ME 330.804: Mass Spectrometry in an “Omics” World...10/21/2012 1 ME 330.804: Mass Spectrometry...

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ME 330.804: Mass Spectrometry in an “Omics” World

Lecture 3MON 27 OCT, 2012Introduction to Proteomics

1ME 330.804: MS2012Mass Spectrometry in an “Omics” World http://www.hopkinsmedicine.org/mams/

Proteomics

DNA

RNA

post‐translationally modified proteins (PTMs)

proteins

biosynthesis of lipids, carbohydrates, small

molecules

enzymes

Genetic predisposition

Expression profiling

Identification of expressed proteins

Location of PTMs and quantitation

Disease biomarkers

proteomics, biomarker discovery and PTM quantitation


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mass/charge

inte

nsi

ty

D A E F R

mass/charge

inte

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D A E F R

mass/charge

inte

nsi

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mass/charge

inte

nsi

ty

mass/charge

inte

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H H V K

mass/charge

inte

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H H V K

trypsin Protein mass fingerprinting

Amino acid sequences

Identification of proteins in mixtures

Location of PTMs

MS

MS/MS

protein database

Most protein/PTM analyses begin by digestion with trypsin to cleave at R/K: “bottom up”


4

b. Using enzymes to determine structure and sequence

• peptide mapping

Chemical and enzymatic cleavage reagents


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c. Peptide mapping: tryptic and other enzymatic digests

Example. -amyloid peptide (A1-40):

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMGGVV

tryptic digest:DAEFR MW = 636.7 A1-5 MH+ observed = 637.8HDSGYEVHHQK MW = 1336.5 A6-16 MH+ observed = 1337.1LVFFAEDVGSNK MW = 1325.7 A17-28 MH+ observed = 1326.7GAIIGLMVGGVV MW = 1085.5 A29-40 MH+ observed = 1086.1

cyanogen bromide:VGGVV MW = 429.6 A36-40 MH+ observed = 431.1VGGVVIA MW = 613.8 A36-42 MH+ observed = 614.2GAIIGLM MW = 673.9 A29-35 MH+ observed = 626.0homoserine MW = 643.8homoserine lactone MW = 625.8

636.7 + 1336.5 + 1325.7 + 1085.5 – 3(18) = 4,329.9

Note that cyanogen bromide digestion revealed a longer amyloid peptide!

The principle of “mass balance”


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Amino acid “residue” masses


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mass/charge

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D A E F R

mass/charge

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D A E F R

mass/charge

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mass/charge

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mass/charge

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H H V K

mass/charge

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H H V K




Location of PTMs

MS

MS/MS

protein database

A mass spectrum of this digest is sufficient for identifying the protein if it has been purified


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%Int.

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3[c].G

92 mV[sum= 27531 mV] Profiles 1-300 Smooth Av 1

AXIMA-QIT Data: myoglobin dig dhb0002.G7 19 Dec 2002 12:12 Cal: combined 19 Dec 2002 12:46 Kratos PC Axima QIT V2.3.1: Mode Positive, Mid 750+, Power: 59

1271.711606.92

1378.90

1854.01

1272.74

1379.881855.01 1982.111607.93

1268.59

1983.08952.53

1192.70

1502.721273.73 1856.001608.90 1984.111380.91953.53 1096.56

1503.75996.52 1120.69 1856.991270.65902.471985.14997.53 1506.96 1609.881143.64 1393.83

A mass spectrum of this digest is sufficient for identifying the protein if it has been purified


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Identification using a database search:mass fingerpintingTaxonomy :Mammalia (mammals) (246067 sequences)

Timestamp : 19 Dec 2002 at 12:18:46 GMTTop Score : 158 forgi|2506462, Myoglobin

Probability Based MowseScore

Protein Summary Report

Switch to Concise Protein Summary Report

To create a bookmark for this report, right click this link: Protein Summary Report (../data/20021219/ FtTtonYe. dat )

Index

Accession Mass Score Description1. gi|2506462 16941 158 Myoglobin2. gi|70561 16940 138 myoglobin [validated] - horse3. gi|2554649 16942 137 Myoglobin (Horse Heart) Mutant WithLeu 104 Replaced ByAsn (L104n)4. gi|494711 16967 118 Myoglobin (Horse Heart) Mutant With His 64 Replaced ByTyr (H64y)5. gi|2914321 16905 118 H64t Variant OfMyoglobin (Horse Heart) Recombinant Wild-Type

