蛋白质代谢的信息处理进展

Neurosciences Research Building关慎恒

Mass Spectrometry Facility/Department of Pharmaceutical Chemistry,Institute for Neurodegenerative Diseases and Department of Neurology,

University of California, San Francisco

第二届中国计算蛋白质组学研讨会

2

Identification (Qualitative)-Peptide/protein IDs

-PTM IDs and site assignment-Interaction partners

Quantification-Expression differences

-PTM occupancies-Interaction strength

Dynamics-Turnover-Transport

-Intrinsic transient behaviors

Biological Insight

More detailed information

HigherThroughput

Isotope labeling isessential

Isotope labeling isnot necessary

3

Study Protein Turnover on A Proteomic Scale

Many neurodegenerative diseases are closely related to protein turnover

•Alzheimer's disease: A aggregation/breakdown of tau in brain•Parkinson’s disease: accumulation of alpha-synuclein•CJD: transmission and accumulation of misfolded prion

Amino Acids Food Source

Waste

Proteins

4

15NInorganic

salt

Label AlgaeWith 15N

feed miceharvesttissues

over time

extractproteins

digestLCMSMS

data processing

GO inference

FunctionLocalizationProcesses

Dynamic Proteomics by 15N Metabolic Labeling

PNAS2010v107p14508

5

Correlations between function and turnover rates

PNAS2010v107p14508

6

Protein Turnover in Human Plasma

AnalyticalBiochemistry2012v420p73

7mcp.M112.021162

Metabolic Labeling Reveals Proteome Dynamics of Mouse Mitochondria

314 and 386 proteins in heart and liver mitochondriaHalf live of heart and liver mitochondria: 17.2 d and 4.26 d

8JBC2010v285p3341

Kinetics of Methylation on Histones

• Marking methyl groups with isotope labeled methionine• Kinetic modeling of isotope incorporation into methylated Lysines

mono-, di-, and trimethylation rates: progressively smalleractive genes = faster rates; silent genes = slower rates

9

MS-based measurement and modeling of histone methylation kinetics (M4K)

• Use SRM to measure labeled co-occupant methylation states• Use labeled arginine to measure protein turnover• Kinetic modeling of co-occupant methylation states

PNAS2012v109p13549

me2me3 rates 100X smaller for H3K27 or H3K36More methyltransferase MMSET, higher rates

10

RAW Files

MSMSPeaklists

PeptideID List

MS2Extract

DatabaseSearch

14N SurveyXIC

fitXIC

15N Distributions

CrossExtract

15N SurveyMS Peaklists

NN LeastSquares

PeptideCurves

ProteinCurves

ProteinTurnover

CurveConstruct

Pep2Prot

fitCurve

Data Processing Pipelinefor Mammalian Protein Turnover Studies

MCP2011v10: M110.005785

LTQFTQ ExactiveSensitivity!

LC alignmentSelectivity!

Compartment (Pool) ModelsAccuracy/Biological significance

11

10 20 30 40 50 60 70-6

-4

-2

0

2

4

6x 10

8

10 20 30 40 50 60 70-6

-4

-2

0

2

4

6x 10

8

Original Basepeak Chromatogram

After LC alignment

LC Alignment for 15N Isotopomer Extraction

12

Protein Turnover - Empirical Modeling

•Mass shift is an independent and fast process

•Incorporation curve may be modeled as a delayed exponential

•The model seems universal applicable (to the whole proteomes)

PNAS2010v107p14508

)1()( )( 0ttketRIA

13

0 5 10 15 20 25 30 350

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Incorporation Time (day)

15N

Rel

ativ

e F

ract

ion

phosphatidylethanolamine-binding protein 1, P70296 in brain

Protein incorporation curve is constructed from 13 peptide curves

14JBiolChem1939v130p703

15PhysMedBiol1957v2p36

Compartment Modeling of Protein Turnover

16

Compartment Modeling and NonCompartmental Analysisin Drug Development

AdvDrugDelivertRew2001v48p249

Pharmacokinetics (PK) studies

Industry Standard Software Package == WinNonLin

17

Compartment (Pool) Modeling “分池模型”

)1(

V

R

dt

d ARA[A]T

RA[A]

V

TA

A

][

][

0],[

0,0

0,][

0,0

0][ 0

tAR

toutput

tAR

tinput

A

A

TA

0),1()(

tett

V

RA

AnalChem2012v84p4014

18

Relative Fractional Label Concentration

938 939 940 941 942 943 944 945 946 947 948 949 950m/z

8.528.02

9.01

4.514.01

5.019.52

5.510.49 10.010.990.00 1.90 7.526.01 10.512.90 3.52 11.016.492.52

7.39

ALFQDVQKPSQDEWGK2+

Nlabel = 2

What is the physical or chemical significance of the SILAC ratio?

