Introduction in Proteomics

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Introduction in Proteomics

Overview

• Proteomics definition

• From genome to proteome

• Aminoacids and proteins

• What proteomics can do?

• Peptide fragmentation

• Proteomics and gel electrophoresis

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• Post translational modifications

• DD NL MS3 for phosphorylation identification

• Intact protein analysis

• Lock mass

• Protein quantification

• RP-HPLC basics

Proteins

= molecules of amino acids that perform much of life’s function

Proteome

Easy to remember

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= set of all proteins in a cell

Proteomics

= study of protein’s structure & function

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Proteomics represents the effort to establish the identities,

quantities, structures, and biochemical and cellular functions of all proteins in an organism, organ, or

organelle, and how these properties vary in space, time, or

Not so easy to remember

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organelle, and how these properties vary in space, time, or

physiological state.

MCP 1.10 pg 675 National Research Council

Steering committee

A true multidiscipline science

• Protein chemistry

• Mass spectrometry

• Genomics

• Bioinformatics

Proteomics

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• Bioinformatics

• Computer science

• Separation science

From Genome to

Proteome

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Proteome

From cell to gene

The genes, parts of the DNA (double helix), are the functional units of the genome.

The human genome is located right in the heart of the cells, in the nucleus.

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They hold all the information necessary to create the proteins you need.

Genes are located along thread-like

structures called chromosomes.

Protein Synthesis

Protein synthesis

is the transcriptionand translation of

specific parts of

DNA to form

proteins.

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Codons set amino acids that are used

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One Genome, multiple Proteomes

tagacgacct ggcccaacgc tgtgcccagt acaagaagga

tggctgtgac ttcgccaaat ggcgttgtgt gctcaagatc ggcaagaaca

ccccctccta ccaagctatc cttgagaatg ccaacgtact ggcacgctat

gcgtccatct gccaatccca gcgcattgtg cccattgtagagcctgaggt

gctgcctgat ggagatcacg accttgacag ggctcagaag

gtcacagaga cagttctggc cgctgtgtac aaggcactca

atgaccacca tgtcttcctg gagggcaccctcctgaagcc caacatggtg

accgcaggac agtcctgctc caagaagtac aattatgagg

acaacgctag agctacagtg ttggccctgt ccagaactgt gccagctgct

gtccctggtgtgactttctt gtcaggaggt cagtcggagg aggatgcctc

tgtcatttgg atgctatcaa caagatc

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tagacgacct ggcccaacgc tgtgcccagt acaagaagga

tggctgtgac ttcgccaaat ggcgttgtgt gctcaagatc

ggcaagaaca ccccctccta ccaagctatc cttgagaatg

ccaacgtact ggcacgctat gcgtccatct gccaatccca

gcgcattgtg cccattgtagagcctgaggt gctgcctgat

ggagatcacg accttgacag ggctcagaag gtcacagaga

cagttctggc cgctgtgtac aaggcactca atgaccacca

tgtcttcctg gagggcaccctcctgaagcc caacatggtg

accgcaggac agtcctgctc caagaagtac aattatgagg

acaacgctag agctacagtg ttggccctgt ccagaactgt

gccagctgct gtccctggtgtgactttctt gtcaggaggt

cagtcggagg aggatgcctc tgtcatttgg atgctatcaa

caagatc

The building polypeptide chain

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Central Dogma of Biology

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genome transcriptome proteome

Amino acids

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Amino acids

Amino acid

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• The human body needs 20 amino acids to be able to

make (or synthesize) its thousands of proteins.

Levels of Protein StructureAminoacid structure

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What are Proteins?

Where we find proteins?Cells and body tissues, hormones, antibodies, and enzymes.

Protein functions?

Proteins are strings of amino acids and are the active elements of cells.

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• structure providing proteins (hair and fingernails).

• help in digestion (stomach enzymes), detoxify poisons, or help fight disease.

• cell membranes proteins and are important in controlling how substances pass

through these membranes.

• enzymes proteins are catalysts for biochemical reactions.

• antibodies proteins react with foreign substances to defend the body.

Sourse of Proteins

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• Grow cells and blow them up (lyses)

• Dissect tissue sample, homogenize and lyse

• Synthesize

Sourse of Proteins ( cell fractionation)

nuclei

+ 0.25M NaClnuclei

+ 0.25M NaCl

cells

+hypotonic buffer

+0.5% NP40

cells

+hypotonic buffer

+0.5% NP40

cytosolic proteins

nucleosolic proteins cytosolic proteins

nucleosolic proteins

nuclei

+ 0.25M NaClnuclei

+ 0.25M NaCl

cells

+hypotonic buffer

+0.5% NP40

cells

+hypotonic buffer

+0.5% NP40

cytosolic proteins

nucleosolic proteins cytosolic proteins

nucleosolic proteins

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+ 0.25M NaCl

chromatin-bound Orc1chromatin-bound Orc1

nuclei

+ 0.32M NaClnuclei

+ 0.32M NaCl

pre-elution step

immunoprecipitationimmunoprecipitation

extracted nucleiextracted nuclei

+ 0.25M NaCl

chromatin-bound Orc1chromatin-bound proteins

nuclei

+ 0.32M NaClnuclei

+ 0.32M NaCl

pre-elution step

immunoprecipitationimmunoprecipitation

extracted nucleiextracted nuclei

Protein Immunoprecipitation

Immunoprecipitation (IP) is the technique of precipitating

a protein antigen out of solution using an antibody that

specifically binds to that particular protein.

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specifically binds to that particular protein.

Biological fractionation

• Necessary!! To decrease complexity before analytical

fractionation

– Abundant protein depletion

– Membrane fraction

– Soluble fraction

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– Soluble fraction

– Organellar fractionation

– Affinity chromatography

– Etc…

Separation techniques versus protein characteristics

• Charge1. Ion exchange chromatography

2. Electrophoresis

3. Isoelectric focusing

• Polarity1. Adsorption chromatography

2. Paper chromatography

3. RP chromatography

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3. RP chromatography

4. Hydrophobic interaction chromatography

• Size1. Dialysis and ultrafiltration

2. Gel electrophoresis

3. Gel filtration chromatography (size exclusion chromatography)

4. Ultracentrifugation

• Specificity1. Affinity chromatography

PI Protocol

• Lyse cells and prepare sample for immunoprecipitation.

• Pre-clear the sample by passing the sample over beads that are not coated with antibody to soak up any proteins that non-specifically bind to the beads.

• Incubate solution with antibody against the protein of interest. Antibody can be attached to solid support before this step (direct method) or after this step (indirect method). Continue the incubation to allow antibody-antigen complexes to form.

• Precipitate the complex of interest, removing it from bulk solution.

• Wash precipitated complex several times. Spin each time between washes or place tube on magnet when using superparamagnetic beads and then remove supernatant. After final wash, remove as much supernatant as possible.

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supernatant. After final wash, remove as much supernatant as possible.

• Elute proteins from solid support (using low-pH or SDS sample loading buffer).

• Analyze complexes or antigens of interest. This can be done in a variety of ways:

– SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) followed by gel staining.

– SDS-PAGE followed by: staining the gel, cutting out individual stained protein bands, and sequencing the proteins in the bands by MALDI-Mass Spectrometry

– Transfer and Western Blot using another antibody for proteins that were interacting with the antigen followed by chemiluminesent visualization.

Two amino acids can undergo a condensation reaction to form a dipeptide. Further condensation reactions result in a polypeptide.