6. gi|999870 16967 117 Myoglobin Mutant With His 93 Replaced ByTyr (H93y)7. gi|1942750 16969 117 Myoglobin (Horse Heart) Mutant WithSer 92 Replaced By Asp (S92d)8. gi|25029635 33033 88 similar to pORF2 [Mus musculus domesticus]9. gi|127664 17226 66 Myoglobin10. gi|127671 17034 65 MYOGLOBIN

Results List

1. gi|2506462 Mass: 16941 Score: 158MyoglobinObserved Mr(expt) Mr(calc) Delta Start End Miss Peptide941.48 940.47 940.47 0.01 146 - 153 1 YKELGFQG1271.67 1270.66 1270.66 0.01 32 - 42 0 LFTGHPETLEK1378.83 1377.82 1377.83 -0.01 64 - 77 0 HGTVVLTALGGILK1502.68 1501.67 1501.66 0.01 119 - 133 0 HPGDFGADAQGAMTK1506.94 1505.93 1505.93 0.00 64 - 78 1 HGTVVLTALGGILKK1606.85 1605.84 1605.85 -0.01 17 - 31 0 VEADIAGHGQEVLIR1815.88 1814.87 1814.90 -0.02 1 - 16 0 GLSDGEWQQVLNVWGK1853.94 1852.93 1852.95 -0.02 80 - 96 0 GHHEAELKPLAQSHATK1885.00 1883.99 1884.01 -0.02 103 - 118 0 YLEFISDAIIHVLHSK1982.02 1981.01 1981.05 -0.04 79 - 96 1 KGHHEAELKPLAQSHATK

No match to: 931.53, 934.49, 949.50, 951.50, 952.52, 968.46, 978.46, 988.49,1006.54


m/e

m/e

ACTGCTGACCTGGTACTGCATGGCAACGTCATGATTCGAAGTCGAAGTCCTAGTCACCTTGTGCAGTTGCTGGATACCGGTCACAATCGTAAGCTGCCATGCAGTACGTACTGACTT

proteins

DNA

Fenselau, C; Demirev, P.A., Characterization of intact microorganisms by MALDI mass spectrometry, Mass Spectrometry Reviews 20 ( 2001) 157-171

Masses are compared with thein silico digest, the calculated masses of all possible tryptic cleavage products for any protein or ORF

Why does “mass fingerprinting” work?


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Table 2. Nine Proteins Identified from HEL Cell CBB 2.D Gel

gel enzyme MW /pI SwissProt protein nameSpot access. No.

Gl trypsin 18012.6/7.68 PO5092 PPIase G2 trypsin 26669.6/6.45 POO938 triosephosphate

isomeraseG3 trypsin 26669.6/6.45 POO938 TIMG8 trypsin 29032.8/4.75 P12324 tropomyosin,

cytoskeletal type G10 trypsin 32575.2/4.64 PO6748 NPMGll trypsin 41737.0/5.29 PO2570 -actinG12 trypsin 61055.0/5.70 P10809 HSP-60G13 trypsin 56782.7/5.99 P30101 ERP60G14 trypsin 47169.2/7.01 PO6733 -enolase

Larger proteins will give more false hits since they have more peptide fragments; restrict search by MW of protein

Wall, et al. Anal. Chem. 72 (2000) 1099-1111.

Identification of gel spots from tryptic maps


mass/charge

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mass/charge

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D A E F R

mass/charge

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mass/charge

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mass/charge

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H H V K

mass/charge

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H H V K




Location of PTMs

MS

MS/MS

protein database

De novo amino acid sequencing or finding PTMs requires tandem mass spectra, or MS/MS


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H2N CH

C NH

CH

C NH

CH

C NH

CH

C OH

R1

O

R2

O

R3

O

R4

O

x3 y3 z3 x2 y2 z2 x1 y1 z1

a1 b1 c1 a2 b2 c2 a3 b3 c3

Biemann, K., Biomed. Mass Spectrom. 16 (1988) 99; Biemann, K. in Methods in Enzymology 193: Mass Spectrometry, McCloskey, J.A., Ed.; Academic Press, San Diego (1990) pp. 886-887.

