Integrative Proteomics 01/24/2001 Mass Spectrometry for Protein Identification Brian Cox Integrative...

Integrative Proteomics01/24/2001

Mass Spectrometry for Protein Identification

Brian Cox Integrative Proteomics

Translation elongation factorEF-Tu


Protein Characterization by Mass Spectrometry

• Protein identification– correlative database matching

• Quality control– recombinant protein, protein modification reactions

• Post-translational modifications– phosphorylation, glycosylation, methylation, etc

• Identification of Domains– Functional structural domains, pro-enzyme to enzyme cleavage


Sarcoplasmic Reticulum Terminal Cisternae Fraction

Myosin PI3 Kinase

TAK-1 TGF beta activated kinase Sarcaluminum precursor

Diamine oxidase 6-Phosphofructokinase, aconitase

GRP 78 HSP 70, GRP 75

Calsequestrin, SK muscle Calsequestrin, SK muscle

SR 53 kDa, ATP synthase alpha

Glyceraldehyde 3-phosphate dehydrogenase Malate dehydrogensase L-lactate dehydrogenase

Calretenin

Succinate dehydrogenase

GTP:AMP Phosphotransferase

SFI SFI

SFI 3-hydroxyacyl-CoA dehydrogenase type II

SFI Myosin light chain

SFI

Myosin regulatory l, light chain two SFI

SFI

SFI

Cytochrome C

Chaperonin 10

Nitric oxide synthase

Calcium transporting ATPase (SERCA1)

6-Phosphofructokinase Glycerol-3-phosphate dehydrogenase ADP, ATP carrier protein

ATP synthase Alpha subunit ATP synthase Alpha/Beta subunit

ATP synthase Beta subunit

Actin SFI SFI

ADP, ATP carrier protein

SFI SFI

SFI

SFI

SFI

Cytochrome C

SFI SFI Complex I-MLRQ

} Similar spectra

} Similar spectra

S P

HSP60, Rabbit serum albumin

ATP synthase beta Creatine kinase, Citrate synthase

Fructose bisphosphate aldolase Aspartate aminotransferase

Actin

Voltage independant pH sensitive K+ channel

3-hydroxyacyl-CoA dehydrogenase type II


0Salt Elution

[NusA]

Ligand

RNA polymerase subunit ’RNA polymerase subunit

Hypothetical ORFRNA polymerase subunit

SUHB

Affinity Chromatography - E. coli NusA


2D Gel Analysis


Identifying and Mapping Protein Domains by Partial Proteolysis

NusA

[Trypsin]

ProteolyticProducts

0


How Do I Identify Isolated Proteins?

• Genetics – applicable to simple organisms

– time consuming

• Antibody screening – identifies only known, usually well characterized proteins

• Edman sequencing – requires large quantities of sample

• Mass Spectrometry – Correlative mass matching (sequence must be known)

– MS/MS fragment data (sequence)


MS Protein Identification

MALDI-ToF

Triple quad

Ion Trap

Q-ToF

FT-MS

Liquid

Solid

time consuming, PTMs, protein sequencing

high throughput, sensitive

Instruments


MALDI-ToF vs ElectrosprayWhich do I use?

• Experimental design – What kind of sample will I have

• Reason for analysis– Unknown protein, quality control or mapping of a

known protein

• Species– Is the genome fully sequenced?


MALDI-ToF MS

100 well sample plate

Bruker DaltonicsReflex III ©

384 well sample plate


MALDI-TOF

• Typically a resolved (isolated) protein• Protein identified by correlative mass mapping of

peptides generated by a known chemistry– Enzymatic or chemical

• Protein sequence must be in a database for identification

• Identification meausred by a statistical matching score


Electrospray MS

Applied Biosystems | MDS SCIEX API 3000™ LC/MS/MS System

Applied Biosystems | MDS SCIEX QSTAR™ Hybrid LC/MS/MS Quadrupole TOF System


Electrospray MS

• Usually requires a chromatographic resolving step as only a limited number of peptides can be analyzed at one time

• Full cell lysate or sub-fraction can be analyzed• Generate MS/MS data (fragmentation of peptide

for de novo sequence or small sequence tags)• Fragmentation can generate enough data that only

one or two peptides are required for a positive identification


MALDI-ToF Schematic

Detectors

Flight tube

Timed ion selectorLaser

Sampleplate

Reflector

Acceleratingfield

++

+

+

+

+ +


In-Gel Sample Preparation

C E Candidate protein bands are excised, reduced, alkylated, and digested overnight with trypsin.

Tryptic peptides are recovered, purified and analyzed by MALDI-ToF mass Spectrometry.


Reflector MALDI Spectrum


Correlative Database Searching

• Based on the principal that each protein generates its own unique “fingerprint” of proteins after cleavage by an enzymatic or chemical method into smaller peptides

• A list of experimental masses can then be compared to lists of calculated masses for each known sequence



• Sequence database used to generate lists of peptide masses based on chemistry rules– Enzymatic (trypsin) or chemical cleavage (CNBr)

• Parameters of search– Chemistry of peptide generation, database restrictions:

mol. wt., taxa, error tolerance for peptide matching



• Mann et al, 1993 used intact mass and peptide masses to identify unknown proteins.