R1-COOH + R2-NH2 R1-CO-NH-R2 + H2O

Peptide bond formation

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Amino acids are linked with the peptide bond, amide bond

The aliphatic amino acids

Glycine, Gly, G Alanine, Ala, A Valine, Val, V Leucine, Leu, L

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Glycine, Gly, G Alanine, Ala, A Valine, Val, V Leucine, Leu, L

Isoleucine, Ile, I Proline, Pro, P

The aromatic amino acids

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Phenylalanine, Phe, F Tyrosine, Tyr, Y Tryptophan, Trp, W

The sulfur containing amino acids

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Cysteine, Cys, C Methionine, Met, M

S-S bond

2Cysteines/oxidation=disulfide bond (only in extracellular and not intracellular proteins)

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Disulfide bonds stabilize protein structure by providing crosslink

The hydroxyl amino acids

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Serine, Ser, S Threonine, Thr, T

The acidic amino acids

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Aspartate, Asp, D Glutamate, Glu, E

The amide amino acids

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Asparagine, Asn, N Glutamine, Gln, Q

The basic amino acids

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Lysine, Lys, K Arginine, Arg, R

The imidazole amino acid

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Histidine, His, H

Amino acids that accept a positive charge

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Histidine

H

His

Lysine

K

Lys

Arginine

R

Arg

Aminoacids clasification

Side chains of aminoacids

Responsible for many of the

uniques properties of proteins

• charged or polar groups

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• charged or polar groups

provide interesting catalytic

groups

• nonpolar amino acids - a

protein folding issue

Amino acid 3LC SLC Average Monoisotopic

Glycine Gly G 57.0519 57.02146

Alanine Ala A 71.0788 71.03711

Serine Ser S 87.0782 87.02303

Proline Pro P 97.1167 97.05276

Valine Val V 99.1326 99.06841

Threonine Thr T 101.1051 101.04768

Cysteine Cys C 103.1388 103.00919

Leucine Leu L 113.1594 113.08406

Isoleucine Ile I 113.1594 113.08406

Monoisotopic and Average Mass

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Isoleucine Ile I 113.1594 113.08406

Asparagine Asn N 114.1038 114.04293

Aspartic acid Asp D 115.0886 115.02694

Glutamine Gln Q 128.1307 128.05858

Lysine Lys K 128.1741 128.09496

Glutamic acid Glu E 129.1155 129.04259

Methionine Met M 131.1926 131.04049

Histidine His H 137.1411 137.05891

Phenyalanine Phe F 147.1766 147.06841

Arginine Arg R 156.1875 156.10111

Tyrosine Tyr Y 163.1760 163.06333

Tryptophan Trp W 186.2132 186.07931

What Proteomics

can do?

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can do?

Types of Experiments; key questions of Proteomics

Protein separation In order identify each protein in the mixture.

Protein identificationMass spectrometry

Antibody based assays can also be used, but are unique to one sequence motif.

Edman degradation used to confirm sequence when MS unavailable.

Protein quantificationGel-based methods, differential staining of gels with fluorescent dyes (difference gel electrophoresis).

Gel-free, various tagging or chemical modification methods, label free methods.

Protein sequence analysisSearching databases.

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Searching databases.

Structural proteomics High-throughput determination of protein structures in three-dimensional space. Methods are x-ray crystallography and

NMR spectroscopy.

Interaction proteomicsThe investigation of protein interactions on the atomic, molecular and cellular levels.

Protein modification Phosphoproteomics and glycoproteomics.

Cellular proteomicsThe goal is to map location of proteins and protein-protein interactions during key cell events. Uses techniques such as

X-ray Tomography and optical fluorescence microscopy.

Methods for protein analysis

High resolution mass spectrometry methodsTop down methods

Intact proteins

Bottom up methods

Digested peptides

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Digested peptides

De Novo Sequencing

Gel based methodsOne/Two-dimensional gel electrophoresis

Protein digestion

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Why Trypsin

• Very Specific R/ K/ except R/P K/P

• Very Active

• Can buy it in a very pure form

– Promega, Sigma, Princeton Scientific

• Resistant to digesting itself

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• Resistant to digesting itself

• K and R residues in an average protein are optimally spaced

– Peptides are usually 600-3000 Da

• For electrospray, fragments are at least doubly charged because of C term basic AA’s K and R

Chemical and enzymatic cleavage reagents

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Proteomics and Mass

Spectrometry

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Spectrometry

Methods for protein analysis

High resolution mass spectrometry methodsTop down methods

Intact proteins

Bottom up methods

Digested peptides

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Digested peptides

De Novo Sequencing

Gel based methods One/Two-dimensional gel electrophoresis

Schematic representation of the top-down approach

Protein mixture

RP-HPLC

ESI-FTICR MS

25.00 50.00 75.00 100.00 125.00 150.00 175.00 200.00 225.00 250.00 275.00 300.00 325.00Time-2

100

%

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m/z

+

++

+

isolate

dissociate

+

+

+

m/z

m/z

Compare experimental

deconvoluted spectra

with predicted

spectra

Protein identification

Peptide fragments

Peptide mass fingerprintingHPLCProtein mixture

1) Denaturation2) Reduction3) Alkylation4) Trypsin digestion

Schematic representation of the bottom-up approach

Mascot

ProFound

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Mass spectrometer

Selected ion

G L F E

MS/MS

Peptide sequence tagging

Protein identification

Database

MS

m/z

m/z

ProFound

MS-Fit

Mascot

XTandem

Sequest

Mass Spectrometry based protein identification

Peptide Mass Fingerprinting (PMF)

• Determine m/z of the peptide ions only (MS)

Product Ion Scanning

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Product Ion Scanning

• Determine the m/z of the peptide ions (Parent ions)

• Fragment peptide ions

• Determine m/z of fragments (Product Ions)

Peptide Mass Fingerprinting

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Finger Print limitations

• You need a mass spectrometer capable of reasonable

accurate masses

• Genome must be pretty small

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Yeast or smaller for good results

• Mixtures of two or more proteins can be a problem

Finger Print Advantages

• Usually can give you better coverage

• Very Fast

• Easy

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• Easy

• Good preliminary screening

Product Ion Scanning

• Digest Protein with trypsin

• Determine the m/z of a peptide ion

– ESI, MALDI

• Isolate the peptide ion from any other ions

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• Fragment the peptide ion

• Determine mass of fragments

• Obtain amino acid sequence data from fragments

NR Database approx

1 Million protein

sequences

50 million tryptic

peptide sequences

SEARCHING....

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=

Match!!!

Time = 15 seconds

200 300 400 500 600 700 800 900 1000 1100 1200 1300m/z0

100

%

733.2870138

263.068777

235.076841

147.12386

270.146333 602.2563

32362.141126

271.15634

565.234920

494.195616

433.1906;7

602.758619

702.276712

733.7970105

734.296074

1104.406645734.8063

31 1033.373324

789.328120

918.347016

972.4068;6

1105.413322

1203.516213

Computer Programs

Thermo Scientific application specific software

- Xcalibur data system (operating platform, Protein

Calculator, Xtract)

- BioWorks protein identification software

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- Proteome Discoverer

- Mass Frontier (structure of compounds)

- MetWorks (drug methabolism software)

- SIEVE (differential expression software)

Search Engines

• 4 general Types

– Automated De novo sequencing

� Peaks

� Lutefisk XP

– Peptide Sequence tags

� Guten Tag

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� Guten Tag

– Cross Correlation

� SEQUEST

– Probability Based

� Mascot

� xTandem 2

� OMSSA

� PROTE_PROBE

Peptide

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Fragmentation

Peptide FragmentationRoepstorff Nomenclature for Possible Peptide Fragments

b1 b2 b3

c3 c2 c1

a3 a2 a1 H+

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NH2 C H

R1

C

OHN CH

R2

C

OHN CH

R3

C

OHN CH COOH

R4

y3 y2 y1

z3 z2 z1

x3 x2 x1

Fragmentation scheme of a tetrapeptide showing

the formation of b and y ions

NH2

CH NH

CH NH

CH NH

CH OH

R1

O

O

O

OR3

R4R2

H+

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NH2

CH NH

CH OH

O

OR3

R4

NH2

CHC

NH

CH

R1

O R2

CO

H++b2

y2

Peptide Fragmentation

(Low-Energy Collision induced fragmentation)

• Under low energy dissociation conditions, peptides primarily fragment at the C-N bond.

• If the charge is retained on the N terminal fragment, the ion is classed as either a, b or c.

• If the charge is retained on the C terminal, the ion type is either x, y or z.

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• A subscript indicates the number of residues in the fragment.

• The loss of CO from the b ion is known as a-type ion

• In addition, peaks are seen for ions which have lost ammonia (-17 Da) denoted a*, b* and y* and water (-18 Da) denoted a°, b° and y°.

