Protein/Peptide characterizationPeptide Mass Fingerprinting

mass position peptide sequence

4541.241 174-213APFTNFDPSTLLPSSLDFWT YPGSLTHPPLYESVTWIICK

2796.291 90-113 LFQFHFHWGSTNEHGSEHTV DGVK

2759.389 228-252 SLLSNVEGDNAVPMQHNNRP TQPLK

2256.042 58-76 EIINVGHSFHVNFEDNDNR

1929.007 40-57 HDTSLKPISVSYNPATAK

1742.907 19-34 LYPIANGNNQSPVDIK

1612.786 114-127 YSAELHVAHWNSAK

1580.791 214-227 ESISVSSEQLAQFR

1186.686 138-149 ADGLAVIGVLMK

1153.479 1-10 ASPDWGYDDK

1026.510 128-137 YSSLAEAASK

985.437 81-89 GGPFSDSYR

970.593 160-168 VLDALQAIK

945.442 11-18 NGPEQWSK

714.378 150-156 VGEANPK

565.282 35-39 TSETK

HUMAN Carbonic anhydrase I

MASPDWGYDDKNGPEQWSKLYPIANGNNQSPVDIKTSETKHDTSLKPISVSYNPATAKEIINVGHSFHVNFEDNDNRSVLKGGPFSDSYRLFQFHFHWGSTNEHGSEHTVDGVKYSAELHVAHWNSAKYSSLAEAASKADGLAVIGVLMKVGEANPKLQKVLDALQAIKTKGKRAPFTNFDPSTLLPSSLDFWTYPGSLTHPPLYESVTWIICKESISVSSEQLAQFRSLLSNVEGDNAVPMQHNNRPTQPLKGRTVRASF

Ion Sources

Matrix-assisted Laser Desorption/Ionization (MALDI)

Mass Analyzers

“MS-only”

Time-of-Flight (Tof)

MALDI plate+ 20,000 V

Nitrogen Laser337 nm

Detector-20,000 V

d

Field-free drift region

+++++

+ ++ ++++ ++

Mass AnalyzersReflectron Tof, orthogonal injection

Reflectron Tof – increase in resolution

Delayed Extraction – increase in resolution

Inte

nsi

ty

Mass to Charge ratio (m/z)432.4 432.6 432.8 433.0 433.2 433.4 433.6 433.8 434.0 434.2 434.4 434.6 434.8