0X8%+1X32%+2X60%Relative Fractional Label Concentration (RF) = -------------------------------------- = 0.76

100% X Nlabel

60%

32%

8%

SILAC labels: Lys 6, Lys 8, Arg 6, or Arg8Stable element labels: 15N, 2H, 13C, etc

total moles percent enrichment (MPE) AnalBiochem2011v412p47

14NAA

15NAA

14NP

15NP

ks’

Ra*H(t) (t)

k0’kb’

(t)

VAA VP

Two-compartment/two rate constant model- Brain Proteins

Free amino acid pool (compartment)

Protein (of interest)pool (compartment)

20

Two-compartment/two rate constant model

]14)[''(]14[

0 NAAkkdt

NAAdV asAA

]14[']14[']14[

NPkNAAkdt

NPdV bsP

][]15)[''(]15[

0 AARaNAAkkdt

NAAdV asAA

]15[']15[']15[

NPkNAAkdt

NPdV bsP

][]15[]14[ AANAANAA

][]15[]14[ PNPNP

Solution of two-compartment/two-rate constant model

tkeAA

NAAt 01

][

]15[)(

b

tk

b

tkb

kk

ek

kk

ek

P

NPt

b

0

0

0

0

1][

]15[)(

AAAA

s

V

k

V

kkk

''' 000

P

bb V

kk

'

][

][

'

'

AA

P

k

k

b

s

0 5 10 15 20 25 30 350

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

t

btk kket b

0,1)(

22

Empirical delayed exponential model

Incorporation Time (day)

988.02

day 23.1

day 0.0563

1)(

1-0

1-

)0(

R

t

k

ety ttk

0 5 10 15 20 25 30 3500.10.20.30.40.50.60.70.80.9

1

RIA

Incorporation Time (day)

15N

Rel

ativ

e F

ract

ion

0 5 10 15 20 25 30 350

0.1

0.2

0.3

0.4

0.5

0.6

0.7

9989.02

day 0.1587

day 0.0373

1)(

1-0

1-

0

0

0

0

R

k

k

ekk

ke

kk

kt

b

tk

b

tk

b

b b

Two-compartment/two rate constant model

Phosphatidyl-ethanolamine-binding

protein 1, P70296 in brain

23

14NAA

15NAA

14NPi

15NPi

ksi

k0a

Ra*H(t)

(t)

14NPt

15NPt

kbi

VPt VAA VPi

k0t

kst

kbt

Three-compartment/five rate constant modelfor Liver Proteins

24

0 5 10 15 20 25 30 350

0.10.20.30.40.50.60.70.80.91

0 5 10 15 20 25 30 350

0.10.20.30.40.50.60.70.80.91

(a) (b)

15N

Rel

ativ

e F

ract

ion

Incorporation Time (day) Incorporation Time (day)

kst =0.713day-1

k0 =2.002day-1

kbt =0.026day-1

kbi =0.317day-1

R2 =0.9995

kb =0.137day-1

k0 =1.62X1010day-1

R2 =0.981

Two-compartment/two rate constant model Three-compartment/four rate constant model

transitional endoplasmic reticulum ATPase (Q01853), liver

Protein incorporation curve is constructed from 37 of a total of 45 peptide curves

25

Compartment Modeling

1. Fit better to experimental data with a minimal

number of parameters

2. Fitting parameters have biological significance

3. Individual rate constants are determined

26

Studies

Cellular models – on goingAging models – on going Disease models: Prion infected - planned

Technical improvement

High sensitivity Instrument - installedLC alignment – implementedProcessing speed and QC – on going

27

John C. Price Sina Ghaemmaghami

Stanley B. Prusiner

Shigenari Hayashi

Alma L. Burlingame

神经退化性疾病研究所

药化系质谱中心