Intro to MS Yates - University of Pittsburgh · 365.23 22.27 444.74 20.58 690.33 21.06 436.76 21.80...

Introduc)on to Mass Spectrometry

Nathan A. Yates University of Pi8sburgh

Biomedical Mass Spectrometry Center

Structural Biophysics Mass Spec Lecture I Pi8sburgh PA, December 3, 2014

1

Overview

•  Informa1on contained in a mass spectrum •  Components of a mass spectrometer •  MS analysis of biomolecules •  Complex mixture analysis and data processing •  Trace level analysis by tandem mass spectrometry (MS/MS) •  Shotgun Proteomics •  Global analysis of complex protein mixtures •  Orbitrap mass analyzers

•  Differen1al mass spectrometry

Focus: Introduction to Mass Spectrometry and Methods for the Structural an Quantitative Analysis of Biomolecules.

2

Mass Spectrometry

3

A Mass Spectrum

m/z =18 (H20)+ since O + H + H = 16 + 1 + 1 = 18 amu m/z=17 (HO)+ since O + H = 16 + 1 = 17 amu m/z=16 (O)+ since O = 16 amu m/z=1 (H)+ since H = 1 amu

4

Electron Impact Method of Ioniza)on

M + e-‐ M+. 2e-‐ +

F+ N + M+.

5

Components of a Mass Spectrometer

Ion Source Mass Analyzer Detector

Data System

6

MS Analysis of Biomolecules?

Simvasta)n 418.6 Da

Myoglobin 16,955 Da

7

Simvasta)n Mass Spectrum (Electron Impact Ioniza)on)

8

9

Proteomic Technology -‐ Electrospray Ioniza)on Produces Gas Phase Ions from Solu)on Phase Analytes (e.g. Pep)des and Proteins)

Electrospray Ioniza)on

John B. Fenn -‐ 2002 Nobel Prize in Chemistry for the development of so` desorp)on ionisa)on methods for mass spectrometric analyses of biological macromolecules

To MS

From µLC (M+H)+

Protonated Molecules

Simvasta)n ESI Mass Spectrum (Electrospray Ioniza)on)

MW = 418.6 Da C25H38O5

ESI-‐

ESI+

10

High and Low Resolu)on MS

C25h38o5 +Na: C25 H38 O5 Na1 p(gss, s/p:40) Chrg 1...

440 445 450m/z

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441.26

442.27

443.27445.28

C25h38o5 +Na: C25 H38 O5 Na1 p(gss, s/p:40) Chrg 1...

442 444 446 448m/z

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441.26

442.26

443.27444.27 445.27

Unit Resolu)on 1 Da @ 10% FWHM

High Resolu)on 0.01 Da @ 10% FWHM

11

Myoglobin Mass Spectrum (Electrospray Ioniza)on)

Myoglobin 16,955 Da

12

Mixture Analysis by (LC-‐MS)

13

What about complex biological mixtures?

14

LC-‐MS Data Analysis C:\Users\yatesn\Desktop\q1652 7/23/2012 11:04:10 AM

RT: 0.00 - 85.01 SM: 3B

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38.60789.43

28.89687.86

29.10687.86 42.77

910.45 63.56687.3228.54

687.86 74.431009.0024.56

844.4431.97442.26

42.92693.87

61.19790.39

64.59724.40

19.53609.31

14.32423.22 70.15

1104.5545.77678.31

58.00876.89 74.64

1455.2020.00466.73 55.79

725.8613.36339.18 53.88

515.79 75.111086.88

7.53402.712.69

312.6980.84445.12

NL:3.05E9Base Peak F: FTMS + p NSI Full ms [300.00-1500.00] MS q1652

q1652 #7077-7835 RT: 19.20-20.72 AV: 91 NL: 2.20E8T: FTMS + p NSI Full ms [300.00-1500.00]

300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z

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609.31466.73

435.27 522.29932.45392.73 819.37

690.33

784.45534.34369.20 847.48671.42 713.42575.30 1217.61645.35 914.44 1047.50946.49

1132.58 1379.661251.66 1462.59

15

Narrow in on a por)on of the mass spectrum (m/z = 400-‐450)