0

1000

2000

3000

4000

5000

6000

7000

8000

432.9226

433.2556

433.5887

m/z = 0.33

m/z = 0.33

Angiotensin: DRVYIHPFHL C62H89N17O14

“Monoisotopic Peak”12C62

1H8914N17

16O14

X + 1 peak12C61

13C11H89

14N1716O14

+12C62

1H8914N16

15N116O14

X + 2 peak12C60

13C21H89

14N1716O14

+12C61

13C11H89

14N1615N1

16O14

+12C62

1H8914N16

16O1318O1

m = 1 Da, z=3

400 500 600 700 800 900 1000 1100 1200 1300m/z, amu

0

8000

Inte

nsity, coun

ts

432.9226

Type   Element   Symbol  Integer  Mass1

 Exact Mass2 

 Abundance  X+1 Factor3 

 X+2 Factor4 

 X  Hydrogen  H  1  1.0078  99.99    

     D or 2H  2  2.0141  0.01    

 X+1  Carbon  12C  12  12.0000  98.91    

     13C  13  13.0034  1.1  1.1nC  0.0060nC2  

 X+1  Nitrogen  14N  14  14.0031  99.6    

     15N  15  15.0001  0.4  0.37nN  

 X+2  Oxygen  16O  16  15.9949  99.76    

     17O  17  16.9991  0.04  0.04nO  

     18O  18  17.9992  0.20    0.20nO

 X Phosphorus 

 P  31  30.9738  100    

 X+2  Sulfur  32S  32  31.9721  95.02    

     33S  33  32.9715  0.76  0.8nS  

     34S  34  33.9679  4.22    4.4nS

 X+2  Chlorine  35Cl  35  34.9689  75.77    

     37Cl  37  36.9659  24.23  32.5nCl  

DRVYIHPFHL C62H89N17O14

[M+3H]3+ = 432.9226 x 3 = 1298.768Measurement mass error = ~50 ppm

C62H89N17O14

X + 1 peak12C61

13C11H89

14N1716O14

+12C62

1H8914N16

15N116O14

+12C62

1H882H1

14N1716O14

68%

6.3% } 75%

432.4 432.6 432.8 433.0 433.2 433.4 433.6 433.8 434.0 434.2 434.4 434.6 434.80

1000

2000

3000

4000

5000

6000

7000

8000

77%

0.9%

Electron = 0.000549 g/molProton = 1.007253 g/molNeutron = 1.00864 g/mol

Protein/Peptide characterizationPeptide Mass Fingerprinting

mass position peptide sequence

4541.241 174-213APFTNFDPSTLLPSSLDFWT YPGSLTHPPLYESVTWIICK

2796.291 90-113 LFQFHFHWGSTNEHGSEHTV DGVK

2759.389 228-252 SLLSNVEGDNAVPMQHNNRP TQPLK

2256.042 58-76 EIINVGHSFHVNFEDNDNR

1929.007 40-57 HDTSLKPISVSYNPATAK

1742.907 19-34 LYPIANGNNQSPVDIK

1612.786 114-127 YSAELHVAHWNSAK

1580.791 214-227 ESISVSSEQLAQFR

1186.686 138-149 ADGLAVIGVLMK

1153.479 1-10 ASPDWGYDDK

1026.510 128-137 YSSLAEAASK

985.437 81-89 GGPFSDSYR

970.593 160-168 VLDALQAIK

945.442 11-18 NGPEQWSK

714.378 150-156 VGEANPK

565.282 35-39 TSETK

HUMAN Carbonic anhydrase I

MASPDWGYDDKNGPEQWSKLYPIANGNNQSPVDIKTSETKHDTSLKPISVSYNPATAKEIINVGHSFHVNFEDNDNRSVLKGGPFSDSYRLFQFHFHWGSTNEHGSEHTVDGVKYSAELHVAHWNSAKYSSLAEAASKADGLAVIGVLMKVGEANPKLQKVLDALQAIKTKGKRAPFTNFDPSTLLPSSLDFWTYPGSLTHPPLYESVTWIICKESISVSSEQLAQFRSLLSNVEGDNAVPMQHNNRPTQPLKGRTVRASF

Electrospray Ionization (ESI)

550 600 650 700 750 800 850 900 950 1000 1050m/z, amu

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

1300

1400

Intensity, counts

719.6340

539.9927

563.9729

684.9795

1079.4690597.6693670.5904

540.0 540.4 540.8 541.2m/z, amu

Inte

nsity, coun

ts

539.9927

540.2492

540.4936

540.7446

540.9927

1079.0 1080.0 1081.0m/z, amu

Inte

nsity, coun

ts

1079.46901078.9731

1079.9691

[M+2H]2+

[M+3H]3+

[M+4H]4+

Protein/Peptide characterization

Collision-activated dissociation (CAD)

N

R1

O

N

R2

O

H

N

R1

O

N

R2

OH H

+

R1

O

+ + N

R2

OH2

“b” “y”

H

H

N

H

R1

O+N

H

R1+N

H

“a”

+TOF Product (432.9): Experiment 2, 15.915 min from fmw_angiotst_030105_2.wiffa=3.55571269460828870e-004, t0=1.03585549568179790e+001

Max. 1654.0 counts.

150 200 250 300 350 400 450 500 550 600 650 700 750 800m/z, amu

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

Inte

ns

ity, c

ou

nts

647.3574

392.7211

382.1879269.1692

378.7201619.3550

513.2865257.1463

132.1045 325.6743 371.2152 500.7836 784.4101569.3014217.1281

D R V Y I H P F H Lb-type 116 272 371 534 647 784 881 1028 1165 1296

y-type 1296 1181 1025 926 763 650 513 416 269 132

y2

y4

y52+y1

b4

b5

b6

b62+

a5a8

2+

y62+

a62+

Introduction to Mass Spectrometry

Amino Acid Residue Masses

Amino Acid3 Letter

CodeSingle Letter

Code 

Residue Mass

 

        Monoisotopic Average

Glycine Gly G   57.02147 57.052

Alanine Ala A   71.03712 71.079

Serine Ser S   87.03203 87.078

Proline Pro P   97.05277 97.117

Valine Val V   99.06842 99.133

Threonine Thr T   101.04768 101.105

Cysteine Cys C   103.00919 103.144

Isoleucine Ile I   113.08407 113.160

Leucine Leu L   113.08407 113.160

Asparagine Asn N   114.04293 114.104

Aspartic Acid Asp D   115.02695 115.089

Glutamine Gln Q   128.05858 128.131

Lysine Lys K   128.09497 128.174

Glutamic Acid Glu E   129.04260 129.116

Methionine Met M   131.04049 131.198

Histidine His H   137.05891 137.142

Phenylalanine Phe F   147.06842 147.177

Arginine Arg R   156.10112 156.188

Tyrosine Tyr Y   163.06333 163.170

Tryptophan Try W   186.07932 186.213

+TOF Product (578.3): Experiment 5, 66.365 min from 03250_1433beta_12hr.wiffa=3.55560354546847490e-004, t0=1.01409857317689070e+001

Max. 5229.0 counts.

