Protein Identification by MALDI Mass Spectrometry Karin Hjernø and Peter Roepstorff Department of Biochemistry and Molecular Biology University of Southern.

Protein Identification by

MALDI Mass Spectrometry

Karin Hjernø and Peter Roepstorff Department of Biochemistry and Molecular Biology

University of Southern Denmark

Denmark

Peptide Mass Fingerprint (PMF)2) Enzymatic digestion (trypsin)

3) Micropurification ( salt, + concentration)

4) Mass spectrometric analysis, data interpretation.

1) Separation of proteins, here by 2D-gels

pI

Mw

http://images.google.dk/imgres?imgurl=http://www.wellesley.edu/Chemistry/chem227/structproteins/stash.jpg&imgrefurl=http://www.wellesley.edu/Chemistry/chem227/structproteins/strctprt.htm&h=320&w=425&sz=29&tbnid=S4ykWHszDEcJ:&tbnh=91&tbnw=122&hl=da&start=7&prev=/images%3Fq%3Dkeratin%2B%26svnum%3D10%26hl%3Dda%26lr%3D

TheoreticalDigestion

Digestion

Protein of Interest

MALDI-MS

...LIHGFYMNKPL...

...LVCDERTFGHG...

...HYIGFREWMKL...

...LIYTSARDEFW...

.................

Databaseof known

Sequences

Experimental Peptide Masses

TheoreticalPeptide Masses

Comparison

679.52 684.34 842.50 856.52 864.48 870.531045.571126.571314.771794.87...

1074.501087.561151.531194.601200.631240.621246.571275.631323.681353.73...

Protein ID

PMF

Enzymes for PMF

www.matrixscience.com

Trypsin•High specificity•Peptides in a mass range compatible with MALDI•Small enzyme

MALDI-MS spectrum

m/z956.0 966.0

Inte

nsi

ty3.50

0

958.46

C:\Documents and Settings\Administrator\Desktop\yeast\b21735al\735calred.massml (10:50 10/29/01)

Description: NoName NoName

m/z2702.0 2711.0

Inte

nsi

ty

2.50

0

2705.2

C:\Documents and Settings\Administrator\Desktop\yeast\b21735al\735calred.massml (11:36 10/29/01)

Description: NoName NoName

1 Da1 Da

842.5 1602.8 2720.2

Three examples of a normal isotopic distribution at different m/z-value.

m/z 838.0 850.0

Intensity

40

0

m/z 1598.0 1612.0

Intensity

1.00

0

m/z 2716.0 2730.0

Intensity

1.80

0

m/z1276.0 1286.0

Inte

nsi

ty

18

0

1277.7 1279.7

C:\Documents and Settings\Administrator\Desktop\backupdisks\Karin\Andrea\Spot # 9341 16.5.00 b14856al.massml (11:27 01/15/02)

Description: Yeast

m/z1852.0 1860.0

Inte

nsi

ty

6.00

0

1852.0

C:\Documents and Settings\Administrator\Desktop\backupdisks\Karin\Andrea\Spot # 8538 16.5.00 b14934al.massml (11:29 01/15/02)

Description: Yeast

?

Isotope distribution

Removal of contaminants

Common contaminants•Keratin•Tryptic autodigest projects (842.5, 2211.1)

•Matrix-clusters

Overlapping matrix (871.94) and peptide (873.50) peaks.

m/z 500 4000

Inte

nsity

60

0

973.

43

1179

.5

Ker

atin

1 K

erat

in1

Ker

atin

1 Ker

atin

9

Ker

atin

9 K

erat

in1

Ker

atin

1 Ke

ratin

1 K

erat

in9

Ker

atin

9 K

erat

in1

Ker

atin

1

Ker

atin

9

Ker

atin

1

C:\Documents and Settings\Administrator\Desktop\keratinrigtspectra.massml (10:52 01/15/02) Description: Yeast

Removal of contaminantsPage selection

Read peak list from clipboard

Copy peak list to clipboard

Perform multipoint calibration

Show/hide graph window

Set window for erazor list

Select erazor list

Select sections in erazor list

Display options

Project name

List information

Mass info

Mass list

Peak Erazor

www.welcome.to/GPMAW

m/z1000 5000

Inte

nsity

25

0

1638

.71475

.6

842.

