteolytic 18O labellingd.SILACe.AQUAf.Label Free quantitation

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Thermo scientific webpage

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ICAT

http://www.imsb.ethz.ch/researchgroup/rudolfa/research

Isotope-Coded Affinity Tags

This part reacts with cysteine

The D-labelled (heavy) reagent witll be 8 mass units heavier than the H-labelled (light) reagent

This part binds specificallyto an avidin affinity column

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ICAT

The heavy and light tags haveidentical chemical propertiesso they will bind to any columnin exactly same manner

Avidin affinity columnbinds specifically to biotinso only ICAT labelled peptides will bind to column

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Separate by ion-exchange and into LC-MS/MS

MS of any peak in the chromatogramTwo peaks differ by 8 mass unitsRatio of light:heavy – tells us relative amountof sample 1 to sample 2

MS/MS of the peak in the MSTo identify the peptide

ICAT

Disadvantages: protein must have cysteinelow sequence coverage – protein is identified often based on only one peptide, often not able to identify PTM

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iTRAQ Isobaric Tag for relative and absolute quantification

Reacts with NH2 groups

N

NCH3

O

NH

O

RAdds tag of mass 145 toterminal NH2 groups and lysines

N

NCH3

CH2+

Rest of molecule +

Reporter ion

MS/MS Fragmentation

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N

NCH3

O

ON

O

O

iTRAQ

Produces an ion of Mw = 117after fragmentation

Mw = 28

N

NCH3

O

ON

O

O


Mw = 29

13CO

N

NCH3

O

ON

O

O

Mw = 30

C18O

etc


13C x 3 15N x 1

13C x 2 15N x 1

13C x 2

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iTRAQ

http://www.broadinstitute.org/scientific-community/science/platforms/proteomics/itraq

Up to 8 different treatments (different types of iTRAQ reagents) – 4 in this example

http://www.iop.kcl.ac.uk/departments/?locator=1031&context=1235

The peptides will elute at the same timebecause they have identical chemical properties

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The MS corresponding to one particular peak in the chromatogramSelect one peak for further fragementation by MS/MS

iTRAQ

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This part gives the sequence information

The low molecular weight region 114-117 contains reporter ionsRatio tells us something about the relative abundance of this proteinin the 4 samples

iTRAQ

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Proteolytic 18O labellling

RNH

NHNH

NH

O

O

O

O

R

R1

R2

R3

If R2 = lysine, arginine

trypsin

H218O

RNH

NH

O

O

OR1

R2

OH

*

* Carboxy oxygens replacedby heavy oxygen

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Proteolytic 18O labellling

Quantitative Protein Analysis Using Proteolytic [18O]Water LabelingKristy J. Reynolds, Catherine Fenselau, Current Protocols in Protein Science, 2004

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SILAC – stable isotope labelling of amino acids in cell culture

Grow cells in media containing isotopically labelled amino acids

Labelling arginine and lysine to ensure all tryptic peptides are labelled (Trypsin cuts at K or R)

Typically e.g. Lys4 – alkly Dx4 subsitution – +4 unitsArg6 – 13Cx6 subsitution – +6 unitsLys8 - 13Cx6 + 15Nx2 substitution +8 unitsArg10 - 13Cx6 + 15Nx4 substitution +10 units

NH

O

NH

NH2

NH2

OH

O

NH2

NH2OH

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SILAC – stable isotope labelling of amino acids in cell culture

Lyse, extract protein, separatetrypsin digest, MS

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AQUA – absolute quantification with reference peptides

Create an isotopically labelled peptide similar to one found in the sampleand spike into the sample

Make an isotopically labelled peptide – in this case Leucine – 13Cx6 +

15Nx1 – +7 mass units

Select a peptide to monitor

Subject this peptide to MS/MSto look at fragmentation pattern

See next slide

(ignore the phosphoprotein part for this lecture)

Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS.Gerber SA, Rush J, Stemman O, Kirschner MW, Gygi SP.Proc Natl Acad Sci U S A. 2003 Jun 10;100(12):6940-5

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1st Quad – allows only one precursor ion through

2nd Quad – trap ions for Collision Induced Decomposition

3rd Quad – allows only one product ion through to be detected

SRM – selective ion monitoring

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e.g. ALELFR is chosen as the peptide to monitor

- Synthesize ALEL*FR (+7 mass units)

-do an MS/MS of ALEL*FR

-Choose one of the high intensity fragment ions to monitor (y4)

- set the LC-MS/MS into the SRM mode so that only m/z 378.3 ions are allowed through 1st Q and 571.2 ions are allowed through the 3rd Q

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Spike sample with the synthesised heavy peptide

The heavy peptide will have identical retention time because chemical properties are identical

Set the LC-MS/MS to the SRM mode to monitorm/z 374.8 → 564.2 to monitor the natural peptide in samplethen m/z 378.3 → 564.2 to monitor the spiked heavy peptideCompare the peak areas to quantitate peptide in sample

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Label-free Quantitation

LC-MS/MS chromatogram

Assume that a protein X is tryptically digested and the peptides elute aspeaks A (12.28 min), B (14.94 min), C (19.72 min) and D (22.69)

A

B CD

Two examples of label-free quantitationXIC – extracted ion chromatogramSC – spectral counting

Avoid isotopes but instrumentation needs to be very reproducible

E

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Label-free Quantitation - XIC

Shows that peak A represents several peptides co-elutingThe peak area of peak A in the chromatogram is an addition of the peak areas in the MS

e.g. the MS corresponding to peak A

e.g. if 660.96 is identified by MS/MS as a peptide coming from protein X – so the peptide from protein X contributes a large part to the peak area of peak Ain the chromatogram

Work out the contribution of the 660.96 to the peak area of peak A by

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Label-free Quantitation - XIC

Work out the contribution of the 660.96 to the peak area of peak A by extracting the ion chromatogram (XIC)

Only one peak bec none of the other peptides have a molecular mass of 660.96Find the peak area

Repeat the same process for all the other peaks B, C and DSum all the peak areas in the XICsConcentration of protein X proportional to total area (need an internal standard)

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Label-free Quantitation – SC

Assuming the LC-MS/MS is set to select only the largest 3 peaks in the MS for collision to produce MS/MS

MS of peak 2

Peak at 660.96 is 2nd highestso selected for MS/MS

Use e.g. protein Prospector to determine that this peptide sequence is derived from protein X

[all data for the this and the next slide are not taken from a real example so do not try to use Protein Prospector to work out the sequence

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Peak at 2116.8 is 3rd highest so selected for MS/MS

MS of peak B

Repeat for all the MS of each peaks

Use e.g. protein Prospector to determine that this peptide sequence is derived from protein X

But e.g. for peak E in the chromatogram, MS/MS of the top 3 peaks in the MS- come from different proteins

Count all the MS/MS spectra which came from a peptide which can be identified as coming from protein X (Spectral Count)

Spectral count proportional to protein concentration (need internal standard)

Label-free Quantitation – SC

lecture 11 some q uantitation methods with lc-ms icat itraq p roteolytic 18 o labelling silac

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