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Protein Valida-on (Sta-s-cal Inference) and Protein

Quan-fica-on


Terminology

•  Pep-de Spectrum Match •  Target / Decoy •  False discovery rate •  Shared pep-de •  Parsimony •  One hit wonders

200 400 600 800 1000 1200 m/z

Rela-ve Ab

undance

3

SPECTRUM

200 400 600 800 1000 1200 m/z

Rel

ativ

e A

bund

ance

H2N-N--S--G--D--I--V--N--L--G--S--I--A--G--R-COOH b2 b3 b4 b5 b6 b7 b8 b9 b10 b11 b12 b13 b14 b1

y13 y12 y11 y10 y9 y8 y7 y6 y5 y4 y3 y2 y1 y14 y-series:

b-series:

Tandem mass spectrometry and pep/de sequence

•  An MS/MS spectrum contains a mixture of b and y ions

4

PEPTIDE SPECTRUM MATCH

Mass spectrum Reference Protein Database

from genomic annotation Pep/de Spectral Match

Nesvizhskii et al Nature Methods -‐ 4, 787 -‐ 797 (2007)

6

•  Visual inspection •  Scoring function f(S,P) measures the quality of the match between spectrum S and peptide sequence P •  Look at fragment ions

–  predicted mass –  expected intensity –  compare to next best –  presence of immonium

ions –  etc.

PEPTIDE SPECTRUM MATCH

Nesvizhskii et al Nature Methods -‐ 4, 787 -‐ 797 (2007)

7

PEPTIDE SPECTRAL MATCH

TARGET-DECOY SEARCH

TARGET-DECOY SEARCH

INPUTS : PEAKLISTS and SEARCH db SEARCHGUI PEPTIDESHAKER 9

Slide from Alexey Nesvizshkii talk at hWp://www.scivee.tv/node/12671

PROTEIN INFERENCE: FROM PEPTIDES TO PROTEINS

10

Nesvizhskii, A. I.; Aebersold, R. Mol. Cell. Proteom. 4.10, 1419-‐1440, 2005

Shared Pep-des

SIQFVDWCPTGFK

Tubulin alpha 6

Tubulin alpha 3

YMACCLLYR

Tubulin alpha 4


85%

85%

85%

Shared

Pep

-des

AEPTIR

IDVCIVLLQHK

NTGDR

Protein

85%

65%

25%


(4%)

0.15 * 0.35 * 0.75 = 0.04 Feng J, Naiman DQ, Cooper B. Anal Chem. 2007 May 15;79(10):3901-‐11.

From Pep-des to Proteins

IDVCIVLLQHK Protein 95%


(5%)

One Hit Wonders

One Hit Wonders

•  Quan-fy as a score: If different pep-des agree: Good! If pep-des are one-‐hit-‐wonders: Bad!

•  Protein Prophet, etc. •  Then can use FDR at protein level



Pep-de / Protein Quan-fica-on


Terminology

•  Absolute Quan-fica-on •  Rela-ve Quan-fica-on •  Label-‐free •  Normaliza-on •  Labelled •  iTRAQ •  Reporter ions


Pep-de / Protein Quan-fica-on •  Absolute – Es-mate the molar amount of protein / pep-de in the biological sample –  PTMs –  Valida-on

•  Rela-ve –Fold change / sta-s-cally significant difference between 2 biological states –  Biological varia-on –  Biomarker studies


Label-‐free

•  Area Under Curve – MS1 –  Integrate XIC

•  Spectral Coun-ng – MS2 –  High abundant proteins

Käll L, Vitek O (2011) Computa-onal Mass Spectrometry–Based Proteomics. PLoS Comput Biol 7(12): e1002277. doi:10.1371/journal.pcbi.1002277


MS Quan-fica-on

•  MS not inherently quan-ta-ve •  Physiochemical proper-es invoke different MS responses

•  MS only samples a small percentage of total pep-des •  Bias and variability

Population Biological

Sample Handling

Instrument


Normaliza-on

•  Remove bias and variability between runs •  Global – commonly used

– Median scale – Total ion current (TIC)

•  Local – very recent development – Proximity-‐based intensity normaliza-on (PIN)


Labeled Quan-fica-on

•  Run samples simultaneously on in a single run •  Add label to samples •  Mix samples together •  Compute ra-os / sta-s-cally significant diffs.