42

2704

.82210

.8

1851

.7

1993

.7

1267

.6

2500

.91307

.5

2202

.9

1765

.5

3338

.333

24.3

3146

.2

2932

.1

1179

.5

2716

.7

2383

.6

2185

.9

<33

55.2

>

1707

.6

2234

.922

24.8

2398

.7

1794

.6

1657

.617

59.7

1280

.6

2283

.9

2149

.8

2444

.9

1715

.6 1837

.7

830.

37

1818

.7

2342

.7

1320

.4

2509

.8

1234

.5

2183

.8

1384

.6

1251

.6

1434

.6

2565

.9

2790

.128

22.0

2748

.126

44.0

1323

.5

2583

.925

89.9

1503

.615

23.6

2807

.0

1036

.4

870.

44

2901

.2

3819

.6

3662

.3

1045

.5

2872

.0

1082

.5

3050

.9

875.

36

3677

.4\\Bmb-filsrv\PRpublic\IngerMZ\PH0508\p54731ic, plug 3.massml (11:21 03/15/05)Description: PH0508, Ag-plug 3

Outlier

Taking advantage of contaminants!

Identification of contaminants

Multipoint calibration on contaminants

< 20 ppm

What!? ... No calibrants..

m/z1000 5000

Inte

nsity

60

0

3905

.9

2568

.5

976.

28

2321

.4

2459

.4

4166

.72851

.5

1592

.3

1336

.3

2550

.5

1576

.3

2865

.5

4182

.6

2582

.5

2305

.4

951.

30

4150

.7

1318

.3 2587

.6

2430

.4

1954

.4

1047

.3

849.

24

1593

.3

2216

.522

90.5

2200

.5

1993

.4

2353

.423

39.4

2704

.4

1953

.4

4294

.6

1608

.3

2525

.5

2103

.521

18.5

2716

.3

1475

.4

1620

.3

1307

.4

1963

.4

1765

.3

1978

.4

919.

25

1794

.3

4032

.7

2601

.6

842.

31

1356

.4

4310

.6

1447

.414

05.3

1277

.4

1106

.3

1658

.4

1063

.3

1464

.4

935.

2599

2.24

820.

23

2444

.4

\\Bmb-filsrv\PRpublic\IngerMZ\PH0507\p54545ic, gelplug 5.massml (11:39 03/15/05)Description: PH0507, gelplug 5

The mass defect: Difference between the monoisotopic and the integer mass value of a given amino acid residue,

The mass defect of a peptide around 8-900 Da is .5, around 2000 Da it is .0 (mass dependent)

< 50 ppm

Filteret and calibrated peak list, what then?!?

Make a database dependent search;

•Open the prefered search program

•Choose the search database

•Choose the search parameters

•Start the search....... Protein candidates

Mascot Interface

927.4719960.5380999.61301026.57271055.53721066.56411072.57671088.60551099.54881129.66791199.67241211.6559

.......

.......

.......

.......1768.94411782.91711867.84181894.96841924.99951982.08342056.98542142.05942310.22162720.3082

Results List

1. gi|6321412 Mass: 76977 Score: 103 tryptophan synthetase; Trp5p [Saccharomyces cerevisiae]Observed Mr(expt) Mr(calc) Delta Start End Miss Peptide 759.43 758.42 758.41 0.01 695 - 700 0 IGWDLR 830.45 829.44 829.44 -0.00 439 - 445 0 QALNVFR 1 Pyro-glu (N-term Q)847.47 846.47 846.47 -0.01 439 - 445 0 QALNVFR 907.51 906.50 906.50 0.00 473 - 479 0 FWVTNLK1003.53 1002.52 1002.57 -0.05 438 - 445 1 RQALNVFR1031.46 1030.45 1030.48 -0.02 273 - 280 0 DEFFAFQK1267.72 1266.71 1266.77 -0.06 385 - 396 0 INNALAQVLLAK

1319.64 1318.63 1318.69 -0.05 207 - 219 0 DTPLAVGFGVSTR1458.70 1457.69 1457.76 -0.07 269 - 280 1 VLSKDEFFAFQK

1484.77 1483.76 1483.79 -0.02 677 - 690 1 GDKDVQSVAEVLPK1583.75 1582.74 1582.79 -0.05 361 - 373 0 LTEHCQGAQIWLK 1607.80 1606.79 1606.85 -0.05 344 - 357 0 SLYSYIGRPSSLHK1664.86 1663.85 1663.89 -0.05 620 - 635 0 AQFIAATDAQALLGFK1702.76 1701.76 1701.79 -0.03 423 - 437 0 FGLTCTVFMGAEDVR

………...