C:\Users\yatesn\Desktop\q1652 7/23/2012 11:04:10 AM

RT: 0.00 - 85.01 SM: 3B

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85Time (min)

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38.60789.43

28.89687.86

29.10687.86 42.77

910.45 63.56687.3228.54

687.86 74.431009.0024.56

844.4431.97442.26

42.92693.87

61.19790.39

64.59724.40

19.53609.31

14.32423.22 70.15

1104.5545.77678.31

58.00876.89 74.64

1455.2020.00466.73 55.79

725.8613.36339.18 53.88

515.79 75.111086.88

7.53402.712.69

312.6980.84445.12

NL:3.05E9Base Peak F: FTMS + p NSI Full ms [300.00-1500.00] MS q1652


400 405 410 415 420 425 430 435 440 445m/z

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435.27

430.28424.25414.75 436.27

440.25424.75423.73407.26 444.74415.25 442.22402.27 431.28428.58 445.24400.56 417.89 437.73425.25408.23

433.22

421.21410.21403.27414.22

16

Plot a Selected Ion Chromatogram (418+) C:\Users\yatesn\Desktop\q1652 7/23/2012 11:04:10 AM

RT: 0.00 - 85.01 SM: 3B

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38.60789.4328.89

687.86 31.81442.26

42.77910.45

63.56687.32

28.54687.86

74.431009.00

24.56844.44

61.19790.39

44.54644.86

19.53609.31

14.32423.22

64.69724.4155.79

725.8611.53389.19

75.111086.88

8.14395.23

34.09417.73 35.12

417.7339.74417.7412.47

418.2218.25417.56

20.46417.88 28.13

418.2442.46417.72

50.96418.25

11.99418.20

NL: 3.05E9Base Peak F: FTMS + p NSI Full ms [300.00-1500.00] MS q1652

NL: 8.47E8m/z= 417.50-418.50 F: FTMS + p NSI Full ms [300.00-1500.00] MS q1652


400 405 410 415 420 425 430 435 440 445m/z

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435.27

430.28424.25414.75 436.27

440.25424.75423.73407.26 444.74415.25 442.22402.27 431.28428.58 445.24400.56 417.89 437.73425.25408.23

433.22

421.21410.21403.27414.22

17

Narrow the chromatographic )me range (19-‐22min) C:\Users\yatesn\Desktop\q1652 7/23/2012 11:04:10 AM

RT: 18.94 - 22.68 SM: 3B

19.0 19.5 20.0 20.5 21.0 21.5 22.0 22.5Time (min)

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19.53609.31

20.00466.73 20.09

466.7319.40392.73

19.66609.31

21.42419.72

21.37419.72 22.39

815.4319.13365.23

22.27444.74

20.58690.33

21.06436.76 21.80

488.73

20.46417.8820.00

417.89 20.85417.88 21.21

417.55

20.11417.89

21.82417.88

19.66418.24

22.14417.72

19.17418.26

22.42417.87

19.31417.74




400 405 410 415 420 425 430 435 440 445m/z

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435.27

430.28424.25414.75 436.27

440.25424.75423.73407.26 444.74415.25 442.22402.27 431.28428.58 445.24400.56 417.89 437.73425.25408.23

433.22

421.21410.21403.27414.22

18

Plot an average spectrum for a chromatographic peak of interest (RT=20.0min)


RT: 18.94 - 22.68 SM: 3B

19.0 19.5 20.0 20.5 21.0 21.5 22.0 22.5Time (min)

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19.53609.31

20.00466.73 20.09

466.7319.40392.73

19.66609.31

21.42419.72

21.37419.72 22.39

815.4319.13365.23

22.27444.74

20.58690.33

21.06436.76 21.80

488.73

20.46417.8820.00

417.89 20.85417.88 21.21

417.55

20.11417.89

21.82417.88

19.66418.24

22.14417.72

19.17418.26

22.42417.87

19.31417.74




400 405 410 415 420 425 430 435 440 445m/z

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428.58 430.22 435.27417.89

408.52

418.23

407.26430.72

408.85435.78

433.22445.73417.24 418.56 436.28421.25409.73 425.20416.21 423.74402.27400.23 411.75 418.90 444.29404.73 436.78 442.26426.20415.24397.25