• Error was from 0.5 to several Daltons for peptides due to poor resolution, average masses only

• Protein with most peptides matching was ranked first, true mass correlation

• Problem was large proteins (>100kDa) generate large number of peptide masses, spurious matches

• Use restriction of maximum protein size to prevent this

• Total protein sequences available was only 26,000



Available number of sequences as of Nov. 1999NCBInr 415408

Available number of sequences as of June 2000NCBInr 510935

Available number of sequences as of Jan. 2001NCBInr 606272



• Improvements in instrument mass accuracy and resolution• Countered by dramatic increase in number of sequences

available to search• Develop new searching algorithms using weighted

statistical value of a peptide, larger mass peptide contains more “unique information”

• Knowledge of distribution frequencies of peptide masses allowed for calculation of probability for a match (Perkins et al 1999 and Eriksson et al 2000) – using database size (number of peptides), error tolerance, number

of matching peptides (weighted)



• More parameters for calculation of a match– Correlation coefficient (r2) for matching peptides to

error tolerance– Amino acid composition from chemical tags or MS/MS

spectrum– Calculation of a Z score for true probability certainty of

the match ”…a Z score is estimated when the search result is compared against an estimated random match population. Z score is the distance to the population mean in unit of standard deviation.”

Proteometrics at the 48th ASMS annual meeting


Reflector MALDI Spectrum


Proteometrics home pageProfound Search


Profound Search


Protein Prospector HomepageMS-Fit Search


MS-Fit Search


Homologous Proteins

Matching peptide to Xenopus protein

Human protein spectrum


Homologous Proteinstrans reg pw ang #36

Residue NumberN 100 200 300 400 500

1

3

5

7

9

11

13

15

17

19

#

# Mass Matching sequence (± error)

1 3100.3 K[1-26]R (+0.35)

2 3123.3 T[27-52]K (+0.21) G[174-201]R (-0.99)

3 1099.1 K[73-81]K (-0.77) T[420-428]K (-0.81)

4 2754.3 G[147-172]K (-1.06)

5 2625.8 E[190-211]K (+0.95)

6 3407.4 S[229-258]K (-2.75)

7 2349.6 Y[273-293]K (+0.20)

8 1648.6 N[294-307]R (+0.18)

9 1355.4 I[308-319]R (+0.12)

10 1677.8 N[320-334]K (+0.01)

11 2855.4 N[320-344]K (-1.24) N[204-228]K (-1.25)

12 1827.0 A[345-361]R (+0.03) E[197-211]K (-1.07) K[128-143]K (+0.16)

13 2154.4 H[377-395]K (-0.07) K[109-127]K (-1.06)

14 2212.4 H[396-412]R (-0.04)

15 2568.7 N[424-444]R (+0.18)

16 1937.2 V[429-444]R (-0.02)

17 1295.1 E[435-444]R (+0.28)

18 1111.0 D[445-454]R (-0.84)

19 1129.0 N[496-504]K (+0.32)

20 1172.0 No matches



















Xenopus Human EST

Linear sequence of protein

Pep

tid

e m

atch

nu

mb

er


MS/MS

• Fragmentation of parent ion (a peptide) to produce sequence dependent data

• Fragmentation induced in MALDI as post source decay

• In electrospray by CID (collision induced decay)• Searching done by similar principles


MALDI-ToF Schematic

Detectors

Flight tube

Timed ion selectorLaser

Sampleplate

Reflector

Acceleratingfield


Peptide Fragmentation

P. Roepstorff and J. Fohlman, Biomed. Mass Spec. 11 (1984) 601. K. Biemann, Biomed. Env. Mass Spec. 16 (1988) 99.



NH3-D R V Y I H P F H L-COOH

P F H L-COOH

Y I H P F H L-COOH

NH3-D R V

NH3-D R V Y I H

R V Y

I H P H

y series

b series


Peptide Fragmentation :FYEEVHDLER

R E L D H V E

F Y E E V H D

HD

VH

D

EV

HD

EE

VH

D

B ions

Y ions


ElectrosprayHPLC UV trace

MS scan (deconvoluted)

MS/MS of selected ion


Electrospray

• Continuous scanning for peptides above a threshold intensity

• Selected peptides under go MS/MS

• Return to scan to find next candidate

• Slow sample introduction for increased peak width off HPLC to allow more time of MS/MS experiments

• MS/MS process less than a few seconds

• Data used for search is a peptide mass plus any sequence tags determined from MS/MS


Unmatched spectrum

PSD candidates


PSDs of candidate peptides


Coverage of ESTNew Sequence

Residue NumberN 50 100 150 200

1

3

5

7

#

# Mass Matching sequence (± error)

1 2789.4 G[32-57]K (-0.35)

2 2903.9 F[58-84]K (-0.73)

3 1665.0 V[90-103]R (-0.10)

4 2959.7 A[104-130]R (-0.43)

5 1429.4 D[119-130]R (+0.10)

6 917.1 I[131-137]R (+0.00)

7 4879.0 C[138-182]K (-0.53)

8 861.5 No matches

9 880.2 No matches

























EST PSD

Linear sequence of protein


Conclusion

• Mass spectrometry will continue to be a powerful tool for the analysis of biopolymers

• Further improvements in instrumentation and computational sciences will increase the utility and versatility of mass spectrometry

• Simplification of process will lead to greater use and availability of this technology– e.g. wide variety molecular biology kits currently

available


Contact Us

Integrative Proteomics is located in Downtown Toronto

For employment inquires please see our web page at

www.integrativeproteomics.com

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