Non-significant matches

1 MSEQLRQTFA NAKKENRNAL VTFMTAGYPT VKDTVPILKG FQDGGVDIIE 51 LGMPFSDPIA DGPTIQLSNT VALQNGVTLP QTLEMVSQAR NEGVTVPIIL 101 MGYYNPILNY GEERFIQDAA KAGANGFIIV DLPPEEALKV RNYINDNGLS 151 LIPLVAPSTT DERLELLSHI ADSFVYVVSR MGTTGVQSSV ASDLDELISR 201 VRKYTKDTPL AVGFGVSTRE HFQSVGSVAD GVVIGSKIVT LCGDAPEGKR 251 YDVAKEYVQG ILNGAKHKVL SKDEFFAFQK ESLKSANVKK EILDEFDENH 301 KHPIRFGDFG GQYVPEALHA CLRELEKGFD EAVADPTFWE DFKSLYSYIG 351 RPSSLHKAER LTEHCQGAQI WLKREDLNHT GSHKINNALA QVLLAKRLGK 401 KNVIAETGAG QHGVATATAC AKFGLTCTVF MGAEDVRRQA LNVFRMRILG 451 AKVIAVTNGT KTLRDATSEA FRFWVTNLKT TYYVVGSAIG PHPYPTLVRT 501 FQSVIGKETK EQFAAMNNGK LPDAVVACVG GGSNSTGMFS PFEHDTSVKL 551 LGVEAGGDGV DTKFHSATLT AGRPGVFHGV KTYVLQDSDG QVHDTHSVSA 601 GLDYPGVGPE LAYWKSTGRA QFIAATDAQA LLGFKLLSQL EGIIPALESS 651 HAVYGACELA KTMKPDQHLV INISGRGDKD VQSVAEVLPK LGPKIGWDLR 701 FEEDPSA

Sequence Coverage: 51%

Significant matchesp < 0.05

m/z500 4000

Inte

nsity

18

0

1891.9

<3810.9><3340.7>

2037.02191.1

1319.6

1882.01475.7

1708.9

2066.0

1307.6

2211.1

1994.0

1393.7

1702.8 1948.0

1435.71179.5

2284.2

1607.81458.7

1277.7

1638.8 2403.22705.2

842.50

1234.61073.6

2717.12249.1

1045.5958.46

2754.4

823.56

847.47759.43

2807.3578.47


Tryptic auto digest

Contaminants


~5500 Da

•Maldi mass range : from ~ 700 Da to ~ 3500

•Suppression effect (preferential ionization of some components at the expence of others)

•Post-translational modifications

Why not 100% sequence coverage

Example – a classical example

Manual evaluation – what to look for?

•Likely/unlikely missed cleavage sites

•Overlapping peptides

•Partial modifications

•Mass accuracy

•(Intensity of the peaks)

•……

N CR R RPKR K

N C

N

Missed cleavage sites

Digestion using trypsin

Missed cleavages•Only missed cleavages of one of the following kind are highlylikely to be observed (relative to other sites): R/KxxxxxxxR/K

xxxxxxxR/KR/KxR/KxxxxxxR/KxxxxE/D R/KxxxxR/KxxxxE/Dx R/KxxxxR/KxxxxR/K E/DxxxxR/KxxxxR/K xE/DxxxxR/KxxxxR/K PxxxxR/K

Basic or acidic residue close to the cleavage site in question


N CR R RPKR K

N C

N

RxE

DR

KR

Manual evaluation




•Mass accuracy


•……


N CR R RPKR K

N C

NDR

KR

M

Mox

Inte

nsity

121

811.

782

(179

4.8

17)

177

9.7

93

178

3.8

00

182

7.7

86

m/z1780 18300

0

113

6.5

m/z1250 1300

Inte

nsity

70

0

128

2.6

38

125

0.6

61

125

4.6

44

126

6.6

41

129

8.6

33

+4

+16 +32

+48

Non-modified

HVFSATQLAAM(ox)FIDK

m/z 1680.0 1700.0

Intensity

16

0

1678.8

1694.8

C:\Documents and Settings\Administrator\Desktop\backupdisks\Karin\Andrea\yeast\b21738al\738calnotred.massml (13:44 01/15/02) Description: NoName NoName

16 Da

HVFSATQLAAMFIDK

m/z830.0 850.0

Inte

nsity

6.00

0

847.47

Tryptic autodigest

QALNVFR

Pyro-QALNVFR

830.45

17 Da

Partial modification

Oxidized tryptophan (W)

Oxidized methionine N-terminal pyro-glutamate

Manual evaluation – what to look for?