De novo sequencing: the fragment ion notation

Sequence ions: a, b, c and x, y, z

Side chain fragmentation ions: d, v, w

Internal ions and immonium ions


H2NHC C N

H

HC C OH

CH3

O

CH3

O

H2NHC C N

H

HC C OH

CH3

O

CH3

O+ H H

H2NHC C N

HC C OH

CH3

O

CH3

OH

H

H2NHC C

CH3

O

H2NHC C OH

CH3

O

+

H2NHC C

CH3

O

- COH2N CH

CH3

carbonium ionleaving group is a stable neutral amine

b –ion

a -ion

acylium ion

heterolytic cleavage

MS/MS can form N-terminal fragment ions

Molecular ion observed by MS

Fragment ions observed by MS/MS


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b-ions for the peptide DAEFR are calculated by summing the masses to the left of each cleavage point:

a-ions are 28 mass units lighter than the b-ions

Calculating the a- and b-ions for a peptide

1 termNresidues


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H2NHC C N

H

HC C OH

CH3

O

CH3

O

H2NHC C N

H

HC C OH

CH3

O

CH3

O+ H H

H2NHC C N

H

HC C OH

CH3

O

CH3

OH

H3NHC C OH

CH3

O

H

HC C OH

CH3

O- NH3

H2NHC C C C OH

CH3

O

CH3

O

N

HH

H

HC C OH

CH3

O

H2NHC C

CH3

O

NH2+

y-ion z-ion

z-ion

hydrogen transfer

Or the z-ion is formed directly

MS/MS can form C-terminal fragment ions

Molecular ion observed by MS

Fragment ions observed by MS/MS


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y-ions for the peptide DAEFR are calculated by summing the masses to the right of each cleavage point including the mass of a hydrogen ion:

Calculating the y-ions for a peptide

19 termCresidues

Protonated molecular ion is a y-ion!


Y-ions

GS

Y+H2O

SHHHHH

H H H H H S S G E N L

B-ions

Example of sequencing by MS/MS


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How do you determine what kinds of ions are in the mass spectrum?

Theoretical spectrum of DAEFR

Things to remember:

1. The molecular ion is a y-ion

2. b-ions are missing a water

3. a-ions are missing a formic acid

4. a-ions are 28 mass units less than b-ions


MS/MS SPECTRA OF

PEPTIDES: NFNRHLHFTLVKDR AND

LLSYDDEAFIRDVAKTimperman, A.T.; Aebersold, R., Anal. Chem. 72 (2000) 4115-4121.

Homework problem: calculate the masses of b and y ions for the two peptides shown and compare with results obtained in their mass spectra.

How good is the mass accuracy in PPM?


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These are known as sequence tags …..

Tandem mass spectrum of a biomarker with a mass of 6710.5

It is generally not necessary to get the whole sequence in order to ID a protein


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Two peptides are found with the same sequence segment, but only one has the correct molecular weight

It is generally not necessary to get the whole sequence in order to ID a protein


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Peptide sequencing can be used to determine post-translational modifications


Post-translational modifications: phosphorylation of SRSGALK from sCMV assembly protein

How do the sequence ion mass spectra differ between the phosphorylated and non-phosphorylated peptide?

For the y-ions:

The b-ions and others can be determined as well, by considering pSer as an amino acid residue with a mass of 167.1


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600 800 1000 1200 1400 1600 1800

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y5y6

y7

y8 y9 y10y11

y12

y13 y14

y14-98

b10b13b12

b10

b11

MH+

MH+ -98y15

*

****

*

FQpSEEQQQTEDELQDK pSer

y14 y13

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%Int.

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100 150 200 250 300 350 400 450 500 550 600

y1 b2

y2

b4

b3

MH+

MH+ -98

y4

*

*

*pThr

b6 y6b7 b8-80

b8

b14

b12

b11

MH+

MH+ -98*

*

**

VSSDGHEpYIYVDPMQLPY

pTyr

b7 b8

b9-80

b9*

b10*

b15*

b16*

MH+ -80

RLEpTRb3

y1y2

b4

y2-98

b4-98

y4-98pThr

600 800 1000 1200 1400 1600 1800

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y5y6

y7

y8 y9 y10y11

y12

y13 y14

y14-98

b10b13b12

b10

b11

MH+

MH+ -98y15

*

****

*


y14 y13

0

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60

80

100

%Int.