The structures of the six singly charged sequence ion

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http://www.matrixscience.com/help/fragmentation_help.html

Example of y and b ions in BioWorks™

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MRFA fragmentation

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M R F A

To better undersand

b1 b2 b3

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HCD vs. CID on MRFAE:\MRFA_CID_HCD

MRFA_CID_HCD #183-198 RT: 3.01-3.27 AV: 16 NL: 1.93E7

T: FTMS + p ESI Full ms2 524.30@hcd40.00 [50.00-600.00]

50 100 150 200 250 300 350 400 450 500

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

271.12236

R=84953

C 11 H 19 O 2 N 4 S

0.12432 ppm

104.05283

R=137349

C 4 H 10 N S

-0.18450 ppm

288.14883

R=82484

C 11 H 22 O 2 N 5 S

-0.13978 ppm

237.12335

R=91142

C 12 H 17 O 3 N 2

-0.09154 ppm

376.19787

R=72159

C 18 H 26 O 4 N 5

-0.16048 ppm

524.26458

R=61277

C 23 H 38 O 5 N 7 S

-0.72984 ppm

453.22758

R=67571

C 20 H 33 O 4 N 6 S

-0.60473 ppm

56.04941

R=187947

C 3 H 6 N

-1.20621 ppm

185.10318

R=103452

C 7 H 13 O 2 N 4

-0.67809 ppm

149.07424

R=118738

C 5 H 13 O N 2 S

-0.45283 ppm

454.23074

R=63433

121.08394

R=122565

HCD

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50 100 150 200 250 300 350 400 450 500

m/z

MRFA_CID_HCD #133-152 RT: 2.18-2.49 AV: 20 NL: 3.56E7

T: FTMS + p ESI Full ms2 524.30@cid35.00 [140.00-600.00]

50 100 150 200 250 300 350 400 450 500

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

271.12230

R=84435

C 11 H 19 O 2 N 4 S

-0.07701 ppm

453.22767

R=65916

C 20 H 33 O 4 N 6 S

-0.38973 ppm

376.19793

R=71240

C 18 H 26 O 4 N 5

0.00283 ppm

489.22757

R=62061

C 23 H 33 O 4 N 6 S

-0.57564 ppm

418.19077

R=67476

C 20 H 28 O 3 N 5 S

0.07745 ppm

229.10048

R=89238

C 10 H 17 O 2 N 2 S

-0.18034 ppm

306.15940R=79572

C 11 H 24 O 3 N 5 S

-0.13604 ppm

181.09710

R=104712

C 9 H 13 O 2 N 2

-0.29719 ppm

153.10215

R=115156

C 8 H 13 O N 2

-0.60042 ppm

CID

0

20

40

60

80

100

Rela

tive A

bundance

524.26473R=124753

C23 H38 O5 N7 S

-0.44024 ppm

525.26739R=120897

C2213C H38 O5 N7 S

-1.77174 ppm 526.26062R=124362

C23 H38 O5 N734S

-0.27554 ppm

NL:1.17E8

MRFA_CID_HCD#252-259 RT: 4.34-4.53 AV: 8 T: FTMS + p ESI SIM ms [514.30-534.30]

Elemental composition on MRFA

measured

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524 525 526 527 528

m/z

0

20

40

60

80

100

0524.26496R=123889

C23 H38 O5 N7 S

0.00001 ppm

525.26769R=120637

C2213C H38 O5 N7 S

-1.19110 ppm 526.26101R=118269

C23 H38 O5 N734S

0.47332 ppm

NL:1.67E4

C23 H38 O5 N7 S: C23 H38 O5 N7 S1

p (gss, s /p:40) Chrg 1R: 124000 Res.Pwr . @FWHM

simulated

0

1

2

3

4

5

6

Re

lativ

e A

bun

da

nce

526.26062R=124362

C 23 H 38 O 5 N 734S

-0.27554 ppm526.27045R=130297

C2113C 2 H 38 O 5 N 7 S

-2.32164 ppm

NL:1.17E8

MRFA_CID_HCD#252-259 RT: 4.34-4.53 AV: 8 T: FTMS + p ESI SIM ms [514.30-534.30]

NL:

Isotopic distribution of 526.26 on MRFA

measured

65

526.25 526.26 526.27 526.28 526.29

m/z

0

1

2

3

4

5

6526.26101R=118269

C 23 H 38 O 5 N 734S

0.47332 ppm526.27075R=96228

C 2113C 2 H 38 O 5 N 7 S

-1.75871 ppm

NL:1.67E4

C 23 H 38 O 5 N 7 S: C 23 H 38 O 5 N 7 S 1

p (gss, s /p:40) Chrg 1R: 124000 Res .Pwr . @FWHM

simulated

Isotopic contribution of 13C and 34S in MRFA

66

Detection Time & Mass Resolution

526.26112

526.27090

526.26112

526.27090

13C2 and 34S isotopes

C23H38N7O534S 526.2607 u 13C2C21H38N7O5S 526.2716 u

∆m = 0.0109 RP (m/∆m) = 48,000

524.0 525.0 526.0 527.0

m/z

0

50

100

Rela

tive A

bundance

524.26489

525.26761

526.26112527.26415

MRFAMolecular ion region

67

526.20 526.25 526.30 526.35

m/z

0

10

20

30

40

50

60

70

80

90

100

Rela

tive A

bundance

526.26551

Detect time: 375 ms; RP: 28,000

Detect time: 750 ms; RP: 55,000

Detect time: 1500 ms; RP: 95,000

„Freedom of fragmentation“ (and detection)

• Combination of three different and complementary

fragmentation techniques CID, HCD, and ETD for sequence

assignments with absolute confidence.

• Most comprehensive solution for

– complex PTM analysis

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– complex PTM analysis

– intelligent sequencing of peptides

– top-down and middle-down analysis

– protein quantitation via stable isotope labelling such as

iTRAQ™ or label-free quantitation

[M + 2H]+•

[M + 2H]2+ + A- [M + 2H]+• + A

Electron Transfer Dissociation (ETD)

[c+H]+ + [z+H]+•

c z••••

69

c z

5

10

15

20

25

30

35

40

Re

lative A

bu

nd

an

ce

1216.75003

1094.416691347.50003899.58335624.58335496.50001

751.41668

856.66669552.50001424.41667 1003.75002 1172.75003

NL: 9.46E4

SubP_SA#1 RT: 4137.28 AV: 1 T: ITMS + p ESI sid=35.00 sa Full ms2 682.60@etd100.00 [185.00-2000.00] with supplemental activation

Unreacted precursor ions

c4 c5 c6 c7c8

c9c10

Importance of supplemental activation for ETD

70

400 500 600 700 800 900 1000 1100 1200 1300

m/z

0

5

10

15

20

25

30

35

40

0856.66669552.50001424.41667 1003.75002 1172.75003

1364.58337

1216.75003

1103.66669 1319.50003899.66669

752.58335 1171.66670624.58335 1001.66669854.66669496.58334

NL: 1.13E5

SubP_noSA#1 RT: 4136.94 AV: 1 T: ITMS + p ESI sid=35.00 Full ms2 682.60@etd100.00 [185.00-2000.00]

without supplemental activation

Charge-reduced

species

c4c5

c6c7

c8 c9

c10

Unreacted precursor ions

Example: Substance P

• ETD has no low mass cut off and provides for a complete interpretation of the spectrum.

• ETD enables sequencing of larger peptides.

• Peptide sequence information from CID and ETD spectra are complementary.

ETD vs CID

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• CID/ETD toggle method improves sequence coverage and increases protein ID confidence.

• With ETD, high quality spectra are obtained for >2+ precursor ions.

• Supplemental activation improves ETD of 2+ ions.

• Digestion with LysC, AspN and GluC may provide additional benefits for proteome analysis with ETD.

CID and HCD of 667.74852+ (Sulfatation)

200 300 400 500 600 700 800 900 1000 1100 1200 1300

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

627.76886z=2

CID

NL: 79.9593qSDDyGHMRF-amid

72

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

646.32288z=1 809.38684

z=1

452.24274z=1

428.14029z=1

589.30151z=1

924.41412z=1

1039.44141z=1

321.20282z=1

136.07515z=1 537.20648

z=2

271.12192z=1 1126.47327

z=1

HCD

m/z

0

2 0

4 0

6 0

8 0

1 0 0

Rela

tive

Ab

und

an

ce

4 9 6 . 7 9 1 6 9

7 7 2 . 4 8 2 3 6

2 3 9 . 1 1 3 5 3 7 0 1 . 4 4 4 8 9 8 7 3 . 5 3 0 3 3

3 8 4 . 2 7 1 2 1

HCD vs CID HTAGFIPRL-amide

CID

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2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0

m / z

0

2 0

4 0

6 0

8 0

1 0 0

01 1 0 . 0 7 0 6 9

7 7 2 . 4 8 2 0 6

8 8 3 . 5 1 4 5 3

7 0 1 . 4 4 4 6 4

4 9 6 . 7 9 1 5 63 6 7 . 2 4 4 4 8

6 0 9 . 3 1 3 1 7

HCDHimm

HCD MS/MS Spectrum of Perisulfakinin

74

Methods for protein analysis

High resolution mass spectrometry methodsTop-down methods

Intact proteins

Bottom-up methods

Digested peptides

75

Digested peptides

De Novo Sequencing

Gel based methods One/Two-dimensional gel electrophoresis

Automated de novo Sequencing of

DPGWNNLKGLWamide using PEAKS™ Software

76

Problems !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

• You have two ladders of overlapping masses

• Cannot tell which is a b ion or which is a y ion

• Incomplete fragmentation

• Chemical noise

• Mass accuracy of some instruments are not good

77

• Mass accuracy of some instruments are not good

enough to determine R groups– Glutamine and lysine differ by 0.036 .