•Mass accuracy


•……

Example – a classical example

Example 2 – a false-positive

m/z800 2200

Inte

nsi

ty

30

0

20

60

.9

19

14

.01

90

1.0

16

77

.9

15

82

.8

14

95

.9

12

87

.6

12

18

.7

10

92

.6

10

18

.5

87

0.5

6

10

47

.6

10

90

.61

12

8.6

11

45

.61

15

1.6

12

17

.71

25

9.6

12

63

.81

29

8.7

14

15

.7 14

39

.81

46

3.8

14

69

.81

45

6.8

15

43

.9

17

79

.8

21

28

.0

22

11

.10

0

10

45

.55

7

12

99

.80

7

22

25

.10

9

84

2.5

09

6

22

83

.13

8

17

94

.84

4

11

46

.62

5

22

33

.10

4

13

09

.67

7

22

98

.16

5

82

4.5

24

3

Spot # 241 8.2.00 b12420al.massml



842.5096927.4719960.5380999.61301026.57271045.57271055.53721066.56411072.57671080.58851088.60551099.54881129.66791197.66231199.67241211.6559

.......

.......

.......

.......1768.94411782.91711867.84181894.96841904.03021924.99951982.08342045.04592056.98542142.05942211.09962310.22162720.30822807.2930

Peak List

Protein mixtures

m/z500 2500

Inte

nsi

ty

25

0

13

61

.76

15

92

.99

12

90

.69

14

46

.78

12

42

.68

16

94

.88

14

50

.74

20

56

.99

16

39

.95

15

67

.77

13

46

.68

14

87

.74

14

67

.87

16

23

.82

12

53

.65

17

68

.94

16

21

.78

12

11

.66

10

88

.61

17

17

.91

11

97

.66

17

82

.92

11

29

.67

19

25

.00

16

87

.87

22

11

.1010

55

.54

10

99

.55

10

45

.57

21

42

.06

18

94

.97

10

72

.58

84

2.5

10

10

80

.59

27

20

.31

20

45

.05

19

04

.03

28

07

.29

96

0.5

38

99

9.6

13

92

7.4

72

23

10

.22

10

26

.57

10

66

.56

11

99

.67

14

39

.80 14

79

.82

17

21

.91

17

38

.90

18

67

.84

19

82

.08

\\Hermes\prgroup\Karin\Andrea!\Spot # IT 2023 20.9.01 b23753al.massml (11:20 11/02/01)Description: Human

Annexin VI

Trypsin autodigest

Protein mixtures

Protein mixtures

m/z500 2500

Inte

nsi

ty

25

0

13

61

.76

15

92

.99

12

90

.69

14

46

.78

12

42

.68

16

94

.88

14

50

.74

20

56

.99

16

39

.95

15

67

.77

13

46

.68

14

87

.74

14

67

.87

16

23

.82

12

53

.65

17

68

.94

16

21

.78

12

11

.66

10

88

.61

17

17

.91

11

97

.66

17

82

.92

11

29

.67

19

25

.00

16

87

.87

22

11

.1010

55

.54

10

99

.55

10

45

.57

21

42

.06

18

94

.97

10

72

.58

84

2.5

10

10

80

.59

27

20

.31

20

45

.05

19

04

.03

28

07

.29

96

0.5

38

99

9.6

13

92

7.4

72

23

10

.22

10

26

.57

10

66

.56

11

99

.67

14

39

.80 14

79

.82

17

21

.91

17

38

.90

18

67

.84

19

82

.08

\\Hermes\prgroup\Karin\Andrea!\Spot # IT 2023 20.9.01 b23753al.massml (11:20 11/02/01)Description: Human

Annexin VI

Hypothetical protein XP_038637(heat shock 70D)

Trypsin autodigest

Protein mixtures

D11m/z1000 3500

Inte

nsi

ty

140

0

19

06

.98

4

14

67

.81

5

20

77

.08

0

22

92

.14

8

16

07

.83

4

13

02

.70

8

17

16

.85

3

14

34

.79

3

11

75

.65

31

12

7.5

62

28

73

.37

72

90

3.4

13

When MS/MS is needed...



