1

Proteomics

George Tsaprailis, DirectorLinda Breci, Associate Director

Arizona Proteomics ConsortiumUniversity of Arizona

What is proteomics and how is Mass spectrometry used

in proteomics ?

2

Proteomics: the study of the Proteome

A collection of proteins, usually comprising

a biological system

Important because (1) proteins perform most cellular

functions, (2) proteins are the major elements of

most cellular structures, and (3) proteins are targets

of drugs/toxicants

Why Proteomics?Mass Spectrometry

Protein ID

3

Protein Chemistry

MassSpectrometry

Computing (+ Bioinformatics)

Proteomics involves-

Protein Chemistry

•Sample isolation/clean-up

•Sample purification

Protein

fractions digest

peptides

4

MassSpectrometry

Key to Proteomics is to obtain peptide masses and/or

sequences

Proteinmixture

MS analysis

MS data

Proteinsseparation

Peptidemixture

digestion

separationPeptides

digestion

MS MS/MS

MassSpectrometry

All types of hardware used in proteomics

5

Computing (+ Bioinformatics)

The Proteomic Approach

Sample

Pre-prep

steps

Protein

DigestPeptides

Mass

Spectrometer

ESI

LC-MS/MS

MALDI

MS

1D PAGE 2D PAGEProtein(s) Solution

HPLC fractions

IP eluent

Protein Id + Informatics

Protein Chemistry

MassSpectrometry

Computing (+ Bioinformatics)

6

What is proteomics and how is Mass spectrometry used

in proteomics ?

Mass Spectrometry

• What is a mass spectrometer and what does it measure?– An instrument that makes ions

– Measures the mass/charge (m/z) of ions

• Mass Spectrometry in proteomics– For proteins and peptides

• whole protein mass measurements

• protein identification based on peptide mass measurement

• protein identification based on peptide structure analysis (fragmentation)

• Need to know some basic principles

7

Protein/peptide relationship

Enzyme

Protein Peptides

Making ions

H2N CH C

CH3

O

HN CH C

CH2

O

CH CH3

CH3

HN CH C

CH2

O

HN CH C

CH2

OH

O

CH2

CH2

CH2

NH2

Ala-Leu-Phe-Lys mass of neutral = 477.3

H+

Ala-Leu-Phe-Lys m/z of singly charged = 478.3

Ala-Leu-Phe-Lys m/z of doubly charged = 239.6

H+

8

Making ions

Ions are made in an ion source

Important methods in Proteomics:

1) MALDI (matrix assisted laser desorption)

2) ESI (electrospray ionization

Electrospray IonizationESI

Matrix Assisted LaserDesorption IonizationMALDI

1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

0

25

50

75

100

5000 10000 15000

0

25

50

75

100

14306.0

Inte

nsity

m/z

+8

1789.00

+9

1590.33

+13

1101.40

+10

1431.47

+111301.53

+12

1193.20

Rela

tive In

tensity

m/z

CalculatedMass Spectrum

10000 20000 30000 40000

0

2000000

4000000

6000000

8000000

10000000

12000000

14000000

16000000

[2M+H]+

14318.68

[M+2H]2+

7157.18

[M+H]+

14318.68

Inte

nsity

m/z

[2M+H] +

28,638.7

9

Analyzing ions

The ion source is coupled to the analyzer

Important analyzers in Proteomics:

1) TOF (time of flight)

2) Ion Trap

Matrix Assisted Laser Desorption (MALDI)

(ion source)

Time of Flight (analyzer)

Pulsed

laser light

Detector

Analyzer

Ion Beam

Sample and matrix

on tip of solid probe

+

+

+ +LASER

FLIGHT TUBE ANALYZER

10

Time of Flight (TOF)

http://www.abrf.org/ABRFNews/1997/June1997/jun97lennon.html

Linear Mode:better sensitivitypoor resolution

Reflectron Mode:less sensitivityhigher resolution

MALDI-TOF spectrum (mix of peptides)

m/z500 2500

90

0

D:\011003_500fmol\Bsaintcal\2Ref\pdata\1\1r (11:26 10/04/01)

x 4.0

Ref

Ref

11

MALDI Reflectron Spectrum Of ACTH

M@LDI13-Nov-2003

2444 2446 2448 2450 2452 2454 2456 2458 2460 2462 2464 2466 2468 2470 2472 2474 2476 2478 2480 2482m/z0

