Salamanca, March 16th 2010

Salamanca, March 16th 2010

Participants:Laboratori de Proteomica-HUVHServicio 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.


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).


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).


HPLC + MS instruments vs. Labeling strategy

Bruker HCT ultra PTM (with ETD) ion trap

iTRAQ reporter ions (114, 115, 116 and117) can not be detected

361.26

426.70

508.26

642.20

741.39 869.52

1069.70

+MS2(650.99), 37.8min #1345

0

1

2

3

4

5

6

5x10Intens.

200 400 600 800 1000 1200 m/z

Discard iTRAQ-based approaches


195.45

272.35

325.33

381.28

480.31

548.29

592.33

647.35

691.43

886.66

967.70

+MS2(620.15), 46.2min #1637

0

1

2

3

4

5x10Intens.

200 400 600 800 1000 m/z

189.49 272.32

319.34

375.27

474.28

548.31 647.33

842.55

880.58

955.66

+MS2(614.15), 46.3min #1638

0

1

2

3

4

5

6

5x10Intens.

200 400 600 800 1000 m/z

445.08

519.11

625.11

677.27

756.95

937.31

1014.81816.46567.73 691.73 792.72

+MS, 19.0-20.9min #(636-691)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

4x10Intens.

400 600 800 1000 1200 1400 m/z

QQMSMS

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.


NH2 NH2

NH2

NH2

25°C

2h

QQMSMS

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

12C/13C-Nicotinoyloxy-succinimide

Light label: X = 12C

Heavy label: X = 13Cm = 6 Da

ICPL-BASED CHEMICAL LABELLING(ICPL: Isotope Coded Protein Labeling)

Nicotinoyloxy-succinimide


X

XN

X

XX X

ON

OO

O

N

ON

N

ON

N

O

N

N

O

N

ICPL Four-plex Labeling


SILACSILAC

ICPL , ICAT, etcICPL , ICAT, etc

iTRAQ, TMTiTRAQ, TMT

LC-MS/MS

Digestion

Sample

“Label Free”

Less risk of experimental error

More risk of Experimental error

Isotopic labeling: schema


16/1816/18OO

Protein extraction

ICPL

ICPL LABELING AT THE PEPTIDE LEVEL INCREASES THE NUMBER OF PROTEINS QUANTIFIED AND THE QUALITY OF THE QUANTIFICATION

Sample A (50g)

Reduction and Alkylation

Sample B (50g)


Label with ICPL-LIGHT(ReagentC12-Nic)

LABEL WITH ICPL-HEAVY(Reagent C13-Nic)

Combine both samples

Proteolytic digestion (Trypsin, Endo Glu-C)

Analyze the samples by LC ESI IT-MS for identification and Quantification


Fractionation at the protein level(if necessary)Fractionation at the protein level(if necessary)

Sample A (50g)


Sample B (50g)


Label with ICPL-LIGHT(ReagentC12-Nic)

LABEL WITH ICPL-HEAVY(Reagent C13-Nic)

Combine both samples


Analyze the samples by LC ESI IT-MS for identification and Quantification


Fractionation at the peptide level(if necessary)Fractionation at the peptide level(if necessary)

ICPLQuantification

Identification(Mascot)

WarpLC(ProteinScape)

ICPL-labeling at thePeptide level

20-25 individual fractions (slices)

ICPL JC 12_1-B,1_01_1086.d: T IC +All

0

2

4

6

9x10Intens.

20 40 60 80 100 120 140 160 T ime [min]

nanoRP-LC ESI-IT( Bruker HCT Ultra)

Experimental Workflow

582.3

673.8

749.6

873.4

928.6986.0

1051.8

1124.3

1320.8

749.6

673.8

+M S, 87.2m in #1983

0.0

0.5

1.0

1.5

2.0

2.57x10

Intens.

400 600 800 1000 1200 1400 1600 m /z

MS

375.2

448.1

503.2

539.1 610.2

655.7

689.2

790.3

841.3

885.3

950.3

1041.3 1136.4

+MS2(674.1), 87.3min #1985

0.0

0.5

1.0

1.5

6x10Intens.

200 400 600 800 1000 1200 m/z

MS2

450.1478.1

553.2

596.7653.2

743.2 800.8

882.6

975.8

1004.3

1047.3

1105.4

1192.31304.2

+MS2(749.5), 87.3min #1984

0

1

2

3

46x10

Intens.

