1.proteomics coursework-3 dec2012-aky

Course B

WHY BOTHER WITH PROTEOMICS?

• Proteins are the machines that drive much

of biology

• Genes are merely the recipe

• The direct characterization of a sample’s

proteins en masse.

• What proteins are present?

• How much of each protein is present?

WHY NOT MICROARRAYS?

Is Proteomics the New Genomics? Jürgen Cox and Matthias Mann, Cell 130, August 10, 2007

ONE GENOME…MANY PROTEOMES

Perhaps not… they

still have a

dynamic

“proteome” code

to break. They

cannot hit a

moving target

AN ANALYTICAL CHALLENGE

Dynamic range of protein

abundances is a challenge for

separation sciences

No equivalent of PCR for

proteins-deal with µ- to nmolconcentrations

Alternate splice forms of

a gene can make different

proteins

>200 Post translational

modifications; cannot be

deduced from a gene or

mRNA

Edman sequencing cannot provide the solutions !!!

TOOLS FOR PROTEOMICS

Sequence databases

DNA

ESTs

Protein

Mass Spectrometry

Ionization

techniques

Analyzers

Software

PMF

MS/MS

De Novo Sequencing

Protein Separation Technology

2D-GE

LCMS

MASS SPECTROMETRY

The PCR for proteins ?

MASS SPECTROMETRY

Analytical method to measure the molecular or atomic

weight of samples

Slide adopted from: Dr.. Ahna Skop. Mass Spectrometry: Methods & Theory

SOFT IONIZATION METHODS

337 nm UV laser

MALDI

cyano-hydroxy

cinnamic acidGold tip needle

Fluid (no salt)

ESI

+

_

Slide adopted from: Nathan EdwardsCenter for Bioinformatics and Computational Biology(UMIACS)

MASS SPECTROMETRY PRINCIPLES

Ionizer

Sample

+

_

Mass Analyzer Detector


MASS SPEC EQUATION (TOF)

mz

2Vt2

=

m = mass of ion L = drift tube length

z = charge of ion t = time of travel

V = voltage

L2

MONOISOTOPIC MASS

www.matrixscience.com

•Mass of the most abundant isotope of a molecule

•Measured in amu or Da

•Usually the lightest isotope of small molecules

UNDERSTANDING A SPECRUM

m/z

Rela

tive I

nte

nsity

853.2 854.3 1200.5

1201.0

+2

+1

(1200.5 × 2) – 2 = 2399.0

MS INSTRUMENTS

A Brief Summary of the Different Types of Mass Spectrometers Used in Proteomics

Methods in Molecular Biology, vol. 484: Functional Proteomics: Methods and Protocols

IDENTIFICATION STRATEGIES

Experimental

masses

Theoretical

Masses

(database)

1. Peptide mass fingerprinting(PMF)

2. MS/MS spectral matching

Experimental spectrumTheoretical spectra

3.De novo sequencing*

72.0 129.0 97.0 101.0 113.1 174.1

A E P T I R H2O

*Adopted from: Brian C. Searle, Proteome Software Inc. Portland, Oregon USA

4. Spectral library search

Nesvizhskii. Journal of Proteomics ,2010

PEPTIDE MASS FINGERPRINTING

A rapid way to identify proteins

PEPTIDE MASS FINGERPRINT

The proteins from a sample are separated on 2D gels

Protein of interest is digested by trypsin (or any other site specific cleavage)

Ionization of peptides in a MALDI mass spectrometer

m/z values detected and plotted as mass spectrum

PMF database search to identify the protein

PROTEASE DIGESTION

trypsin

PEPTIDE MASS FINGERPRINT

m/z

Rela

tive I

nte

nsity

PMF DATABASE SEARCH

450.2201

609.3667

698.3100

1007.5391

1199.4916

2098.9909

PEAKLIST

>gi|2924450|emb|CAA17750.1| PROBABLE FATTY-ACID-CoA LIGASE FADD18 (FRAGMENT) (FATTY-ACID-CoA

SYNTHETASE) (FATTY-ACID-CoA SYNTHASE) [Mycobacterium tuberculosis H37Rv]