100

%

ACTHResCk 3 (0.098) Cm (1:5) TOF LD+ 6.57e32467.344

2466.324

2451.525

2468.330

2469.316

2470.337

2471.290

Electrospray (ESI) (ion source)

Ion Trap (analyzer)

Detector

Analyzer

Ion Beam

Liquid sample sprayed

from needle or capillary

++

+

++

+

++

+

Dry gas

or Heat

4500 V

HPLC

ESI

ION TRAP ANALYZER

12

ESI-Ion Trap Spectrum

200 400 600 800 1000 1200 1400 1600 1800 2000

0

20

40

60

80

100

Re

lative In

ten

sity

m/z

476.9

952.4

1903.4

[M+H]+ = 951.4 + 1 = 952.4

[M+2H]2+ = (951.4 + 2) / 2 = 476.7

[2M+H]+ = (951.4 x 2) + 1 = 1903.8

[M+2H]2+

[2M+H]+

[M+H]+

View an ion trap animation

• Exercise 1

13

Resolution and mass accuracy

varies by instrument

MASS RANGE Resolution Accuracy (Error)

m/z (at m/z 1,000) (at m/z 1,000)

2,000 (full scan)

10,000 (zoom scan)

0.006% (60 ppm) Ext. Cal.

0.003% (30ppm) Int.Cal.

INSTRUMENT

to 4,000FTICR

MALDI/TOF to 400,000

LCQ (Ion Trap)

15,000 (reflectron)

0.0001% (1ppm)

0.03% (300 ppm)to 2,000

500,000

610)(

×−

=

lMWTheoretica

MeasuredMWlMWTheoreticappm

Resolution

http://www.matrixscience.com

Resolution

30,000

10,000

3,000

1,000

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MALDI Reflectron Spectrum Of ACTH

M@LDI13-Nov-2003

2444 2446 2448 2450 2452 2454 2456 2458 2460 2462 2464 2466 2468 2470 2472 2474 2476 2478 2480 2482m/z0

100

%

ACTHResCk 3 (0.098) Cm (1:5) TOF LD+ 6.57e32467.344

2466.324

2451.525

2468.330

2469.316

2470.337

2471.290

You must know the resolution of your

instrument to analyze the data!

– We need to know the possible error in the measurement

– Is the peak monoisotopic?

– Is the peak average?

15

Analysis of whole proteins

by MALDI-TOF and ESI-Ion trap

• MALDI-TOF = measure with 1 or 2 protons

– large molecules like Proteins require Linear mode

(much lower resolution)

• ESI-Ion Trap = measure with many protons (high charge state)

– mass of the protein can be calculated from the

multiply charged peaks

Mass Spec measures isotopesExcel calculated example: Carbon is 12.000

For every 12C there is 1.1% 13C

10 carbons

100

10.8

0.5

0

20

40

60

80

100

120

1 2 3 4 5 6 7

isotopes add up10 carbons = 11% 13C Peak

for 100 carbons, the 13C peakis larger than the 12C peak

100 carbons

92.5

100

53.5

18.8

51 0.2

0

20

40

60

80

100

120

1 2 3 4 5 6 7

16

Proteins have very large isotope widthsTheoretical Isotope distribution of Lysozyme