– Phenylalanine and oxidized methionine differ by 0.033

– Gly + Val = 156.090 u and Arg = 156.101

Big Problem

• Can take you a very long time to “sequence” a “good”

product ion spectra without a computer

– 30 minutes if your good

– 1-2 days to never if you are not

78

– 1-2 days to never if you are not

• One experiment can generate 10,000 MS/MS spectra

• Proteins due to their nature are very complex

eg. cysteine – cysteine cross-linking, tertiary structure

formation and post-translational modifications)

Beside the amount...

79

• Proteins can be very large in size and hard to handle.

Decrease complexity

• Separate proteins (before digestion)– 2-d electrophoresis– 1-d electrophoresis– Chromatography (RP, cation/anion exchange, size excision)– Immunoafinity– Molecular weight-centrifugation, ultrafiltration, solvent precipitation

• Separate peptides (after digestion)– Multidimensional chromatography (MUDPIT)

80

– Multidimensional chromatography (MUDPIT)

Reducing agents – detergents (SDS, CHAPS), salts (urea, guandine), acids (formic)

Break sulfur bridges – DTT and use iodoacetamide to keep them from reforming

Heat

Proteomics and Gel

81

Electrophoresis

Methods for protein analysis

High resolution mass spectrometry methodsTop down methods

Intact proteins

Bottom up methods

Digested peptides

82

Digested peptides

De Novo Sequencing

Gel based methodsOne/Two-dimensional gel electrophoresis

2-D electrophoresis

2-DE is a powerful separation technique, which

allows simultaneous resolution of thousands of

proteins.

83

2-D electrophoresis

Disadvantages– Modest detection limit

– High abundance proteins

– Protein bias (pI)

– Difficult to automate

– Labor intensive

– Requires many more mass

84

– Requires many more mass spec runs

Advantages– High resolution separation

– Can get quantitation by staining

– Good “snapshot” of the Proteome!

Approach used to identify a spot from a 2-D gel analysis

Excision ofprotein spot

2-D gel

Peptide fragments

Mass spectrometer

Peptide mass fingerprinting

Gel spot

DigestionExtraction

MS

85

spectrometer

Selected ion

G L F E

MS/MS

Peptide sequence taggingSearch

Protein identification

Database(NR, EST)Database(NR, EST)

Search

1-D electrophoresis

• Disadvantages– Difficult to automate

– Very limited expression profiling

• Advantages– No staining necessary

In gel trypsin digestion

[65

-78

]

[57

7-5

96

]

M IS S W1 W2 W3 E1 E2 E3

94

67

45

M IS S W1 W2 W3 E1 E2 E3

94

67

45

A

In gel trypsin digestion

[65

-78

]

[57

7-5

96

]

[65

-78

]

[57

7-5

96

]

M IS S W1 W2 W3 E1 E2 E3

94

67

45

M IS S W1 W2 W3 E1 E2 E3

94

67

45

A

86

– No staining necessary

– High protein recovery

– Decreased runs needed

• 2-50 proteins can be identified per band or section

1200 1700 2200 2700 m/z

[20

8-2

15

][6

16

-623

][2

35

-242

][7

49

-756

]

[38

-46

]

[75

7-7

67

]

[47

-55

]

[42

0-4

30

][3

92

-403

][4

7-5

7]

[27

1-2

83

]

[33

7-3

51

]

[46

4-4

78

]

[37

2-3

91

]

[47

9-4

95

]

[18

-35

]

[19

5-2

15

]

[47

9-5

03

]

B

1200 1700 2200 2700 m/z

[20

8-2

15

][6

16

-623

][2

35

-242

][7

49

-756

]

[38

-46

]

[75

7-7

67

]

[47

-55

]

[42

0-4

30

][3

92

-403

][4

7-5

7]

[27

1-2

83

]

[33

7-3

51

]

[46

4-4

78

]

[37

2-3

91

]

[47

9-4

95

]

[18

-35

]

[19

5-2

15

]

[47

9-5

03

]

1200 1700 2200 2700 m/z

[20

8-2

15

][6

16

-623

][2

35

-242

][7

49

-756

]

[38

-46

]

[75

7-7

67

]

[47

-55

]

[42

0-4

30

][3

92

-403

][4

7-5

7]

[27

1-2

83

]

[33

7-3

51

]

[46

4-4

78

]

[37

2-3

91

]

[47

9-4

95

]

[18

-35

]

[19

5-2

15

]

[47

9-5

03

]

B

Typical MUDPIT Preparation

(Multidimensional Protein Identification Technology)

Protein digest mixtures

SCX fractionation

CID

Micro

electrospray

ionization

RPfractionation

• Advantages

– Dynamic Range 105

– Sensitivity! Low

abundance proteins

– Minimized protein bias

– Highly automated

87

MS-1 MS-2

Auto MS/MS detection

Tandem Mass Spectra

Database searchprotein I.D.

• Disadvantages

– Poor isoform &

modification distinction

– Still overwhelms the

mass spectrometer

– Does not give you a

very good “snapshot”

Post Translational

Modification

88

Modification

After Translation

Post-translational modifications

• Phosphorylation – Ser, Thr, Tyr

• Methylation – Lys

• Oxidation – Met

89

• Oxidation – Met

• Deamidation – Asn, Gln

• Glycosylation

PTM Scanning

• Can search data for unexpected modifications from the

unimod database

www.unimod.org

90

Ways to look for PTM’s

• Mass spectrometry– Good for PTM’s with a stable mass

• Phosphorylation

• Methylation

• Acetylation

– Difficult to analyze large modifications

• Large carbohydrates

91

• Large carbohydrates

• Edman Sequencing• Phosphorylation

• Radio-labeling– Very sensitive

– Usually hard to get the identity or AA with PTM

The Trinity of Protein Phosphorylation Analysis

II. Identify the sites of phosphorylation

I. Identify the kinase responsible for phosphorylation

� Predict potential phosphorylation sites with the Scansite program

(http://scansite.mit.edu)

� Gel kinase assay

92

II. Identify the sites of phosphorylation

III. Identify the function of phosphorylation

� Generate mutants forms of protein for in vitro testing

� Separate phosphoproteins by SDS-PAGE

� Digest in-gel the phosphoproteins to generate (phospho)peptides

� MS/MS analysis

Most important part of finding PTM’s

• Protein Coverage!

93

Problems with Mass spec approach

• Cannot prove a negative

– Just because you do not see your PTM does not

mean it is not there!

94

Some common PTM’s

95From Jensen et al. nature march 2003 vol 21

Data Dependent NL MS3 method setup for

96

method setup for phopshorylation

identification

LTQ Orbitrap MS LTQ Orbitrap MS

Instrument configuration settings

97

Configuration window: important settings

98

Data Dependent NL MS3

99

NL in MS2 trigger MS3

100

Scan events

1. First scan

event is the

reference scan

(usually a FTMS

full scan)

2. All subsequent

scans events may

be dependent on

scan event 1.

101

scan event 1.

3. A scan

depending on a MS

scan will produce a

MS/MS spectrum. A

scan depending on

a MS/MS scan will

produce a MS3

spectrum

Scan event 2

102

Scan event 2. Settings

103

NL masses

104

Scan event 2 determined from Scan event 1

.

105

Scan event 3 (MS3 of the peaks which lost phosphate)

106

Monoisotopic precursor selection toggle…

see next slide

107

… And What It Does

• Problem: monoisotope signal is lower abundant than first isotope⇒ wrong precursor ion mass would be used for db searches ⇒ wrong results

• Monoisotope Toggle reads the

108

• Monoisotope Toggle reads the charge state…

• …calculates a theoretical pattern for the measured m/z…

• …determines the correct monoisotope signal ⇒ correct results from db search

Non-Peptide Monoisotopic Recognition…

see next slide

109

…And What It Does

• determines a charge state (isotope distance) for a

detected signal cluster

• looks for a signal left from the most abundant signal

• this signal needs to have at least 60% intensity of the

110

• this signal needs to have at least 60% intensity of the

highest one

• the toggle will only work if the isotope cluster follows a

‚standard intensity deviation‘ → ‚Cl‘ or ‚Br‘ containing

cluster will not work

New features

• Use m/z values as Mass

– Triggers only one charge state out of a CS distribution

• Chromatography Trigger

– Peak apex triggering

111

– Peak apex triggering

• NL Triggered HCD

– Performs HCD fragmentation on precursors, which carry modifications, which result in CID spectra with NL

“Use m/z values as Mass”

112

� triggers only the most intense charge state of a charge state distribution

“Use m/z values as Mass”

60

70

80

90

100

Rela

tive A

bu

nd

an

ce702.11792

z=4

561.89581z=5

539.08704z=5

113

400 500 600 700 800 900 1000

m/z

0

10

20

30

40

50

Rela

tive A

bu

nd

an

ce

673.85785z=4 722.35474

z=3

935.82166z=3803.94409

z=2388.22092z=2

897.80676z=3

Neutral Loss Triggered HCD

OT-FS

OT-CID1

OT-CID2

NL?OT-HCDno

yes

114

OT-CID2

OT-CID3

OT-HCD

OT-HCD

NL?