100 150 200 250 300 350 400 450 500 550 600 650 700 750Mass/Charge

100 150 200 250 300 350 400 450 500 550 600

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100

%Int.

100 150 200 250 300 350 400 450 500 550 600 650 700 750Mass/Charge

100 150 200 250 300 350 400 450 500 550 600

y1 b2

y2

b4

b3

MH+

MH+ -98

y4

*

*

*pThr

b6 y6b7 b8-80

b8

b14

b12

b11

MH+

MH+ -98*

*

**

VSSDGHEpYIYVDPMQLPY

pTyr

b7 b8

b9-80

b9*

b10*

b15*

b16*

MH+ -80

RLEpTRb3

y1y2

b4

y2-98

b4-98

y4-98pThr

(a)

(c)

(b)

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%Int.

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600 800 1000 1200 1400 1600 1800

y5y6

y7

y8 y9 y10y11

y12

y13 y14

y14-98

b10b13b12

b10

b11

MH+

MH+ -98y15

*

****

*


y14 y13

0

20

40

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100

%Int.

100 150 200 250 300 350 400 450 500 550 600 650 700 750Mass/Charge

100 150 200 250 300 350 400 450 500 550 600

0

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%Int.

100 150 200 250 300 350 400 450 500 550 600 650 700 750Mass/Charge

100 150 200 250 300 350 400 450 500 550 600

y1 b2

y2

b4

b3

MH+

MH+ -98

y4

*

*

*pThr

b6 y6b7 b8-80

b8

b14

b12

b11

MH+

MH+ -98*

*

**

VSSDGHEpYIYVDPMQLPY

pTyr

b7 b8

b9-80

b9*

b10*

b15*

b16*

MH+ -80

RLEpTRb3

y1y2

b4

y2-98

b4-98

y4-98pThr

600 800 1000 1200 1400 1600 1800

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600 800 1000 1200 1400 1600 1800

y5y6

y7

y8 y9 y10y11

y12

y13 y14

y14-98

b10b13b12

b10

b11

MH+

MH+ -98y15

*

****

*


y14 y13

0

20

40

60

80

100

%Int.

100 150 200 250 300 350 400 450 500 550 600 650 700 750Mass/Charge

100 150 200 250 300 350 400 450 500 550 600

0

20

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60

80

100

%Int.

100 150 200 250 300 350 400 450 500 550 600 650 700 750Mass/Charge

100 150 200 250 300 350 400 450 500 550 600

y1 b2

y2

b4

b3

MH+

MH+ -98

y4

*

*

*pThr

b6 y6b7 b8-80

b8

b14

b12

b11

MH+

MH+ -98*

*

**

VSSDGHEpYIYVDPMQLPY

pTyr

b7 b8

b9-80

b9*

b10*

b15*

b16*

MH+ -80

RLEpTRb3

y1y2

b4

y2-98

b4-98

y4-98pThr

(a)

(c)

(b)

Phosphorylation:

Some examples of a phosphoserine, phosphothreonine and phosphotyrosine

Note: while fragmentation is sufficient to determine the positions of phosphorylation, the major fragmentation is the loss of the labile phosphate group.

Note: here the phosphate group appears in the b-series, y-series or both.


Note cleavages at aspartate residues

Wang, D.; Thompson, P.; Cole, P.A. and Cotter, R.J., Structural Analysis of a Highly Acetylated Protein Using a Curved-Field Reflectron Mass Spectrometer, Proteomics 5 (2005) 2288-96.

Generally not necessary to see the entire amino acid sequence to identify a peptide or to locate a post-translational modification

K1337

K1473 K1499

K1542

K1554

K1637

K1546K1549K1550K1551

K1555K1558K1560

K1337

K1473 K1499

K1542

K1554

K1637

K1546K1549K1550K1551

K1555K1558K1560

Post-translational modifications: acetylation


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Anderson, N.L.; Anderson, N.G., The Human Plasma Proteome: History, Character, and Diagnostic Prospects, Mol Cell Proteomics 1 (2002) 845-867].

albumin

biomarkers from tissue

Proteomics in the real world: the plasma proteome


Proteomics Approaches

Top-down methods• Pre-separation at the protein level using SEC or HPLC• On-line protein separation using HPLC or HILIC• Direct mass spectrometric sequencing of proteins