9th Isotope14313.906

1st Isotope14304.885

Isotope # m /z % M ax im um

0 14304.885 0.2

1 14305.888 1.2

2 14306.891 4.6

3 14307.893 12.8

4 14308.896 26.9

5 14309.898 46.3

6 14310.900 67.6

7 14311.902 86.3

8 14312.904 97.7

9 14313.906 100.0

10 14314.908 93.5

11 14315.910 80.4

12 14316.912 64.2

13 14317.914 47.8

14 14318.916 33.4

15 14319.918 21.8

16 14320.920 13.2

17 14321.922 7.5

18 14322.924 3.9

19 14323.925 1.8

20 14324.927 0.7

21 14325.929 0.2

Lysozyme by MALDI/TOF

Average mass = 14,314

10000 20000 30000 40000

0

2000000

4000000

6000000

8000000

10000000

12000000

14000000

16000000

[2M+H]+

14318.68

[M+2H]2+

7157.18

[M+H]+

14318.68

Inte

nsity

m/z

[2M+H]+

28638.68

[M+H]+

14316.24

[2M+2H]2+

7157.18

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Lysozyme by ESI-Ion Trap

Average mass = 14,314

1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

0

25

50

75

100

5000 10000 15000

0

25

50

75

100

14306.0

Inte

nsity

m/z

+8

1789.00

+9

1590.33

+13

1101.40

+10

1431.47

+11

1301.53

+12

1193.20

Re

lative

In

ten

sity

m/z

CalculatedMass Spectrum

14318.2

So we’ve made measurements

Now What?

• A lot of information is available on-line about proteins and/or the gene

• We will explore protein information in general

• We will then use the available info to perform data analysis

18

MALDI-TOF analysis of Alkaline

phosphatase – Computer Exercise #2

0 25000 50000 75000 100000

2.00E+008

4.00E+008

6.00E+008

8.00E+008

1.00E+009

1.20E+009

Inte

nsity

m/z

[M+2H]2+

23445.54

[M+H]+

47155.22

[2M+H]+

94472.97

Protein identification – Two strategies

• single stage mass spectrometry (MS)

– called “peptide mass mapping”

– measure all peptides in one spectrum

– MALDI-TOF

– produces low confidence results

• tandem mass spectrometry (MS/MS)

– measure peptides as they elute from an HPLC

– ESI-Ion Trap

– produces high confidence results

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Single Stage – Peptide mass mapping

Using MALDI-TOF

MALDI-TOF Spectrum of tryptic digest

m/z500 2500

90

0

D:\011003_500fmol\Bsaintcal\2Ref\pdata\1\1r (11:26 10/04/01)

x 4.0

Ref

Ref

20

MALDI Reflectron Spectrum Of ACTH

M@LDI13-Nov-2003

2444 2446 2448 2450 2452 2454 2456 2458 2460 2462 2464 2466 2468 2470 2472 2474 2476 2478 2480 2482m/z0

100

%

ACTHResCk 3 (0.098) Cm (1:5) TOF LD+ 6.57e32467.344

2466.324

2451.525

2468.330

2469.316

2470.337

2471.290

Data Analysis for peptide mass mapping

• Important data

– multiple peaks

– mass accuracy

– confirming information

(pI, approx. mass,

organism, etc.)

?MS

MS Peptide MWFound in Selected

DatabasesNDALYFPT...

SWDLTAL...

PTDLDVSY...

protein peptides identify

rank

for example:Measured Peptide = 1274.5183

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Data Analysis for peptide mass mapping

>gi|27807105|ref|NP_777037.1| solute carrier family 6 (neurotransmitter transporter,