NL?

RT: 0.00000 - 39.96544

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38

Time (min)

0

20

40

60

80

100

Re

lative

Ab

un

da

nce

30.99966478.31247

31.07783478.31296

11.53153589.80402

13.32670495.75748

31.23676478.31235

15.00021717.86932

28.56122353.1505717.43568

678.8215310.94827

410.19772 31.38696478.31326

24.80037499.22260

21.24148367.12732

2.21873311.98303

39.76065519.13251

4.55575519.13116

36.70453519.13214

NL: 7.07E7

Base Peak F: FTMS + p NSI Full ms [300.00-1400.00] MS ft051117_DEK_16_MDM_TBB_K25_ph

ft051117_DEK_16_MDM_TBB_K25_ph #746 RT: 12.53 AV: 1 NL: 1.40E6T: FTMS + p NSI Full ms [300.00-1400.00]

50

100

Re

lative

Ab

un

da

nce

633.36344R=65778

z=3475.27383R=88272

z=4

802.42220R=51415

z=11097.15152

R=32148z=3

707.28542R=57530

z=3

380.22460R=136210

z=5

944.39175R=44574

z=2

533.26548R=101970

1144.00233R=29975

z=?

984.36977R=35330

z=?

852.89723R=34052

z=?

x10

FTMS

Base Peak Chromatogram

*-1.02 ppm

Identification of phosphorylated Ser-303 in DEK

protein using NLMS3 method

115

300 400 500 600 700 800 900 1000 1100 1200 1300 1400

m/z

0

Re

lative

Ab

un

da

nce

z=3z=3

R=136210z=5

z=2R=101970

z=2 z=?R=35330

z=?R=34052

z=?

ft051117_DEK_16_MDM_TBB_K25_ph #750 RT: 12.57 AV: 1 NL: 4.70E3T: ITMS + c NSI d Full ms2 944.39@cid35.00 [250.00-1900.00]

300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800

m/z

0

20

40

60

80

100

Re

lative

Ab

un

da

nce

895.4

886.5

926.5 1289.1599.2470.2 1418.1877.4 1191.11003.2714.2 822.4581.2 1118.2 1741.31630.31532.4433.1

967.2

823.7

345.2309.0

ft051117_DEK_16_MDM_TBB_K25_ph #751 RT: 12.58 AV: 1 NL: 1.39E2T: ITMS + c NSI d Full ms3 944.39@cid35.00 895.43@cid35.00 [235.00-1805.00]

300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800

m/z

0

20

40

60

80

100

Re

lative

Ab

un

da

nce

1191.1886.4

1320.2599.2 877.4

470.1 714.2 787.1 1192.21076.2822.4 1003.2581.31643.21532.21321.31173.2765.6

1118.3

1302.41004.3 1661.4985.3700.6

600.1

474.1 1626.31514.5

916.3

1445.4373.2943.2

327.3 1334.4

1463.1

529.7

407.0

ITMS2 (944.39)

ITMS3 (895.43)

299KESEpSEDSSDDEPLIK314

299KESEDhaEDSSDDEPLIK314

[M-H3PO4+2H]2+

299KESEpSEDSSDDEPLIK314

pm -H3PO4

pm -H3PO4 -H2O

Identification of phosphorylated Ser-303 in DEK protein

using NLMS3 method and Bioworks

IT MS2

116

299KESEDhaEDSSDDEPLIK314

pm -H2O

pm -2H2O

IT MS3

Identification of the phosphorylation site Ser-39 from

eIF3c subunit using neutral loss/MS3 method

117

Multiphosphorylation of the N-terminal part of eIF3c

118

Neutral Loss Scanning

119

(A) elimination of phosphate from phosphothreonine to produce dehydroamino-2-butyric acid (scheme isalso valid for phosphoserine where dehydroalanine forms) and phosphoryc acid

(B) absence of the same mechanistic pathway for b-elimination of phosphate from phosphotyrosine.

Mechter_1880_3+_ETD_FT_Recal #1-20 RT: 0.01-0.31 AV: 20 NL: 3.18E5T: FTMS + p NSI Full ms2 459.22@etd100.00 [120.00-1500.00]

55

60

65

70

75

80

85

90

95

100

Re

lative A

bu

nd

an

ce

459.2254z=3

LTQ Orbitrap XL ETD for Phosphopeptide Analysis

RpSRGGKLGpSLGK

[M+3H]3+

[M+3H]2+•

120

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400

m/z

0

5

10

15

20

25

30

35

40

45

50

55

Re

lative A

bu

nd

an

ce

623.7946z=2

1076.4759z=1

688.8367z=2

554.2560z=1

1360.6494z=1

341.1339z=1

908.4700z=1798.3886

z=1 1036.5426z=1

739.3722z=1497.2346

z=1

301.2004z=1

1247.5928z=1

1343.6226z=1

1150.5386z=1

258.1456z=?

426.5608z=3

938.5614z=1

∆pS

∆pS

[M+3H]2+•

174.1356

z=1

Phosphopeptide standards courtesy of Karl Mechtler, IMP Vienna, Austria

LTQ Orbitrap XL ETD for Phosphopeptide Analysis

using ETD

121Phosphopeptide standards courtesy of Karl Mechtler, IMP Vienna, Austria

CID and HCD of 667.74852+ (Sulfatation)

200 300 400 500 600 700 800 900 1000 1100 1200 1300

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

627.76886z=2

CID

NL: 79.9593qSDDyGHMRF-amid

122

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

646.32288z=1 809.38684

z=1

452.24274z=1

428.14029z=1

589.30151z=1

924.41412z=1

1039.44141z=1

321.20282z=1

136.07515z=1 537.20648

z=2

271.12192z=1 1126.47327

z=1

HCD

m/z

Intact protein

analysis

123

analysis

C1080 H1697 N268 O325 P5 S6

Beta-casein (5x phosphorylated)

Analysis of intact phosphoproteins reveals the

number of phosphorylation sites

124

experimentalsimulated∆∆∆∆m = 1.3 ppm

RELEELNVPGEIVESLSSSEESITRINKKIEKFQSEEQQQ

TEDELQDKIHPFAQTQSLVYPFPGPIPNSLPQNIPPLTQT

PVVVPPFLQPEVMGVSKVKEAMAPKHKEMPFPKYPVEPFT

ESQSLTLTDVENLHLPLPLLQSWMHQPHQPLPPTVMFPPQ

SVLSLSQSKVLPVPQKAVPYPQRDMPIQAFLLYQEPVLGP

VRGPFPIIV

1

209

- 5 HPO3

Alkaline Phosphatase

Dephosphorylation by AP treatment reveals the number of

phosphorylation sites

125

Amino acid sequence of bovine Carbonic Anhydrase II

SHHWGYGKHNGPEHWHKDFPIANGERQSPVDIDTKAVVQDPALKPLALVY

GEATSRRMVNNGHSFNVEYDDSQDKAVLKDGPLTGTYRLVQFHFHWGSSD

DQGSEHTVDRKKYAAELHLVHWNTKYGDFGTAAQQPDGLAVVGVFLKVGD

ANPALQKVLDALDSIKTKGKSTDFPNFDPGSLLPNVLDYWTYPGSLTTPP

LLESVTWIVLKEPISVSSQQMLKFRTLNFNAEGEPELLMLANWRPAQPLK

Ac-

126

LLESVTWIVLKEPISVSSQQMLKFRTLNFNAEGEPELLMLANWRPAQPLK

NRQVRGFPK

Sum Formula: C1312H1996N358O384S3

Monoisot. Mass: 29,006.682670

N-Terminus: N-Acetylation

Full FTMS of the Bovine Carbonic Anhydrase II

700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400

m/z

0

50

100

Rela

tive A

bundance

937.25580R=80000

854.64618R=84404

1001.82526R=80500807.22192

R=843001076.03418

R=71501 1161.95667R=70104

1210.32898R=73100

745.25647R=91604

1320.17651R=65704 1383.28064

R=65304

0

50

100

Rela

tive A

bundance

937.25580R=80000

907.99872R=78304

968.46460R=76400

1001.82526R=80500

880.54437R=77500

951.14423R=82400

1007.26550R=83300

127

∆∆∆∆m = 0.44 ppm

880 900 920 940 960 980 1000

m/z

0

Rela

tive A

bundance

28955 28960 28965 28970 28975 28980 28985 28990 28995 29000 29005 29010

m/z

0

50

100

Rela

tive A

bundance

29006.66975

29010 29015 29020 29025 29030 29035

m/z

0

50

100

Rela

tive A

bundance

29023.7186329026.72841

29020.7163529028.73130

29018.71561 29030.72314

29032.7259629016.71637

CAIII_SIM_1001 #1 RT: 38.6 AV: 1 NL: 1.21E6T: FTMS + p ESI Full ms3 1001.83@cid0.00 1001.83@cid0.00 [275.00-1400.00]