Digestion

Protein of Interest

MALDI-MS

(MALDI-) MS/MS

RAWGYVLE

Protein ID

MS and MS/MS

Verification of protein ID, analysis of unassigned peaks

Tandem mass spectrometer

Mass analyzer

Ion Source DetectorMass

analyzerCC

Precursor selection

Collision Cell, fragmentation

of ionsSeparationof fragments

MS of a peptide mixture

899.013

MS/MS of a peptide2+

(collision energy 10 eV)899.013

MS/MS of a Peptide (collision energy 15 eV)

899.013

MS/MS of a Peptide(collision energy 18 eV)

899.013

Peptide fragmentation

Roepstorff and Fohlman, 1984

Biemann, 1988

Paizs and Suhai, 2004

Formation of b- and y-ions

Identification of the peptide LLQVVEEPQALAAFLR

Y1Y2

Y3Y4

Y5

Y6

Y7Y8

Y10

Y11

Y12

Y13

Y9899.013

Y13E E V V

Jens Andersen, Odense, Denmark

69.0 444.6 820.2 1195.8 1571.4 1947.0

Mass (m/z)

2.9E+4

0

10

20

30

40

50

60

70

80

90

100

% Inte

nsity

4700 MS/MS Precursor 1844.93 Spec #1 MC=>NF0.7[BP = 120.1, 29008]

120.0661

86.0839

201.06831035.360284.0678 792.2761

1225.3296

348.1187 907.2828417.1365

1053.3120620.2520173.0822 435.13651150.3596

235.08091297.4041

102.0426 330.11821497.4641

288.0954 1716.5573530.161474.0513 1410.4763735.2722 1827.02581110.3387401.1511 1597.5017191.0951 574.1970303.1168 1282.3550836.1899 949.2646 1468.4775

Manuel Interpretation

115

Amino Acid 3 LetterCodeSingle Letter

Code Residue Mass

Monoisotopic

Glycine Gly G 57.02147

Alanine Ala A 71.03712

Serine Ser S 87.03203

Proline Pro P 97.05277

Valine Val V 99.06842

Threonine Thr T 101.04768

Cysteine Cys C 103.00919

Isoleucine Ile I 113.08407

Leucine Leu L 113.08407

Asparagine Asn N 114.04293

Aspartic Acid Asp D 115.02695

Glutamine Gln Q 128.05858

Lysine Lys K 128.09497

Glutamic Acid Glu E 129.04260

Methionine Met M 131.04049

Histidine His H 137.05891

Phenylalanine Phe F 147.06842

Arginine Arg R 156.10112

Tyrosine Tyr Y 163.06333

Tryptophan Try W 186.07932

69.0 444.6 820.2 1195.8 1571.4 1947.0

Mass (m/z)

2.9E+4

0

10

20

30

40

50

60

70

80

90

100

% Inte

nsity


120.0661

86.0839

201.06831035.360284.0678 792.2761

1225.3296

348.1187 907.2828417.1365

1053.3120620.2520173.0822 435.13651150.3596

235.08091297.4041

102.0426 330.11821497.4641

288.0954 1716.5573530.161474.0513 1410.4763735.2722 1827.02581110.3387401.1511 1597.5017191.0951 574.1970303.1168 1282.3550836.1899 949.2646 1468.4775

D


69.0 444.6 820.2 1195.8 1571.4 1947.0

Mass (m/z)

2.9E+4

0

10

20

30

40

50

60

70

80

90

100

% Inte

nsity


120.0661

86.0839

201.06831035.360284.0678 792.2761

1225.3296

348.1187 907.2828417.1365

1053.3120620.2520173.0822 435.13651150.3596

235.08091297.4041

102.0426 330.11821497.4641

288.0954 1716.5573530.161474.0513 1410.4763735.2722 1827.02581110.3387401.1511 1597.5017191.0951 574.1970303.1168 1282.3550836.1899 949.2646 1468.4775

D

128146



Code Residue Mass

Monoisotopic





















146??