glycine), member 9 [Bos taurus]�gi|1279843|gb|AAB01159.1| glycine transporter

MAAAQGPVAPSKLEQNGAVPSEATKSDQNLGQGNWRNQIEFVLTSVGYAVGLGNV

WRFPYLCYRNGGGAFMFPYFIMLIFCGIPLFFMELSFGQFASQGCLGVWRISPMFK

GVGYGMMVVSTYIGIYYNVVICIAFYYFFSSMTPVLPWTYCNNPWNTPDCMSVLDN

PNITNGSQPPALPGNVSQALNQTLKRTSPSEEYWRLYVLKLSDDIGNFGEVRLPLLG

CLGVSWVVVFLCLIRGVKSSGKVVYFTATFPYVVLTILFIRGVTLEGAFTGIMYYLTPQ

WDKILEAKVWGDAASQIFYSLGCAWGGLVTMASYNKFHNNCYRDSVIISITNCATSV

YAGFVIFSILGFMANHLGVDVSRVADHGPGLAFVAYPEALTLLPISPLWSLLFFFMLILL

GLGTQFCLLETLVTAIVDEVGNEWILQKKTYVTLGVAVAGFLLGIPLTSQAGIYWLLLM

DNYAASFSLVIISCIMCVSIMYIYGHQNYFQDIQMMLGFPPPLFFQICWRFVSPAIIFFIL

IFSVIQYQPITYNQYQSSQTGLPLFTCQIAPAHVPQPLSGARTPSPKPWSVRVSVLRA

PLCSDSPGRAASNPL

MAAAQGPVAPSK = 1127.5883LEQNGAVPSEATK = 1343.6807SDQNLGQGNWR = 1274.5878

Measured Peptide = 1274.5183

1274.5878 theoretical

1274.5183 measured

0.0695 difference

error = 55 ppm

Data Analysis for peptide mass mapping

• Important data

– multiple peaks

– mass accuracy

– confirming information (pI, approx. mass, organism, etc.)

?MS

MS Peptide MWFound in Selected

DatabasesNDALYFPT...

SWDLTAL...

PTDLDVSY...

protein peptides identify

rank

22

Computer Exercise #4

Analyze peptide mass mapping data

• 4 lists of peptide masses provided on

worksheet

– (Alternate address of excel data):

http://www.chem.arizona.edu/facilities/msf/index.html

Problems with whole protein analysis

• Peaks are broad

– large groups of isotope peaks

– peaks further broadened by adducts (contaminants, salts)

• Proteins are often modified

– Instrument may not resolve the mass difference

– No information regarding which amino acid is modified

• Proteins are in a complex matrix

– background stuff

– other proteins (too complex!!!)

Therefore proteins are identified from peptides!

23

How are proteins separated

• Proteins from biological organisms are a complex mixture

• Separating proteins

– 1D SDS-PAGE

• Cross linking controls MW separated

• Low resolution technique, spot can contain 10's to

100's of proteins

– 2D SDS-PAGE

• Best for complex protein mixtures (IEF + SDS-PAGE)

• Other methods

– Chromatography (reverse phase, size exclusion, ion

exchange, affinity)

– Preparative isolectric focusing (IEF)

Protein Mixture

or IP eluant

1D SDS-PAGE

�Great clean-up tool (rid of salts, detergents, etc…)

�Great concentration tool

�Biological analytes

�Various stains available – various detection limits

�USE PRECAST GELS (polymer issue) if possible

�Various size gels (spatial resolution)

�Various MW ranges

1D Electrophoresis

24

http://www.biorad.com

1D Electrophoresis

Separation on the basis of intrinsic charge (pKa)

isoelectric focusing

Se

pa

ratio

n o

n t

he

ba

sis

of

Siz

e

PA

GE

(S

DS

ge

l e

lectr

op

ho

resis

)

(1)

(2)

2D Electrophoresis

25

Protein Mixture or IP eluant

or Cell/tissue2D SDS-PAGE

�Great clean-up tool (rid of salts, detergents, etc…)

�Various stains available – various detection limits

�Protein profiling

�Various pH ranges

�2D gels are very much sample related (sample may require further clean-up

prior to 2D gel

Avoid excess salts in sample (not focus, IPGs burn, 30-40 mM max salt)

�Often Automated w/ robotics–high throughput (MALDI-TOF)

�Often good for visualizing PTMs

2D Electrophoresis

+ –pH 3 pH 7.5 pH 10

+ –

pH 3 pH 7.5 pH 10

+ –

pH 3 pH 7.5 pH 10

+ –

pH 3 pH 7.5 pH 10

The 1st D: Isoelectric Focussing

26

+ –

pH 3 pH 7.5 pH10

–pH 3 pH 7.5 pH10

+ –pH 3

pH 7.5pH10

Proteins

migrate

through the gel

at a rate

proportional to

their size

Smallest

proteins travel

the furthest

distance

charge

size

The 2nd D: SDS-PAGE

• Do Computer Exercise #3

• Laboratory tour