0

20

40

60

80

100

Rela

tive A

bundance

1001.85901R=79200

1033.56897R=63604

951.12561R=67704

436.66791R=108704

866.92560R=39704

1155.01184R=48704

1320.69739R=45804

527.01331R=88404

337.77481R=104904

673.80261R=72301

Isolation of the [M+29H]29+ ion of the Bovine Carbonic

Anhydrase II

128

300 400 500 600 700 800 900 1000 1100 1200 1300 1400

m/z

CAIII_SIM_1001 #1 RT: 38.6 AV: 1 NL: 1.21E6T: FTMS + p ESI Full ms3 1001.83@cid0.00 1001.83@cid0.00 [275.00-1400.00]

1001.0 1001.5 1002.0 1002.5 1003.0 1003.5

m/z

0

20

40

60

80

100

Rela

tive A

bundance

1001.85901R=79200

1001.96216R=78904

1001.68750R=76604

1002.03101R=77304

1001.61847R=77104 1002.10010

R=75904

1002.54681R=70604

1003.20111R=75104

1002.20349R=69404

1002.75409R=71704

1001.51563R=724041001.24005

R=67204

1003.44348R=755041000.99670

R=67204

Higher Energy Collision Dissociation (HCD) of the [M+34H]34+ ion

T: FTMS + p ESI Full ms2 854.00@hcd14.00 [100.00-1400.00]

18

20

22

24

26

28

30

Re

lative

Ab

un

da

nce

539.28272z=1

159.09247z=1 519.04469

HCD (854.0)

129

200 400 600 800 1000 1200 1400

m/z

0

2

4

6

8

10

12

14

16

Re

lative

Ab

un

da

nce

z=1 519.04469z=5 836.80323

z=?

337.18795z=1

HCD Fragmentation Details using ProSight PTM

130

HCD Fragmentation Details using ProSight PTM

131

T: FTMS + p ESI Full ms2 854.00@cid20.00 [235.00-1400.00]

60

70

80

90

100

Re

lative

Ab

un

da

nce

951.14404z=8

592.93689z=5

807.23987z=19

1095.21057z=8

CID (854.0)

Collision Induced Dissociation (CID) of the [M+34H]34+ ion

132

400 600 800 1000 1200 1400

m/z

0

10

20

30

40

50

Re

lative

Ab

un

da

nce

1251.52588z=7

1134.52502z=1

CID Fragmentation Details using ProSight PTM

133

CID Fragmentation Details using ProSight PTM

134

Ubiquitin_ETD_11 #1-106 RT: 0.01-6.13 AV: 106 NL: 1.10E3T: FTMS + p ESI Full ms2 778.50@etd30.00 [210.00-2000.00]

55

60

65

70

75

80

85

90

95

100

Re

lative A

bu

nd

an

ce

277.1328z=1

948.1839z=3

1136.6501z=1

390.2168z=1 869.7391

z=4

ETD spectrum of Ubiquitin, 12+ charge state, with

Orbitrap detection

*

135

400 600 800 1000 1200 1400 1600 1800

m/z

0

5

10

15

20

25

30

35

40

45

50

55

Re

lative A

bu

nd

an

ce

z=4

537.2850z=1

1347.2301z=2790.4653

z=2636.3535

z=11012.2238

z=3764.4484

z=1

1108.7627z=6

1159.3174z=3

1273.6870z=4

669.3921z=2 1421.7778

z=6433.2516z=2

1702.7334z=5

1486.8083z=5

305.1328z=?1

* http://upload.wikimedia.org/wikipedia/commons/a/ac/Ubiquitin_cartoon.png

FUNDAMENTAL: Mass Accuracy

Parts per million = [Masstheor - Massexp ]

Massx 106

(PPM)

136

Masstheor(PPM)

Avandia_pos_05a #670 RT: 17.24 AV: 1 NL: 8.27E6F: FTMS + p ESI Full ms [100.00-1000.00]

55

60

65

70

75

80

85

90

95

100

Rela

tive

Abundance

358.1217

Mass Accuracy and Resolving Power

N NO

NH

S

O

O

Rosiglitazone

C18H19N3O3S

[M+H]+ 358.12199

error: 0.77 ppm

R 60,000 @m/z400

⇒⇒⇒⇒ R = 77,000

137

358.2 358.4 358.6 358.8 359.0 359.2 359.4 359.6 359.8 360.0 360.2

m/z

0

5

10

15

20

25

30

35

40

45

50

55

Rela

tive

Abundance

359.1249

360.1175

Mass Accuracy and Resolution: Clozapine

Clozapine_Mix #2104 RT: 25.47 AV: 1 NL: 2.03E5T: FTMS + p NSI Full ms [90.00-800.00]

326.5 327.0 327.5 328.0 328.5 329.0 329.5

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative A

bu

nd

an

ce

327.1374R=9300

329.1340

R=9300328.1407

R=9200

Clozapine_Mix #813 RT: 2.93 AV: 1 NL: 4.15E5T: FTMS + p NSI Full ms [150.00-800.00]

326.5 327.0 327.5 328.0 328.5 329.0 329.5

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative A

bu

nd

an

ce

327.1371R=18400

329.1338R=18800

328.1406R=18600

R 7,500 Scan 0.3 sec R 15,000 Scan 0.3 sec

error: 0.9 ppm error: <0.1 ppm

138

Clozapine_Mix #849 RT: 3.27 AV: 1 NL: 2.15E5T: FTMS + p NSI Full ms [150.00-800.00]

326.5 327.0 327.5 328.0 328.5 329.0 329.5

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative A

bu

nd

an

ce

327.1369R=74500

329.1340R=74600

328.1402R=73600

Clozapine_Mix #875 RT: 3.93 AV: 1 NL: 1.65E5T: FTMS + p NSI Full ms [150.00-800.00]

326.5 327.0 327.5 328.0 328.5 329.0 329.5

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative A

bu

nd

an

ce

327.1368

R=147700

329.1339R=146300

328.1402R=148900

R 60,000 Scan 1 sec R 100,000 Scan 1.8 sec

error: 0.6 ppmerror: 0.9 ppm

Different scan speeds and resolution – Mass accuracy < 1ppm

Mass spectrometers do not measure mass, but m/z!

� If a peptide with a mass of 700 Da gets ionized and

acquires 2 protons at pH 3.0

� The mass spectrometer sees

700+2 = 702/2 = 351.00

Mass to charge (m/z)

139

700+2 = 702/2 = 351.00

� This mass is referred to as [M+2H]2+

100

60

80

648.82

649.32

Determining Charge State

649.32-648.82=0.5

1/0.5=2 (charge state)

140

648 649 650 651

m/z

0

20

40

649.81

650.31

(648.82*2)-2 = M 1295.64

m/z= 648.82

Lock Mass

141

Lock Mass

Ions which can be used as lock masses during

online LC-MS analyses

The

polydimethylcyclosiloxane

(PCM) ions generated in the

electrospray process from

ambient air (protonated

(Si(CH3)2O))6; m/z 445.120025) were used for

internal recalibration in real

142Olsen, Mann et al. Mol. Cell. Proteomics 2005, 4: 2010-2021

“Parts per million mass accuracy on an orbitrap mass

spectrometer via lock-mass injection into a C-trap.”

internal recalibration in real

time.

Detection

Internal calibration = Lock Mass

Injection of the calibrantInjection of analyte

Mixing of ion populations and ejection

143

Olsen, Mann et al. Mol. Cell. Proteomics 2005, 4: 2010-

2021

“Parts per million mass accuracy on an orbitrap mass

spectrometer via lock-mass injection into a C-trap.”