Labeled

•  Isobaric – MS2, iTRAQ –  Number of samples

•  Synthe-c Pep-des – MS1 –  Absolute (AQUA)

•  Metabolic – MS1, SILAC –  Not higher life forms

Käll L, Vitek O (2011) Computa-onal Mass Spectrometry–Based Proteomics. PLoS Comput Biol 7(12): e1002277. doi:10.1371/journal.pcbi.1002277

Isobaric Tag Total mass = 305

Reporter Group 113 –119, 121 m/z

Balance Group (?) Mass 184, 186 – 192 m/z

Amine specific pep/de reac/ve group (NHS) N-‐hydroxysuccinimide

N

N

O

ON

O

O

N

N

O

ON

O

O

~~~~~~~

N+

N

N+

N

13CH2

15N+

N

13CH2

13CH2

13CH215N+

N

13CH2

13CH2

13CH215N+

15N

13CH3

13CH2

13CH215N+

15N

13CH2

13CH3

13CH2

13CH215N+

N

13CH2

13CH215N+

13CH2

13CH2

15N

13CH2

13CH3

113 114 115 116

117 118 119 121

Applied Biosystems has granted permission to use this slide.

Isob

aric Rep

orter G

roup

s 113 –119, 121 m

/z

iTRAQ® 8-‐Plex Reagent Chemical Structure


Reduce, alkylate Cysteines

Trypsin Digest


Trypsin Digest


Trypsin Digest


Trypsin Digest

Proteoly-c Diges-on

iTRAQ TAG 114

iTRAQ TAG 115

iTRAQ TAG 116

iTRAQ TAG 117

Label pep-des with iTRAQ® Reagents

Tissue Images: Rosas HD et al, (2002) Neurology, 58, 695

MIX

2D LC-‐MS/MS

Normal Brain Tissue-‐1

Normal Brain Tissue-‐2

Obtain protein-‐containing sample, extract protein

Huntington’s Disease-1

Huntington’s Disease-2

iTRAQ Experiment


+TOF Product (513.3): Experiment 2, 33.388 min from keyxx001_perez061_fxn13_queb2_1004.wiffa=3.56649482550429800e-004, t0=4.09124284567078580e+001

Max. 1224.0 counts.

50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900m/z, amu

0

100

200

300

400

500

600

700

800

900

1000

1100

1200In

ten

sity

, co

un

ts216.2

255.1

145.1

428.3

114.1

244.2 782.5513.3

400.2117.1

175.1315.2

598.4499.3185.1212.1

881.6711.4272.2

70.1383.386.1 527.4

199.1 343.2 412.3230.2 326.2440.3

a1

b1

y9

I

b2

y8

a3

b3

y7

I

a4

b4

y6

I

a5y5

I

y4

y3

y2I

y1

y9(+2)

y9

Inte

nsity

, cou

nts

Peptide = VAIVVGAPRMW = 1024.6249Protein ID = platelet membrane

glycoprotein 11b

“Reporter Ion Mass Tags” from which quantitation is calculated

Pep-de iTQVAIVVGAPR MWmono 1024.62 Protein Match Platelet

membrane glycoprotein 11b

Pep-de match is made from product ions, e.g., b-‐ and y-‐ion series

iTRAQ Experiment MS2 Spectrum


112.72493 113.91559 115.10626 116.29692 117.48759 118.67826

Mass (m/z)

976.9

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity

4700 MS/MS Precursor 1289.72 Spec #1 MC[BP = 1127.5, 1180]

115.13

114.13 116.13 117.13

113.11No change disease:control

control disease

iTRAQ Results

•  Reporter ion intensi-es reflect rela-ve pep-de amounts


112.74415 113.88269 115.02123 116.15977 117.29831 118.43685

Mass (m/z)

1.1E +4

0

10

20

30

40

50

60

70

80

90

100

% Int

ensit

y


116.14117.14

115.14

114.14

113.12

112.52129 113.84865 115.17602 116.50338 117.83075 119.15811

Mass (m/z)

3070.1

0

10

20

30

40

50

60

70

80

90

100

% Int

ensit

y


115.12

114.12

116.12117.12

113.11

control

control disease

disease

Increase disease:control

Decrease disease:control

iTRAQ Results •  What fold changes are significant?

•  Do they represent biological relevance as opposed to experimental variability?



From Käll L, Vitek O (2011) Computa-onal Mass Spectrometry–Based Proteomics. PLoS Comput Biol 7(12): e1002277. doi:10.1371/journal.pcbi.1002277

Labe

led & Label-‐free

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