406.74

19

Narrow in on a por)on of the mass spectrum (m/z = 415-‐420)


RT: 18.94 - 22.68 SM: 3B

19.0 19.5 20.0 20.5 21.0 21.5 22.0 22.5Time (min)

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19.53609.31

20.00466.73 20.09

466.7319.40392.73

19.66609.31

21.42419.72

21.37419.72 22.39

815.4319.13365.23

22.27444.74

20.58690.33

21.06436.76 21.80

488.73

20.46417.8820.00

417.89 20.85417.88 21.21

417.55

20.11417.89

21.82417.88

19.66418.24

22.14417.72

19.17418.26

22.42417.87

19.31417.74




415.0 415.5 416.0 416.5 417.0 417.5 418.0 418.5 419.0 419.5 420.0m/z

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417.89

418.23

417.24 418.56416.21

418.90416.71416.26 419.23415.24 416.89415.74 417.81 419.70

20

Plot an average spectrum for a chromatographic peak of interest (RT=20.8min)


RT: 18.94 - 22.68 SM: 3B

19.0 19.5 20.0 20.5 21.0 21.5 22.0 22.5Time (min)

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19.53609.31

20.00466.73 20.09

466.7319.40392.73

19.66609.31

21.42419.72

21.37419.72 22.39

815.4319.13365.23

22.27444.74

20.58690.33

21.06436.76 21.80

488.73

20.46417.88

20.00417.89 20.85

417.8821.21417.8920.11

417.8921.84417.88

22.12417.88



q1652 #7896 RT: 20.85 AV: 1 NL: 4.31E7T: FTMS + p NSI Full ms [300.00-1500.00]

415.0 415.5 416.0 416.5 417.0 417.5 418.0 418.5 419.0 419.5 420.0m/z

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417.88

418.22

418.55

416.23 418.89417.23419.20417.73415.23 416.73 417.26415.74 416.96414.91 418.26

21

Narrow in on a por)on of the mass spectrum (m/z = 418.1-‐418.3)


RT: 18.94 - 22.68 SM: 3B

19.0 19.5 20.0 20.5 21.0 21.5 22.0 22.5Time (min)

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19.53609.31

20.00466.73 20.09

466.7319.40392.73

19.66609.31

21.42419.72

21.37419.72 22.39

815.4319.13365.23

22.27444.74

20.58690.33

21.06436.76 21.80

488.73

20.46417.88

20.00417.89 20.85

417.8821.21417.8920.11

417.8921.84417.88

22.12417.88




418.10 418.12 418.14 418.16 418.18 418.20 418.22 418.24 418.26 418.28 418.30 418.32 418.34m/z

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418.22

418.20

418.23 418.2622

Plot a selected ion chromatogram for m/z 418.20 C:\Users\yatesn\Desktop\q1652 7/23/2012 11:04:10 AM

RT: 18.94 - 22.68 SM: 3B

19.0 19.5 20.0 20.5 21.0 21.5 22.0 22.5Time (min)

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19.53609.31

20.00466.73 20.09

466.7319.40392.73

19.66609.31

21.42419.72

21.37419.72 22.39

815.4319.13365.23

22.27444.74

20.58690.33

21.06436.76 21.80

488.73

20.83418.20

20.92418.20

19.66418.20 22.40

418.2019.73418.20




418.10 418.12 418.14 418.16 418.18 418.20 418.22 418.24 418.26 418.28 418.30 418.32 418.34m/z

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418.22

418.20

418.23 418.26

23

Expand view to see full )me range C:\Users\yatesn\Desktop\q1652 7/23/2012 11:04:10 AM

RT: 0.00 - 85.01 SM: 3B

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85Time (min)

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38.60789.4328.89

687.86 31.81442.26

42.77910.45

63.56687.32

28.54687.86

74.431009.00

24.56844.44

61.19790.39

44.54644.86

19.53609.31

14.32423.22

64.69724.4155.79

725.8611.53389.19

75.111086.88

8.14395.23

20.83418.20

14.96418.20 20.92

418.201.71

418.20 11.38418.20 23.20

418.202.04

418.20 60.04418.2038.32

0.0039.62418.20




418.10 418.12 418.14 418.16 418.18 418.20 418.22 418.24 418.26 418.28 418.30 418.32 418.34m/z

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418.22

418.20

418.23 418.26

24

How do you separate really complex mixtures?

