Protein/Peptide characterization
Download
1 / 24

Protein/Peptide characterization - PowerPoint PPT Presentation


  • 124 Views
  • Uploaded on

Protein/Peptide characterization. Peptide Mass Fingerprinting. HUMAN Carbonic anhydrase I.

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about ' Protein/Peptide characterization' - haamid


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript

Protein/Peptide characterization

Peptide Mass Fingerprinting

HUMAN Carbonic anhydrase I

MASPDWGYDDKNGPEQWSKLYPIANGNNQSPVDIKTSETKHDTSLKPISVSYNPATAKEIINVGHSFHVNFEDNDNRSVLKGGPFSDSYRLFQFHFHWGSTNEHGSEHTVDGVKYSAELHVAHWNSAKYSSLAEAASKADGLAVIGVLMKVGEANPKLQKVLDALQAIKTKGKRAPFTNFDPSTLLPSSLDFWTYPGSLTHPPLYESVTWIICKESISVSSEQLAQFRSLLSNVEGDNAVPMQHNNRPTQPLKGRTVRASF


Ion Sources

Matrix-assisted Laser Desorption/Ionization (MALDI)


Mass Analyzers

“MS-only”

Time-of-Flight (Tof)

Nitrogen Laser

337 nm

+

+

+

+

+

+

+

+

+

+

+

+

+

+

d

Field-free drift region

Detector

-20,000 V

MALDI plate

+ 20,000 V


Mass Analyzers

Reflectron Tof, orthogonal injection


Reflectron Tof – increase in resolution

Delayed Extraction – increase in resolution


432.9226

8000

7000

433.2556

6000

5000

4000

433.5887

3000

2000

1000

0

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

432.9226

8000

Intensity, counts

0

400

500

600

700

800

900

1000

1100

1200

1300

m/z, amu

Angiotensin: DRVYIHPFHL C62H89N17O14

Dm/z = 0.33

Dm = 1 Da, z=3

Dm/z = 0.33

Intensity

Mass to Charge ratio (m/z)

“Monoisotopic Peak”

12C621H8914N1716O14

X + 2 peak

12C6013C21H8914N1716O14

+

12C6113C11H8914N1615N116O14

+

12C621H8914N1616O1318O1

X + 1 peak

12C6113C11H8914N1716O14

+

12C621H8914N1615N116O14


8000

77%

7000

6000

5000

4000

3000

2000

1000

0

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

Electron = 0.000549 g/mol

Proton = 1.007253 g/mol

Neutron = 1.00864 g/mol

DRVYIHPFHL C62H89N17O14

[M+3H]3+ = 432.9226 x 3 = 1298.768

Measurement mass error = ~50 ppm

X + 1 peak

12C6113C11H8914N1716O14

+

12C621H8914N1615N116O14

+

12C621H882H114N1716O14

68%

C62H89N17O14

}

75%

6.3%

0.9%


Protein/Peptide characterization

Peptide Mass Fingerprinting

HUMAN Carbonic anhydrase I

MASPDWGYDDKNGPEQWSKLYPIANGNNQSPVDIKTSETKHDTSLKPISVSYNPATAKEIINVGHSFHVNFEDNDNRSVLKGGPFSDSYRLFQFHFHWGSTNEHGSEHTVDGVKYSAELHVAHWNSAKYSSLAEAASKADGLAVIGVLMKVGEANPKLQKVLDALQAIKTKGKRAPFTNFDPSTLLPSSLDFWTYPGSLTHPPLYESVTWIICKESISVSSEQLAQFRSLLSNVEGDNAVPMQHNNRPTQPLKGRTVRASF


1079.4690

1078.9731

1079.9691

Intensity, counts

1079.0

1080.0

1081.0

m/z, amu

539.9927

540.2492

719.6340

540.4936

1400

Intensity, counts

540.7446

1300

540.9927

1200

539.9927

1100

540.0

540.4

540.8

541.2

m/z, amu

1000

900

800

Intensity, counts

700

563.9729

600

500

684.9795

400

1079.4690

597.6693

300

670.5904

200

100

0

550

600

650

700

750

800

850

900

950

1000

1050

m/z, amu

Electrospray Ionization (ESI)

[M+3H]3+

[M+4H]4+

[M+2H]2+


Protein/Peptide characterization

Collision-activated dissociation (CAD)