Internal Calibration

• Lock masses are isolated in the LTQ with a reduced

inject time.

• The C-Trap is filled with them. The reduced inject time

adjusts the lock mass intensity to appr. 5%.

• The LTQ is filled for a second time using the full inject

time.

144

time.

• Ions from the second filling are moved to the C-trap and

mixed with the previous ion population of the calibrant.

• Ions are shot into the orbitrap.

• After the transient acquisition and data processing an

existing calibration function is adjusted based on the

measured frequencies of the lock masses.

Getting the most from your lock mass

• For a specific compound of interest use a lock mass

close in mass

• Note: if you have a lock mass at low m/z but your

compound has high m/z, it might be better to use

external calibration

• Generally: we recommend using 1 lock mass only

145

• Generally: we recommend using 1 lock mass only

• If you put more lock masses, you need fill time for each

of them, and it does not necessarily improve the result

• If no lock mass is found the system applies the last

external calibration

• Thus, you should keep your orbitrap externally well

calibrated anyway!

Internal Calibration: Tune Setup

146

1. Locking ON

2. Enter accurate mass of a known compound

Internal Calibration: Method Setup

• Enable lock mass in the current method ‘L’

• Can edit lock masses

• A lock mass from the ‘method’ take preference over the

lock mass from ‘Tune’ file

147

Ionisation and desorption in ESI mass spectrometry

Electrospray Solvated

Macromolecular Ion

Aerosol droplets Desolvated

Macromolecular Ion

spray

Electrospray Solvated

Macromolecular Ion

Aerosol droplets Desolvated

Macromolecular Ion

spray

148

Ions are formed by spraying the solution from a fine needle into an electric field at

atmospheric pressure.

The droplets pick up charges, in the form of protons; solvent evaporates as the

droplets enter the lower pressure regions of the instrument leaving “naked” intact

peptides with varying numbers of charges

(z =1,2,3,4…n).

Protein

Quantification

149

Quantification

Why Quantitate on the protein level

• There can be a big disconnect between the mRNA level

and the protein level

– Protein Turn over

– Post-translational modifications

150

• Gel based

• LC-MS based

• Label free approaches

• Stable isotope labelling

Quantitation in Proteomics

151

• Stable isotope labelling

Differential proteomics

Comparison of the relative amounts of proteins between

two or more biological samples

- Diseased vs healthy

- Larva vs pupa vs adult

152

- Larva vs pupa vs adult

- Sepsis - die vs live

• Samples to be run in tandem

• Labeled and unlabeled peptides to elute together (LC)

• Labeled and unlabeled peptides ionize similarly (ESI)

Labeling methods

Allow to:

153

• Labeled and unlabeled peptides ionize similarly (ESI)

Quantitation with Isotope Labels

• Incorporate a ‘mass tag’ into protein or peptide

– Create heavy and light version

• Mix the labeled samples

• Analyse together

• Use mass spectrometer to differentiate the heavy from

light versions

154

• Compare to get the ratio

Light (L)

1036 1037 1040 1043 1044 10450

10

20

30

40

50

60

70

80

90

100

Rela

tive

Abundance

1039.5125z=2

1043.0231z=2

1038 1039

1038.5087z=2

1039.0102z=2

1038.0074z=2

Heavy (H)

1041 1042m/z

1041.0171z=2

1042.0200z=2

1042.5215z=2

1041.5186z=2

Ratio

H:L = 2.1

General idea: Differential proteomics

Enzymatic digestion (Trypsin)

LC/MS/MS

In the LC, heavy and light co-elute

MS

155m/z

rela

tive a

bundance

Normal (12C)

Abnormal (13C)

Peptide found to be

up regulated in

abnormal sample

MS/MS

rela

tive a

bundance

MS/MS

Fragment ion masses

help to identify

peptide sequence

belonging to a

specific protein

General idea: Differential proteomics

fragment ions

156

m/z

rela

tive a

bundance

specific protein

Protein determined to

be up regulated in

abnormal sample

Stable isotope labeling methods

- Isotope-Coded Affinity Tagging (ICAT)

- Hydrogen vs deuterium (8 Da separation)

- 13C vs 12C (9 Da separation)

- Isotope Tagging for Relative and Absolute protein Quantitation (iTRAQ)

- Four isobaric reagents

157

- Four isobaric reagents

- Compare quantity of reporter molecule in MS/MS

- Stable Isotope Labeling of Amino acids in Cell culture (SILAC)

-Cell growth in stable isotope-enriched media (13C Glucose, 15N Ammonium, 2H Water)

Isotope-Coded Affinity Tagging (ICAT)

X = Hydrogen (Light)

or

Deuterium (Heavy)

8 Da difference

158

http://dir.niehs.nih.gov/proteomics/emerg2.htm

Binds to and modifies

cysteine residues

(alkylation)

Used to affinity

capture reacted

cysteine containing

peptides (avidin)

ICAT

- Analyze only the peptides containing cysteine

- Reduce complexity of sample

- Cys relative abundance 2.5%

- Problems with getting the hydrogen and deuterium labeled -

- peptides to co-elute

159

- Changed linker chain so that carbons were isotopically

labeled (13C vs 12C, 9 Da separation)

- 13C and 12C peptides co-elute

- Reducing complexity also means missing a lot of peptides

and reduction in confidence in protein identification

Stable Isotope Labeling of Amino acids in Cell

culture (SILAC)

� SILAC uses in vivo metabolic incorporation of “heavy” 13C- or 15N-labeled amino acids into proteins followed by mass spectrometry-based analysis.

� ‘Kits’ commercially available

160

Everly, P.A., et al. (2004).Quantitative cancer proteomics: Stable isotope labeling with amino acids (SILAC) as a tool for prostate cancer research. Mol & Cell Proteomics. 3.7: 729-735.

Mann, M. (2006). Functional and quantitative proteomics using SILAC. Nature Reviews. 7:952-959.

SILAC workflow

161Mann, M. (2006). Functional and quantitative proteomics using SILAC. Nature Reviews. 7:952-959.

Isotope Tagging for Relative and Absolute protein

Quantitation (iTraq)

162

iTraq is a registered Trademark od Applied Biosystems

iTraq-Chemistry

163

covalently links an iTRAQ™ reagent

isobaric tag with each lysine side chain

and N-terminal group of a peptide

ensures that an iTRAQ™ reagent-labeled peptide,

whether labeled with iTRAQ™reagent 114, 115,

116, or 117, displays at the same mass

RT: 0.00000 - 47.89528

60

65

70

75

80

85

90

95

100

Rela

tive A

bundance

10.06990721.39038

21.85394778.72766

16.06159854.79303

26.23417883.45465

6.41333446.94089

Base peak chromatogram and MASCOT identification

of proteins from the PRG study sample

*

******

*

1640 5 10 15 20 25 30 35 40 45

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

Rela

tive A

bundance

446.94089

11.21418677.84949

5.96203317.22058

16.67878855.12280

30.468841204.97388

5.68283384.24384

40.22073432.23828

34.65009404.207492.31799

323.16766

C:\Documents and Settings\...\ft050930_5 9/30/2005 3:43:18 PM itraq_1

RT: 0.00000 - 47.89528

0 5 10 15 20 25 30 35 40 45

Time (min)

0

50

100

0

50

100

Re

lative

Ab

un

da

nce 10.06990

721.3903821.85394

778.7276616.06159854.79303

26.23417883.45465

6.41333446.94089 30.46884

1204.97388 40.22073432.23828

34.65009404.20749

45.88895308.22272

2.31799323.16766

21.42729844.45465

NL: 2.83E7

Base Peak F: FTMS + p NSI Full ms [300.00-1600.00] MS ft050930_5

NL: 7.87E6

m/z= 844.44786-844.46474 F: FTMS + p NSI Full ms [300.00-1600.00] MS ft050930_5

ft050930_5 #2288 RT: 21.43 AV: 1 NL: 7.72E6F: FTMS + p NSI Full ms [300.00-1600.00]

50

100

Re

lative

Ab

un

da

nce

844.45465R=49220

z=2563.30560R=74662

z=3896.09943R=47783

z=31132.62744 1522.14624672.07874 1388.82874831.95197 1001.13818545.85303 1206.24377436.46356380.55933

MS2 identification of an iTRAQ labeled tryptic peptide from BSA

and quantification of the tag in a subsequent MS3 scan

FTMS

*

165

300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600

m/z

0

Re

lative

Ab

un

da

nce

z=31132.62744

R=35651z=?