- electron-transfer dissociation (ETD) on an LTQ/Orbitrap- electron-capture dissociation (ECD) on an FTMS

Bottom-up methods• Off-line separation of proteins using SEC or HPLC (C4)• Digestion of each protein fraction with trypsin• On-line HPLC (C18) mass spectrometry• MS sequencing of peptides using collision-induced dissociation (CID)


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mass/charge

inte

ns

ity

D A E F R

mass/charge

inte

ns

ity

D A E F R

mass/charge

inte

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mass/charge

inte

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mass/charge

inte

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H H V K

mass/charge

inte

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trypsin HPLC + MS

“Bottom up” methods in proteomics

Protein database

MS/MS

Mascot or Sequest

Identification of parent protein and/or PTM sites


Removal of albumin and other high abundance proteins

Protein isolation • immunoprecipitation

• by organelle• phosphorylated proteins

• etc.

Protein fractionation• size exclusion chromatography

• electrophoresis• reverse phase chromatography

Tryptic digestion of proteins

Peptide fractionation

MS and MS/MS of peptides

“Bottom-up” strategies for identifying proteins


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Generally begin with removal of albumin and other high abundance proteins

BioMag® ProMax Albumin Removal Kit

QIAGEN - QproteomeAlbumin/IgG Depletion Kit

Calbiochem® ProteoExtract™ Removal Kits

42% EtOH/ 0.1M NaCl1 hr. @ 4oC

then 45 min @ 16,000g

Lipid Depleted Serum

15 minutes@ 15,000 x g

IgG Depleted Serum

Protein G resin

Whole Serum

Albumin Enriched

Supernatant

Lipid/IgG/HSA Depleted

Serum Pellet


then 45 min @ 16,000g


then 45 min @ 16,000g





IgG Depleted Serum

Protein G resin

IgG Depleted Serum

Protein G resin

Whole Serum

Albumin Enriched

Supernatant

Albumin Enriched

Supernatant


Serum Pellet


Serum Pellet

Fu et al., Proteomics 5 (2005) 2656 – 2664Colantonio et al., Proteomics 5 (2005) 3831-3835

Chemical extraction method


Biological sample

Size exclusion chromatography

Immuno‐precipitation

1D or 2D gel separation

Reversed phase HPLC

Tryptic digestion of each fraction

MS/MS

Protein separation

Peptide separation

Proteomics analyses generally require separations at the protein and peptide level


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200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600Mass/Charge

1

1054.63

587.031055.64

955.59458.04

588.071056.65

1036.64

570.051037.64858.53368.95 956.56 1592.92759.45

702.21

587.40

1452.89

1053.77

1567.95895.56459.08 799.46 1169.67 1269.73343.15 682.14215.48

1455.881361.83 1568.73

0

10

20

30

40

50

60

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90

100

%Int.

300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250Mass/Charge

1

3.7 mV[sum= 1114 mV] Prof iles 1-300 Smooth Av 5 -Baseline 100

Data: sample HM4 1260ms20001.N1 19 Aug 2002 17:04 Cal: Oklahoma 19 Aug 2002 16:10 Kratos PC Kompact MALDI 7 V2.3.0a: Mode PosMidMass, Power: 120

973.63

303.97

1243.71

566.94 715.27659.11 974.60314.96469.98 539.01400.95 756.43652.09355.97 955.56 1244.69697.15 842.51442.95 555.99 1102.65296.98 781.45

Need at least 2 peptide sequence spectra to identify a protein

MS/MS of 1611

MS/MS of 1260

Generally these will NOT be from the same chromatographic fraction


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Mascot Search Results

Search title : digestMS data file : C:\Program Files\Kompact\data\Customers\Oklahoma\mass lists\HM4 1611 ms2.txtDatabase : NCBInr 20020814 (1030915 sequences; 326041867 residues)Taxonomy : Drosophila (fruit flies) (28122 sequences)Timestamp : 19 Aug 2002 at 16:03:04 GMTSignificant hits: gi|5921205 ATP synthase alpha chain, mitochondrial precursor (Protein bellwether)

1. gi|5921205 Mass: 59384 Total score: 48 Peptides matched: 1 1 1611.15 1610.14 1609.87 0.27 0 48 1 TGAIVDVPVGDELLGR