160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500m/z, amu

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

2600

Inte

ns

ity, c

ou

nts

329.1613

255.1456175.1256

272.1750

312.1307

157.1041

158.0911 270.1291257.1671 330.1647 442.2415344.1971212.1467

245.1295217.1389183.1487 354.2145 425.2125313.1268 470.3016383.1911195.1234

+TOF Product (578.3): Experiment 5, 66.365 min from 03250_1433beta_12hr.wiffa=3.55560354546847490e-004, t0=1.01409857317689070e+001

Max. 5229.0 counts.

500 550 600 650 700 750 800 850 900 950 1000m/z, amu

50

100

150

200

250

300

350

400

450

500

550

600

650

Inte

ns

ity, c

ou

nts

714.4382

686.3713570.8088

785.4351757.4333827.5163

658.3663

740.3993 856.4853641.3310548.8108 617.3710 669.3386884.4812 1000.5370768.3821

R G D L P F V V P R157 214 329 442 539 686 785 884 981 1155

1155 999 942 827 714 617 470 371 272 175 y

b

m/z300 600 900 1200 1500

12

3

45

MS Scan

200 400 600 800 1000 1200m/z

Similar to LIM and SH3 protein 1

MS/MS #1

400 800 1200 16001800m/z

???

MS/MS #2

200 400 600 800 1000 1200m/z

Acyl-CoA-binding protein

MS/MS #3

200 400 600 800 1000 1200m/z

hypothetical protein MGC2477

MS/MS #4

200 400 600 800 1000 1200m/z

skeletal muscle tropomyosin

MS/MS #5

MS scan

MS/MS#1 MS/MS#2 MS/MS#3 MS/MS#4 MS/MS#5

~10 seconds

Mass AnalyzersQuadrupole Mass Spectrometer

+

+--

0 = U – V cos t

Ion source

lens

lens

quadrupole detector

U = fixed (DC) potentialV cos t = applied RF, amplitude V and frequency t

Mass AnalyzersTriple Quadrupole

“Single quad”

“Triple quad”

Ion source

lens

lens

quadrupole detector

lens

quadrupole

lens

quadrupole

Q1 Q2 Q3

Why triple quadrupole? - MS/MS analysis of peptides

lens quadrupole detectorquadrupole quadrupole

Q1 Q2 Q3

Pass 1 m/z valueFragment, pass all fragment ions

scan m/z values (single quad)

Mass Analyzers

Quadrupole Ion Trap

Thermo-Finnigan LTQ mass spectrometer

Quadrupole Time-of-Flight

ITMS + c ESI Full ms

400 500 600 700 800 900 1000 1100 1200

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

816.63

432.30

783.95

778.23701.98

593.28

743.58 857.95 1223.77

526.87 1166.32887.06620.07453.33 1114.11954.77 1024.82

1140 1145 1150 1155 1160 1165 1170 1175 1180

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

1166.32

1174.80

1167.211143.44

1175.88

1168.221144.31

1176.781173.91

1153.831165.691148.21 1156.90 1182.83

LTQ Ion trap

+2? +3?

+4

+3+5

0.87

420440460480500520540560580600620640660680700720740760780800820840860880m/z, amu

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

900

950

997

Inte

nsity, coun

ts

496.8928

445.2570

543.7190451.2044

539.5885

487.5576

744.8367

493.0928

601.6043 730.8341591.2612

487.0488.0489.0490.0491.0492.0493.0494.0495.0496.0497.0498.0499.0500.0501.0502.0m/z, amu

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

900

950

997

Inte

nsity, coun

ts

496.8928

497.2254

487.5576

497.5597487.8905

493.0928

488.2278497.8955

+3

+3

+1

0.33

0.33

Quadrupole Tof

Fourier Transform Ion Cyclotron Resonance Mass Spectrometer(FTMS)

resolution = 5,000,000

Introduction to Mass Spectrometry

432.4 432.6 432.8 433.0 433.2 433.4 433.6 433.8 434.0 434.2 434.4 434.6 434.80

1000

2000

3000

4000

5000

6000

7000

8000

Dynamic Range

400 450 500 550 600 650 700 750 800 850 900 950 1000m/z, amu

0500

1000150020002500300035004000450050005500600065007000750080008490

Intensity, counts

432.9226

382.2313 463.7657648.8623

513.2979 858.5471

636.0 637.0m/z, amu

0

49

Intensity, counts

635.7516

636.2614

resolution = 11,000

Resolution

resolution = 5,000,000

m/mm = FWHM