400 600 800 1000 1200 1400 m/z

MS2


ICPL-labeling at theprotein level

HUVH LC-MS run A-1 / B-1 A-2 / B-2 A-3 / B-3 A-4 / B-4

Total Sample A/B

(Combined A1-4 / B1-4 runs)**

1 Number of MS/MS spectra acquired 2031 2057 2023 2027 2034.52 Number of total assigned peptides id. 374 368 349 356 361.753 Number of unique peptides id. 334 329 304 323 5074 Number of E Coli proteins id. (total) 142 137 130 129 1715 Number of E Coli single hit- proteins id 74 69 70 58 746 Number of Spiked proteins id. 4 4 4 4 47 FDR* <1% <1% <1% <1% <1%

8 Total Number of proteins quantitated 50 46 44 43 669 Number of proteins quantitated > 3 peptides 4 6 4 5 1710 Number of proteins quantitated > 2 peptides 14 12 11 12 2311 Number of proteins quantitated 1 peptide 32 28 29 26 26

12 Average of A/B ratios for E Coli proteins 1.23***13 Standard deviation A/B ratios 0.51***14 % CV A/B ratios E Coli proteins 41.5***


CNB LC-MS run AB-1 AB-2 AB-3 AB-4

Total Sample A-B

(Combined AB1-4 runs)**

MASCOT1 Number of MS/MS spectra acquired 3438 3508 3380 3342 34172 Number of total assigned peptides id. 377 449 479 475 4453 Number of unique peptides id. 192 220 229 231 3714 Number of E Coli proteins id. (total) 60 75 69 70 1055 Number of E Coli single hit- proteins id 34 39 40 38 696 Number of Spiked proteins id. 2 3 2 37 FDR* 3.40% 2.60% 2.70% 3.00% 2.93%

8 Total Number of proteins quantitated 60 75 69 70 1059 Number of proteins quantitated > 3 peptides 23 28 29 31 3710 Number of proteins quantitated > 2 peptides 13 16 13 9 3611 Number of proteins quantitated 1 peptide 24 31 27 30 32

12 Average of A/B ratios for E Coli proteins 0.92 0.99 0.88 1.03 0.9613 Standard deviation A/B ratios 0.36 0.56 0.28 0.7814 % CV A/B ratios E Coli proteins 39.13 56.57 31.82 75.73

protein # runs Mascot score

Phenyx Score

Ratio 1 Score 1

# peptides used for quant 1

Ratio 2 Score 2


Ratio 3 Score 3 # peptides

used for quant 3

Ratio 4 Score 4


Average ratio

%CV p-value

CYC_HORSE 8 534.33 66.85 0.62 438.42 13 0.58 465.11 12 0.55 490.13 10 0.61 534.33 13 0.59 5.36 0.30MYG_HORSE 8 450.57 62.45 2.46 409.46 6 2.18 450.57 5 2.05 323.15 5 2.23 336.29 6 2.23 7.67 0.06ALDOA_RABIT 4 72.29 6.59 2.16 72.29 2 1.12 52.15 1 1.64 44.84 0.10

ALBU_BOVIN

CYC_HORSE 4 113.00 0.56 113 1 0.44 79 1 0.77 86 2 0.80 48 1 0.64 26.889 0.28MYG_HORSE 4 242.00 1.59 165 2 1.27 242 2 1.14 161 1 2.17 208 1 1.54 30.027 0.11ALDOA_RABIT 4 283.00 2.44 208 1 2.38 283 1 2.44 216 1 2.70 179 1 2.49 5.788 0.04ALBU_BOVIN 4 365.00 349 278 306 365

not detected

0,59

2,23

1,64

0

0,64

1,54

2,49

0

0,67

2,60

2,00

0,20

0

0,5

1

1,5

2

2,5

3

CYC_HORSE MYG_HORSE ALDOA_RABIT ALBU_BOVIN

avg 1

avg2

expected



0,00

10,00

20,00

30,00

40,00

50,00

60,00

70,00

80,00

0 10 20 30 40 50 60 70

ICPL (pept label)

ICPL (prot label)

Peptide number

CV%

CV%

0,00

1,00

2,00

3,00

4,00

5,00

6,00

0 10 20 30 40 50 60 70

ICPL (pept label)

ICPL (prot label)

Peptide number

Ratio

n>2

0,00

5,00

10,00

15,00

20,00

25,00

30,00

35,00

40,00

45,00

50,00

0-5% 6-10% 11-20% >20%

CNB1 %(n=70)

CNB2 % (n=88)

HUVH1 % (n=133)