Mascot Search Results

Search title : digestMS data file : C:\Program Files\Kompact\data\Customers\Oklahoma\mass lists\HM4 1260 ms2.txtDatabase : NCBInr 20020814 (1030915 sequences; 326041867 residues)Taxonomy : Drosophila (fruit flies) (28122 sequences)Timestamp : 19 Aug 2002 at 16:17:32 GMTSignificant hits: gi|5921205 ATP synthase alpha chain, mitochondrial precursor (Protein bellwether)1. gi|5921205 Mass: 59384 Total score: 38 Peptides matched: 1 1 1260.70 1259.69 1259.64 0.06 0 38 1 SAEISNILEER

MS/MS of 1611

MS/MS of 1260

In this example both MS/MS spectra give the same ID


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Discovery Mode (biomarkers)

Samples• Tissue, CSF, cells better than blood• May be pooled samples in discovery mode

Analysis• Separation at protein level using HPLC (C4 column) or SEC• Tryptic digestion of 10-15 protein fractions (grouped?)• On-line HPLC (C18 column) MS analysis• Data-dependent MS/MS analysis of major molecular species

Data analysis and bioinformatics• Peptide/protein ID using MASCOT or PROTEIN DISCOVERER• Other software, eg. Protein Center• Relative quantitation by spectral counting


Output: protein identifications from peptides


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Quantitative global analyses using spectral counting


38

Quantitation using isotope tags for relative and absolute quantitation (iTRAQ)


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Quantitation using isotope tags for relative and absolute quantitation (iTRAQ)


Validation and Quantitation

Prior to analysis• candidate peptides (proteins) from discovery analysis• candidate peptides from literature• candidate peptides from biological systems analysis• build SRM scheme (which transitions?) from:

- experimental data from discovery- software tools: PinPoint or Skyline

LCMS and Selected Reaction Monitoring (MRM)• individual (non-pooled) patent and control samples• triple quadrupole mass spectrometry

Biostatistics• Number of patient and control samples


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y11

y9

y7

Selected reaction monitoring (SRM)

GAGQNIIPASTGAAK

X

X

X

Triple quadrupole mass spectrometer


42

dc

volt

age

dc

volt

age

time time

dc

volt

age

dc

volt

age

time time

Product ion (normal) scan:

Multiple reaction monitoring (MRM) scan:

Reconstructed mass chromatograms?

dc

volt

age

dc

volt

age

time time

Reconstructed mass chromatogram:

Ion traps and TOFs scan whole spectrum in MS2

No sensitivity or selectivity advantage gained from plotting only the ions of interest

Quadrupoles allow only the selected ions through; increased selectivity and sensitivity for low abundance species


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sp|Q92876|KLK6_HUMAN

LSELIQPLPLER

704.413934 965.577842 y8704.413934 852.493778 y7704.413934 724.435201 y6

Surrogate biomarker peptide and its transitions


Can multiplex using programmed transition times


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“Top down” methods in proteomics

Protein database

MS/MS using ECD or ETD

ProSightPC

Identification of parent protein and/or PTM sites

Mass/charge

HPLC + MS

Mass/charge


Håkansson, Chalmers, M.J.; Quinn, J.P.; McFarland, M.A.; Hendrickson, C.L.; Marshall, A.G., Combined Electron Capture and Infrared Multiphoton Dissociation for Multistage MS/MS in a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer, Anal. Chem. 75 (2003) 3256-3262.

ECD on a Fourier transform mass spectrometer can be used for “top down” proteomics


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Figure 1. MS-based platform for the detection of individual histone H3 codes. The most abundant form contained H3.2K9me2 and H3.2K27me2; a less abundant form contained H3.2K9me1 and H3.2K27me3.

Benjamin A Garcia, James J Pesavento, Craig A Mizzen & Neil L Kelleher, Pervasive combinatorial modification of histone H3 in human cells, Nature Methods - 4, 487 - 489 (2007)

Identification of H3 isoforms by top-down methods

+18 intact H3.2 from HeLa cells +8 residues 1-50 H3.2

HILIC chromatography

+8 residues 1-50 Fraction 7


ME 330.804: Mass Spectrometry in an “Omics” World...10/21/2012 1 ME 330.804: Mass Spectrometry...

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