69.0 444.6 820.2 1195.8 1571.4 1947.0

Mass (m/z)

2.9E+4

0

10

20

30

40

50

60

70

80

90

100

% Inte

nsity


120.0661

86.0839

201.06831035.360284.0678 792.2761

1225.3296

348.1187 907.2828417.1365

1053.3120620.2520173.0822 435.13651150.3596

235.08091297.4041

102.0426 330.11821497.4641

288.0954 1716.5573530.161474.0513 1410.4763735.2722 1827.02581110.3387401.1511 1597.5017191.0951 574.1970303.1168 1282.3550836.1899 949.2646 1468.4775

D

Q/K


69.0 444.6 820.2 1195.8 1571.4 1947.0

Mass (m/z)

2.9E+4

0

10

20

30

40

50

60

70

80

90

100

% Inte

nsity


120.0661

86.0839

201.06831035.360284.0678 792.2761

1225.3296

348.1187 907.2828417.1365

1053.3120620.2520173.0822 435.13651150.3596

235.08091297.4041

102.0426 330.11821497.4641

288.0954 1716.5573530.161474.0513 1410.4763735.2722 1827.02581110.3387401.1511 1597.5017191.0951 574.1970303.1168 1282.3550836.1899 949.2646 1468.4775

D

Q/K

172



Code Residue Mass

Monoisotopic





















172?!?

69.0 444.6 820.2 1195.8 1571.4 1947.0

Mass (m/z)

2.9E+4

0

10

20

30

40

50

60

70

80

90

100

% Inte

nsity


120.0661

86.0839

201.06831035.360284.0678 792.2761

1225.3296

348.1187 907.2828417.1365

1053.3120620.2520173.0822 435.13651150.3596

235.08091297.4041

102.0426 330.11821497.4641

288.0954 1716.5573530.161474.0513 1410.4763735.2722 1827.02581110.3387401.1511 1597.5017191.0951 574.1970303.1168 1282.3550836.1899 949.2646 1468.4775

D

Q/K

172

57



Code Residue Mass

Monoisotopic





















69.0 444.6 820.2 1195.8 1571.4 1947.0

Mass (m/z)

2.9E+4

0

10

20

30

40

50

60

70

80

90

100

% Inte

nsity


120.0661

86.0839

201.06831035.360284.0678 792.2761

1225.3296

348.1187 907.2828417.1365

1053.3120620.2520173.0822 435.13651150.3596

235.08091297.4041

102.0426 330.11821497.4641

288.0954 1716.5573530.161474.0513 1410.4763735.2722 1827.02581110.3387401.1511 1597.5017191.0951 574.1970303.1168 1282.3550836.1899 949.2646 1468.4775

D

Q/KG

115


69.0 444.6 820.2 1195.8 1571.4 1947.0

Mass (m/z)

2.9E+4

0

10

20

30

40

50

60

70

80

90

100

% Inte

nsity


120.0661

86.0839

201.06831035.360284.0678 792.2761

1225.3296

348.1187 907.2828417.1365

1053.3120620.2520173.0822 435.13651150.3596

235.08091297.4041

102.0426 330.11821497.4641

288.0954 1716.5573530.161474.0513 1410.4763735.2722 1827.02581110.3387401.1511 1597.5017191.0951 574.1970303.1168 1282.3550836.1899 949.2646 1468.4775

D

Q/KG

D


69.0 444.6 820.2 1195.8 1571.4 1947.0

Mass (m/z)

2.9E+4

0

10

20

30

40

50

60

70

80

90

100

% Inte

nsity


120.0661

86.0839

201.06831035.360284.0678 792.2761

1225.3296

348.1187 907.2828417.1365

1053.3120620.2520173.0822 435.13651150.3596

235.08091297.4041

102.0426 330.11821497.4641

288.0954 1716.5573530.161474.0513 1410.4763735.2722 1827.02581110.3387401.1511 1597.5017191.0951 574.1970303.1168 1282.3550836.1899 949.2646 1468.4775

F

DQ/K

GD

DL


69.0 444.6 820.2 1195.8 1571.4 1947.0

Mass (m/z)

2.9E+4

0

10

20

30

40

50

60

70

80

90

100

% Inte

nsity


120.0661

86.0839

201.06831035.360284.0678 792.2761

1225.3296

348.1187 907.2828417.1365

1053.3120620.2520173.0822 435.13651150.3596

235.08091297.4041

102.0426 330.11821497.4641

288.0954 1716.5573530.161474.0513 1410.4763735.2722 1827.02581110.3387401.1511 1597.5017191.0951 574.1970303.1168 1282.3550836.1899 949.2646 1468.4775

F

DQ/K

GD

DL


69.0 444.6 820.2 1195.8 1571.4 1947.0

Mass (m/z)