MAASLSENLSCHSSNMCRLSGNAATNLERPGEEPPGDRCTRRQAVRPARTLAKKGNIPVGYYKDEKKTAETFRTINGVRYAIPGD

YAQVEEDGTVTMLGRGSVSINSGGEKVYPEEVEAALKGHPDVFDALVVGVPDPRY

GQQVAAVVQARPGCRPSLAELDSFVRSEIAGYKVPRSLWFVDEVKRSPAGKPDYRWAKEQTEARPADDVH

AGHVTSGS

>gi|15610649|ref|NP_218030.1| fatty-acid-CoA ligase [Mycobacterium tuberculosis H37Rv]

MAASLSENLSCHSSNMCRLSGNAATNLERPGEEPPGDRCTRRQAVRPARTLAKKGNIPVGYYKDEKKTAE

TFRTINGVRYAIPGDYAQVEEDGTVTMLGRGSVSINSGGEKVYPEEVEAALKGHPDVFDALVVGVPDPRY

GQQVAAVVQARPGCRPSLAELDSFVRSEIAGYKVPRSLWFVDEVKRSPAGKPDYRWAKEQTEARPADDVH

AGHVTSGS

Protein FASTA

database450.2017 (P21234)

609.2667 (P12345)

664.3300 (P89212)

1007.4251 (P12345)

1114.4416 (P89212)

1183.5266 (P12345)

1300.5116 (P21234)

1407.6462 (P21234)

1526.6211 (P89212)

1593.7101 (P89212)

1740.7501 (P21234)

2098.8909 (P12345)

in silico

digestion

OUTPUT:

2 Unknown masses

1 hit on P21234

3 hits on P12345

RESULT:

protein is P12345

22

MODIFICATIONS

Fixed modifications: will be present on any

occurrence of the affected amino acid.Eg.+57@C

Variable modifications: may be present on some

or all positions of the affected amino acid.

Eg.+16@M


TANDEM MASS SPECTROMETRY

Peptide Sequencing by two stage MS

PRECURSOR SELECTION

m/z

Rela

tive I

nte

nsity

MS1

Tandem MS or MS/MS or MS2

Unfragmented

parent/precursor ion

COLLISION INDUCED DISSOCIATION

CID in presence of inert gas

26

FRAGMENTATION

PEPTIDE

MW ion ion MW

98 b1 P EPTIDE y6 703

227 b2 PE PTIDE y5 574

324 b3 PEP TIDE y4 477

425 b4 PEPT IDE y3 376

538 b5 PEPTI DE y2 263

653 b6 PEPTID E y2 148

SHOTGUN PROTEOMICS & DATABASE

SEARCH

The pros and cons of peptide-centric proteomics. Mark W. Duncan, Ruedi Aebersold, Richard M. Caprioli

Nature Biotechnology, Vol. 28, No. 7. (01 July 2010), pp. 659-664

DATABASE SEARCH ALGORITHMS

SEQUEST

Mascot

X!Tandem

OMSSA

ProbID

Phenyx

Myrimatch

MassWiz

DE NOVO SEQUENCING

Sequencing a peptide from scratch

30

DE NOVO INTERPRETATION

100

0250 500 750 1000

m/z

% I

nte

nsi

ty


31


100

0250 500 750 1000

m/z

% I

nte

nsi

ty

E L


32


100

0250 500 750 1000

m/z

% I

nte

nsi

ty

E L F

KL

SGF G

E DE

L E

E D E L


33

SUMMARY

Proteomics is large-scale study (qualitative and

quantitative) study of proteins by mass spec.

Mass spectrometry + sequence databases

represent a huge leap for protein (bio-)chemistry.

ProteinSeparation - 2DGE and HPLC

Ionization - MALDI and ESI

Identification - PMF, MS/MS and de novo

sequencing

Sample prep, instruments and algorithms still

maturing, much work to be done.

NEXT…

Significance Assessment of database matches

False Discovery rate

Protein Inference

1.proteomics coursework-3 dec2012-aky

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