H

R1

H

H

O

R1

O

R1

O

N

H2

N

+

N

N

+

N

N

+

O

R2

O

R2

O

R2

H

H

H

“b”

“y”

R1

R1

+

N

N

+

O

H

H

“a”

b-type 116 272 371 534 647 784 881 1028 1165 1296

D R V Y I H P F H L

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

b5

b62+

a62+

y62+

y2

a82+

b4

a5

y4

y1

y52+

b6


Introduction to Mass Spectrometry

Amino Acid Residue Masses


b

157 214 329 442 539 686 785 884 981 1155

R G D L P F V V P R

1155 999 942 827 714 617 470 371 272 175

y


MS Scan

Similar to LIM and SH3 protein 1

hypothetical protein MGC2477

MS/MS #1

MS/MS #4

3

400

800

1200

1800

1600

5

m/z

1

2

4

300

600

900

1200

1500

200

400

600

800

1000

1200

200

400

600

800

1000

1200

m/z

m/z

skeletal muscle tropomyosin

Acyl-CoA-binding protein

???

MS/MS #5

MS/MS #2

MS/MS #3

200

400

600

800

1000

1200

200

400

600

800

1000

1200

m/z

m/z

~10 seconds

MS scan

MS/MS#2

MS/MS#5

MS/MS#1

MS/MS#4

MS/MS#3

m/z


+

-

-

+

Mass Analyzers

Quadrupole Mass Spectrometer

lens

Ion source

lens

quadrupole

detector

F0 = U – V cos wt

U = fixed (DC) potential

V cos wt = applied RF, amplitude V and frequency wt


Mass Analyzers

Triple Quadrupole

“Single quad”

“Triple quad”

lens

lens

lens

Ion source

lens

quadrupole

quadrupole

quadrupole

detector

Q1

Q3

Q2

Why triple quadrupole?

- MS/MS analysis of peptides

lens

quadrupole

quadrupole

quadrupole

detector

Q1

Q3

Q2

scan m/z values (single quad)

Pass 1 m/z value

Fragment, pass all fragment ions


Mass Analyzers

Quadrupole Ion Trap




816.63

100

95

997

90

950

85

900

80

850

75

800

70

750

65

700

60

650

55

600

50

550

Intensity, counts

Relative Abundance

45

500

40

450

35

400

432.30

350

30

783.95

300

25

778.23

701.98

250

20

200

15

593.28

150

743.58

10

857.95

1223.77

100

5

50

0

0

997

950

900

850

800

750

700

650

600

550

Intensity, counts

500

450

400

350

300

LTQ Ion trap

Quadrupole Tof

ITMS + c ESI Full ms

496.8928

445.2570

543.7190

451.2044

539.5885

+4

487.5576

+3

744.8367

+5

493.0928

601.6043

730.8341

591.2612

526.87

1166.32

887.06

620.07

453.33

1114.11

954.77

1024.82

420

440

460

480

500

520

540

560

580

600

620

640

660

680

700

720

740

760

780

800

820

840

860

880

400

500

600

700

800

900

1000

1100

1200

m/z, amu

m/z

1166.32

100

1174.80

95

+3

90

496.8928

85

0.33

80

75

70

497.2254

65

+2? +3?

60

0.33

55

50

+3

1143.44

Relative Abundance

45

1167.21

0.87

40

+1

1175.88

487.5576

35

497.5597

30

487.8905

25

493.0928

250

20

200

1144.31

1168.22

15

488.2278

150

497.8955

10

1176.78

1173.91

100

1153.83

5

50

1165.69

1148.21

1156.90

1182.83

0

0

487.0

488.0

489.0

490.0

491.0

492.0

493.0

494.0

495.0

496.0

497.0

498.0

499.0

500.0

501.0

502.0

1140

1145

1150

1155

1160

1165

1170

1175

1180

m/z, amu

m/z




8000

7000

6000

5000

4000

3000

2000

1000

0

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

635.7516

49

Intensity, counts

636.2614

0

636.0

637.0

m/z, amu

Introduction to Mass Spectrometry

Resolution

m/Dm

Dm = FWHM

resolution = 11,000

resolution = 5,000,000

Dynamic Range

432.9226

8490

8000

7500

7000

6500

6000

5500

5000

Intensity, counts

4500

4000

3500

3000

2500

2000

1500

382.2313

463.7657

1000

648.8623

858.5471

500

513.2979

0

400

450

500

550

600

650

700

750

800

850

900

950

1000

m/z, amu


ad