2.9E+4

0

10

20

30

40

50

60

70

80

90

100

% Inte

nsity


120.0661

86.0839

201.06831035.360284.0678 792.2761

1225.3296

348.1187 907.2828417.1365

1053.3120620.2520173.0822 435.13651150.3596

235.08091297.4041

102.0426 330.11821497.4641

288.0954 1716.5573530.161474.0513 1410.4763735.2722 1827.02581110.3387401.1511 1597.5017191.0951 574.1970303.1168 1282.3550836.1899 949.2646 1468.4775

GDG


69.0 444.6 820.2 1195.8 1571.4 1947.0

Mass (m/z)

2.9E+4

0

10

20

30

40

50

60

70

80

90

100

% Inte

nsity


120.0661

86.0839

201.06831035.360284.0678 792.2761

1225.3296

348.1187 907.2828417.1365

1053.3120620.2520173.0822 435.13651150.3596

235.08091297.4041

102.0426 330.11821497.4641

288.0954 1716.5573530.161474.0513 1410.4763735.2722 1827.02581110.3387401.1511 1597.5017191.0951 574.1970303.1168 1282.3550836.1899 949.2646 1468.4775

Immonium ions, ”diagnostic” ions

72 83 94 105 116 127

Mass (m/z)

2.9E+4

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity


120.0661

86.0839

84.0678

102.0426

74.0513101.0969

72.0 100.2 128.4 156.6 184.8 213.0

Mass (m/z)

2.9E+4

0

10

20

30

40

50

60

70

80

90

100

% Int

ensit

y


120.0661

86.0839

201.0683

84.0678 129.0862

173.0822

102.0426

74.0513147.0961 191.0951101.0969 183.0528 203.0798175.0881159.0696

K/Q

I/L

T

F

K/Q

E

Immonium ions, ”diagnostic” ions

69.0 444.6 820.2 1195.8 1571.4 1947.0

Mass (m/z)

2.9E+4

0

10

20

30

40

50

60

70

80

90

100

% Inte

nsity


120.0661

86.0839

201.06831035.360284.0678 792.2761

1225.3296

348.1187 907.2828417.1365

1053.3120620.2520173.0822 435.13651150.3596

235.08091297.4041

102.0426 330.11821497.4641

288.0954 1716.5573530.161474.0513 1410.4763735.2722 1827.02581110.3387401.1511 1597.5017191.0951 574.1970303.1168 1282.3550836.1899 949.2646 1468.4775

1. Ion series: DGD(Q/K)DFL

2. Ion series: GDG

Immonium ions: F,T,(I/L),E,(Q/K)

Proline-induced fragmenation (N-terminal to Proline; Xaa|Pro)

Most abundant when the Xaa is Val, His, Ile, Leu, Asp

Not abundant when Xaa is Pro or GlyBreci, 2003; Kapp, 2003

Fragmentation of VVAASLNPVDFK, singly charged ion

Fragmentation of VPTVDVSVVDLTVR , singly charged ion

No y13???

Peptide with proton predominantly associated with basic residue

The proton is mobilized through by collisional activation

NH2

NN

N

O

R1

R2

OR3

O

N

R4

OB

CO2H

H+

NH2

NN

N

O

R1

R2

OR3

O

N

R4

OLys

CO2HH+

Proton mobility

If an arginine is present (most basic residue), then the proton is sequestered (non-mobile) and require more energy for mobilization

NH2

NN

N

O

R1

R2

OR3

O

N

R4

OArg

CO2H

H+

CID or PSD

NH2

NN

N

O

R1

R2

OR3

O

N

R4

OArg

CO2H

H+

H+

If more protons than arginines are present, then a mobile proton will be present

MALDI ESI

Acidic-induced fragmentation

Paizs and Suhai

Arg

Asp

Also Glu, but to a lesser degree

Charge-remote fragmentation in contrast to Charge-induced fragmentation

Fragmentation of VPTVDVSVVDLTVR , singly charged ion

y4y9

Dominating fragmentation-pathways for singly charged MALDI-peptide ions

LSMOXTNDPLEAAR

Y6 Y3 Y1

64

64

Y6

Y3Y1

Verification of MALDI-TOF/TOF search result

Peptides containing ArgParent mass Intensity parent mass Sequence Modification Expected MS/MS ions

1479.15 37417 LGEYGFQNALIVR 175.12 300.16 1180.651566.86 138278 DAFLGSFLYEYSR 175.12 116.03 425.21 1143.54 1452.72928.45 78882 YLYEIAR 175.12 359.24 569.26