1522.14624R=26857

z=?

672.07874R=71282

z=4

1388.82874R=28179

z=?

831.95197R=57961

z=2

1001.13818R=38096

z=?

545.85303R=70488

z=?

1206.24377R=28228

z=?

436.46356R=78460

z=?

380.55933R=61628

z=?

ft050930_5 #2268 RT: 21.27 AV: 1 NL: 3.68E4T: ITMS + c NSI d Full ms2 844.45@cid35.00 [220.00-1700.00]

300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700

m/z

0

50

100

Re

lative

Ab

un

da

nce

965.2723.3866.3291.1

822.4652.2 1083.41036.3719.3605.2 1397.5966.2

1183.3724.3 867.2406.2 1212.4 1282.31084.4 1543.5505.3 969.4821.6653.2345.1 1442.5868.3466.1 534.2 1545.51085.5 1526.51019.4 1214.5246.1 761.3 1661.4

ft050930_5 #2272 RT: 21.30 AV: 1 NL: 4.62E3T: ITMS + c NSI d Full ms3 844.45@cid35.00 291.00@cid35.00 [70.00-595.00]

100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290

m/z

0

50

100

Re

lative

Ab

un

da

nce

246.1

116.9 274.1113.9 144.9

230.2163.0

228.0147.0 273.1100.8

569TVMENFVAFVDK580

ITMS2

ITMS3

FTMS

y1

MS/MS identification of the iTRAQ labeled peptide 569TVMENFVAFVDK580

166

Precursor Ion 1319.144 (2+)

HCD MS/MS of iTRAQ labelled Peptide

167

50

60

70

80

90

100

Re

lativ

e A

bu

nd

an

ce

Carbonic Anhydrase (1 : 0.5 : 0.2 : 0.03)

10

20

30

40

50

60

70

80

90

100

Rela

tive

Ab

und

an

ce

114.11012

115.10724

116.11040

117.11372

# bSeq

.y #

1 244.1778 V 9

2 357.2618 L 1018.5901 8

3 472.2888 D 905.5060 7

4 543.3259 A 790.4791 6

168

200 400 600 800 1000 1200

m/z

0

10

20

30

40

50

Re

lativ

e A

bu

nd

an

ce

114 115 116 117

m/z

0 5 656.4099 L 719.4420 5

6 771.4369 D 606.3579 4

7 858.4689 S 491.3310 3

8 971.5530 I 404.2989 2

9 K 291.2149 1

HCD/iTRAQ

• No „low mass cutoff“ with HCD fragmentation

• Good quantitation in the low mass region

• Good sensitivity

169

• Good sensitivity

�No limits to detect reporter ions and

fragments in one spectrum

�Good peptide ID

�Good quantitation

Label-Free Approach

Sample 1

Sample 2

170

• Align chromatographic traces (The quality of the alignment software is a key parameter in the comparative LC-MS procedure)• Match peptide mass/charge state• Quantify based on elution profile of each peptide• Identify using MS/MS spectra

171Comparative LC-MS: A landscape of peaks and valleys, Proteomics, 2008Antoine H. P. America and Jan H. G. Cordewener

ABRF Competition PRG 2007

Thermo Fisher: BRIMS (LTQ Orbitrap)

Thermo Fisher: SJ Proteomics Marketing (LTQ Orbitrap)

172

RP-HPLC basics

173

RP-HPLC basics

Analysis and purification of

proteins and peptides by Reversed-Phase HPLC

• Separation of peptide fragments from enzymatic digests.

• Purification of natural and synthetic peptides.

• Study enzyme subunits and research cell functions.

174

• Study enzyme subunits and research cell functions.

• Analysis of protein therapeutic products.

• To verify conformation and to determine degradation

products in intact proteins.

Mechanism of interaction between polypeptides and

RP-HPLC columns

175

RP-HPLC separates polypeptides based on subtle differences in

the “hydrophobic foot” of the polypeptide being separated.

Isocratic versus gradient elution

Shallow gradients can be used very effectively to separate

similar polypeptides where isocratic separation would be

impractical.

176

The role of the column in polypeptide separations

by RP-HPLC

• The HPLC column provides the hydrophobic surface onto which the polypeptides adsorb.

• Columns consist of stainless

177

• Columns consist of stainless steel tubes filled with small diameter, spherical adsorbent particles, generally composed of silica whose surface has been reacted with silane reagents to make them hydrophobic.

Adsorbent Pore DiameterPolypeptides must enter a pore in order to be adsorbed and separated.(pores of about 300 Å) Some peptides (<~2,000 MW) may also be separated on particles of

100 Å pores.

Adsorbent Particle Size

The role of the column in polypeptide separations

by RP-HPLC

178

Smaller diameter particles generally produce sharper peaks and better resolution.

Adsorbent Phase TypeHydrophobic phase is usually a linear aliphatic hydrocarbon of eighteen

(C18), eight (C8) or four (C4) carbons.

Column Dimensions: Length

• Column length does not significantly affect separation and resolution of proteins.

• Consequently, short columns of 5–15 cm length are often used for protein separations.

179

often used for protein separations.

• Columns of 15–25 cm length are often used for the separation of synthetic and natural peptides and enzymatic digest maps.

• Column back-pressure is directly proportional to the column

length.

Column back-pressure

180

• In case of viscous solvents, such as isopropanol, shorter

columns will result in more moderate back-pressures.

Column dimensions: diameter

When the diameter of an HPLC column is reduced

1. flow rate is decreased

2. solvent used decreased

3. detection sensitivity is increased (smaller amounts

181

3. detection sensitivity is increased (smaller amounts of polypeptide can be detected).

Very small diameter HPLC columns ((75 µm Diameter)

are particularly useful when coupling HPLC with mass spectrometry (LC/MS).

Columns, example

182

Organic modifier

The organic modifiers solubilizes and desorbs the polypeptide from the hydrophobic surface.

183

Organic solvents used in LC/MS

Acetonitrile (ACN)

■ It is volatile and easily removed from collected fractions.

■ It has a low viscosity, minimizing column back-pressure.

■ It has little UV adsorption at low wavelengths.

184

■ It has little UV adsorption at low wavelengths.

■ It has a long history of proven reliability in RP-HPLC

polypeptide separations.

Isopropanol

■ Used for large or very hydrophobic proteins.

disadvantage of isopropanol is its high viscosity.

Organic solvents used in LC/MS

185

■ To reduce the viscosity of isopropanol - use a mixture of 50:50 acetonitrile: isopropanol. Adding 1–3% isopropanol to acetonitrile has been shown to increase protein recovery in some cases.

Ethanol

■ Elute hydrophobic, membrane-spanning proteins and is

used in process purifications.

Methanol

Organic solvents used in LC/MS

186

Methanol

Dichloromethane

Clorofprm

Hexane

The ion-pairing reagents or buffers sets the eluent pH and interacts with the polypeptide to enhance the separation.

Ion-pairing reagents and buffers

187

Ion-pairing reagents and buffers

Trifluoroacetic acid

■ It is volatile and easily removed from collected fractions.

■ It has little UV adsorption at low wavelengths.

■ It has a long history of proven reliability in RP-HPLC

188

■ It has a long history of proven reliability in RP-HPLC

polypeptide separations.

■ Enhancement of chromatographyc resolution.

■ Adverse effect on ions formation in LC/MS

Heptafluorobutyric acid (HFBA)

■ Is effective in separating basic proteins.

Triethylamine phosphate (TEAP)

■ Has been used for preparative separations.

Ion-pairing reagents and buffers

189

Formic acid (FA)

■ In concentrations of 10 to 60%, has been used for the chromatography of very hydrophobic polypeptides.

Ion exchange chromatography orthogonal analytical

techniques

The benefits of ion exchange chromatography

■ high sample loading capacity.

■ crude solutions can be loaded

The benefits of Reversed Phase chromatography

■ a high degree of selectivity based on differences in hydrophobicity or molecular conformation.

190

■ crude solutions can be loaded onto ion-exchange columns.

■ addition of urea, acetonitrile or

non-ionic detergents to break-up

complexes.

■ optimization of elution selectivity

■ use of volatile buffers or ion-pairing agents.

■ freedom from interferences by

salt or buffers from ion exchange.

LC/MS additives and buffers summary

Protons donors

Acetic acid

Formic acid

Proton acceptors

Amonium hidroxid

Amonia solutions

191

Ion-Pair reagents

Trichloroaceticacid (low than 0.02% v/v)

Trifloroaceticacid

Triethylamine

Trimethylamine

Buffers

Amonium acetate

Amonium formate

How to make a gradient

192

193