HUVH2 % (n=134)

sequence coverage %

N /

tot

al %


CNB1 (n) CNB1 % CNB2 (n) CNB2 % HUVH1 (n) HUVH1 % HUVH2 (n) HUVH2 %0-5% 21 30.00 27 30.68 54 40.60 61 45.526-10% 17 24.29 23 26.14 38 28.57 34 25.3711-20% 16 22.86 19 21.59 29 21.80 24 17.91>20% 16 22.86 19 21.59 12 9.02 15 11.19total 70 88 133 134


P09373 KTHNQGVFDVYTPDILR ICPL_L_K(1) KTHNQGVFDVYTPDILR ICPL_L_K(1)KSGVLTGLPDAYGR ICPL_L_K(1) KSGVLTGLPDAYGR ICPL_L_K(1)KSGVLTGLPDAYGR ICPL_H_K(1) KTHNQGVFDVYTPDILR ICPL_H_K(1)KTHNQGVFDVYTPDILR ICPL_L_K(1) KSGVLTGLPDAYGR ICPL_H_K(1)TPEYDELFSGDPIWATESIGGMGLDGR TPEYDELFSGDPIWATESIGGMGLDGRFNSLTKEQQQDVITR ICPL_L_K(1) FNSLTKEQQQDVITR ICPL_L_K(1)FNSLTKEQQQDVITR ICPL_L_K(1) VDDLAVDLVERKSGVLTGLPDAYGR ICPL_L_K(1) KSGVLTGLPDAYGR ICPL_L_K(1)KSGVLTGLPDAYGR ICPL_L_K(1) AGAPFGPGANPMHGRAGAPFGPGANPMHGR EMLLDAMENPEKYPQLTIR ICPL_H_K(1)VDDLAVDLVER LREEIAEQHRLREEIAEQHR DAIPTQSVLTITSNVVYGKKTGNTPDGR ICPL_H_K(2)DAIPTQSVLTITSNVVYGKKTGNTPDGR ICPL_H_K(2)CNB1 CNB2

P09373 GAVASLTSVAK ICPL_H_K(1),ICPL_H_Nterm(1) GAVASLTSVAK ICPL_H_K(1),ICPL_H_Nterm(1)DAIPTQSVLTITSNVVYGK ICPL_L_K(1),ICPL_L_Nterm(1) DAIPTQSVLTITSNVVYGK ICPL_H_K(1),ICPL_H_Nterm(1)LATAWEGFTK ICPL_L_K(1),ICPL_L_Nterm(1) IVGLQTEAPLKR ICPL_H_K(1),ICPL_H_Nterm(1)THNQGVFDVYTPDILR ICPL_L_Nterm(1) SGVLTGLPDAYGR ICPL_L_Nterm(1)SGVLTGLPDAYGR ICPL_L_Nterm(1) IVGLQTEAPLKR ICPL_L_K(1),ICPL_L_Nterm(1)IVGLQTEAPLKR ICPL_H_K(1),ICPL_H_Nterm(1) VDDLAVDLVER ICPL_L_Nterm(1)VDDLAVDLVER ICPL_L_Nterm(1) VDDLAVDLVER ICPL_L_Nterm(1)IVGLQTEAPLKR ICPL_H_K(1),ICPL_H_Nterm(1) LATAWEGFTK ICPL_L_K(1),ICPL_L_Nterm(1)THAPVDFDTAVASTITSHDAGYINK ICPL_L_K(1),ICPL_L_Nterm(1) LATAWEGFTK ICPL_H_K(1),ICPL_H_Nterm(1)ALIPFGGIK ICPL_H_K(1),ICPL_H_Nterm(1) SGVLTGLPDAYGR ICPL_L_Nterm(1)ALIPFGGIK ICPL_L_K(1),ICPL_L_Nterm(1) IVGLQTEAPLKR ICPL_L_K(1),ICPL_L_Nterm(1)NYTPYEGDESFLAGATEATTTLWDK ICPL_L_K(1),ICPL_L_Nterm(1) IVGLQTEAPLKR ICPL_H_K(1),ICPL_H_Nterm(1)

ALIPFGGIK ICPL_H_K(1),ICPL_H_Nterm(1)ALIPFGGIK ICPL_L_K(1),ICPL_L_Nterm(1)ALIPFGGIK ICPL_H_K(1),ICPL_H_Nterm(1)LPLNFK ICPL_H_K(1),ICPL_H_Nterm(1)

HUVH 2HUVH 1

CNB1 CNB2 HUVH1 HUVH2ICPL 378 462 114 112TOTAL 378 462 344 327


Peptide labeling Protein labeling


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.


Thank you for your attention

Questions?

Salamanca, March 16th 2010

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