1193.86 170361 DTHKSEIAHR missed cleavage 175.12 116.03 496.30 698.31 1078.58712.97 12683 SEIAHR 175.12 217.08 496.30

1477.16 20315 FwGKYLYEIAR 2-W_Ox2/ 175.12 359.24 1119.51 1313.651084.16 18261 YLYEIARR missed cleavage 175.12 569.26 515.341053.21 13897 CCTKPESER 175.12 175.12 391.19 662.26 878.341283.69 14464 HPEYAVSVLLR 175.12 364.16 920.561439.31 264249 RHPEYAVSVLLR missed cleavage 175.12 520.26 920.56

Peptides containing LysParent mass Intensity parent mass Sequence Modification Expected MS/MS ions

1566.86 138278 LKPDPNTLcDEFK 9-C_Cysteic_acid/ 242.19 454.27 1114.48 1326.561249.70 22359 FKDLGEEHFK missed cleavage1163.95 1448 LVNELTEFAK1143.01 1510 KQTALVELLK missed cleavage886.56 46360 DDSPDLPK 244.17 318.09 569.33 643.26

Prediction of intense ions

The human proteomeIsoelectric point (pI)

Molecular weight (Mw)

Marked spots are differentially expressed between normal red strawberries and a white mutant (half up-regulated and half down-regulated).

(fragaria ananassa)

Strawberry (not sequenced)

Hjernoe et al, 2005, Proteomics, in press

Difference in protein expression visual by eye

Difference in protein expression detected by the differential analysis software (here DeCyder)

Red contra white strawberries

Number of spots

Proteins found to be down regulated

Protein identified based on homology to

1 Flavanone 3- hydroxylase Onobrychis viciifolia

1 Dihydroflavonol reductase Arabidopsis thaliana

4 O-methyltransferase Fragaria x ananassa

4 Chalcone synthase Fragaria x ananassa

.... .... ....

All four proteins are known to be involved in the flavonoid biosynthesis pathway. One of the functions of flavonoids is to act as a pigment, giving colour to fruits.

MS/MS

MS

Search combining both MS and MS/MS spectra

Example of protein identification based on MS/MS spectra from a MALDI-TOF/TOF instrument

Search against all green plants

Significant hit

Details from the search result

4-sulfophenyl-isothiocyanate (SPITC)

Marakov et al, J. Mass Spectrom. (2003) 38; 373-377

Wang et al., Rapid.Commun. Mass. Spectrom (2004), 18(1); 96-102

SPITC-derivatization, an N-terminal Sulfonation

The peptide needs a net-charge of +1 in order to be detected

Derivatization of peptides

m/z500 2000

Inte

nsi

ty

10.00

0 + SPITC

- SPITC

R E AG I T I V Q G D P L E Y N

MS/MS Fragmentation of NYELPDGQVITIGAERFound in gi|21538, actin [Solanum tuberosum]

215 Da

1774

1774

1989

Karin Hjernø

Fragaria x ananassa

MALDI-TOF/TOF spectra obtained at Applied Biosystems 4700 Proteomics Analyzer

b2 b3

y1

y1

y2y3

y4

y5

y6

y7

y8 y9

y10

y11

y12

y13y14

y15

y10

y12

y13

898

920 1051

963

903 905

880

825834

418433 455 465 478 487

664 663 708 715

745

780 784

96

118 157161

212213277

281

297 301324325 334 349

352396

397404409

416

1112

Chalcone synthase

O-methyltransferase

Dihydroflavonol reductaseFlavanone 3-hydroxylase

Red

919

1026

899

294

499 509

The Bet v 1-homologous strawberry allergen, Fra a 1

898

9201051

919

898

9201051

919

777

Red

White

Mw

pI

Strawberry allergen-containing spots

K* L L T G G H P A S V L SPITC

K* L V T G H P A S V L SPITC

K* L V T G G H P A S V L SPITC

m/z200 1400

Inte

nsi

ty

01392

1435

1449

One spot, three distinct isoforms

The End....

Protein Identification by MALDI Mass Spectrometry Karin Hjernø and Peter Roepstorff Department of Biochemistry and Molecular Biology University of Southern.

Documents

ppm slide

gpmaw slide

mw slide

isotope distribution

gi6321412 mass

mass dependent

mass defect

protein id pmf slide