25

Trace Analysis by Tandem Mass Spectrometry

26 758A . ANALYTICAL CHEMISTRY.V OL. 57. NO. 7, , l l ,NE 1985

Reac1on Ioniza1on

Collision-‐ Induced Dissocia1on (CID)

Tandem Mass Spectrometry

27

Collision-‐induced Dissocia)on (CID)

•  Kine)c energy of parent ions is increased •  Parent ions undergo energy conver)ng collisions •  Parent ions fall apart into product ions and neutrals •  Also referred to collision-‐ac)vated dissocia)on (CAD)

+ +

28

+

+

analyte ion m/z 400

matrix ion m/z 400

+

+

analyte product ion m/z 325

matrix product ion m/z 250

MS/MS (CID)

MS/MS (CID)

•  Different compounds with same m/z but different structures •  MS/MS allows interference-‐free quan)ta)on of analyte in matrix

Single Reac)on Monitoring (SRM)

29

30

Factors that determine what you can observe:

Where you look (samples) astronomy – region of space proteomics – sub frac1on of the proteome

The objects themselves (analytes) astronomy – luminosity, distance, composi1on proteomics – abundance, sequence, physical proper1es, PTM’s

Things that interfere (background) astronomy – sunlight, atmosphere, interstellar objects proteomics – abundant proteins, sample matrix, un-‐resolved proteins

Parallels between Astronomy and Proteomics

How you look (technology / method) astronomy – telescope, catalog space, search for phenomena proteomics – mass spectrometer, shotgun, targeted, differenBal mass spectrometry

31

Technology Enhances the “View” of the Cosmos

The Palomar telescope Courtesy California Ins)tute of Technology 200-‐inch telescope – 1949

M17 -‐ The Omega Nebula 7M X luminous than the Sun Credit & Copyright: 2MASS image mosaic by E. Kopan; courtesy the University of Massachuse8s and the Infrared Processing and Analysis Center/Caltech.

Charge Coupled Device "No method of advancing science is so produc)ve as the development of new and more powerful instruments and methods of research.” George Ellery Hale

Proteomic Technology -‐ Quadrupole Ion Traps

32

Electromagne)c Traps for Charged and Neutral Par)cles Wolfgang Paul, Nobel Prize, December 8, 1989

Proteomic Technology -‐ Modern Quadrupole Ion Traps Sequence Proteomic “Space”

33

Linear Quadrupole Ion Trap

MS/MS

Pep)de Sequence Informa)on

MS

Molecular Weight Informa)on

Proteomic Approach #1 -‐ Shotgun Proteomics

34

Modern Shotgun Proteomics Technologies

35


300 400 500 600 700 800 900 1000m/z

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460.26324.87486.79

365.23

559.33 729.45417.24 651.34972.58

494.75

759.47 864.49 1025.50

q1652 #6847 RT: 18.69 AV: 1 NL: 6.51E6T: FTMS + p NSI d Full ms2 [email protected] [110.00-775.00]

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460.29347.20

633.33487.30443.26

374.21

597.29


300 400 500 600 700 800 900 1000m/z

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357.07

665.23373.28682.25439.23 536.28 612.30

751.27

RT: 0.00 - 85.01

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38.6028.91

74.4629.09 38.72 61.21 63.5728.52 74.6764.5742.90

14.31 58.0047.8813.8955.76 75.1113.36

11.5377.38

NL:3.20E9Base Peak MS q1652

85 minutes and 39,556 spectra later…

Example Shotgun Proteomics Results 319 Proteins (2492) Pep)des Iden)fied from a Polyacrylamide Gel Band

36

Example Data Dependent LC-‐MS/MS Analysis

37


RT: 0.00 - 85.01 SM: 3B

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85Time (min)

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38.60789.4328.89

687.86 31.81442.26

42.77910.45

63.56687.32

28.54687.86

74.431009.00

24.56844.44

61.19790.39

44.54644.86

64.59724.40

19.53609.31

14.32423.22 55.79

725.8611.53389.19

75.111086.88

7.35402.71

42.53910.4511.82

346.8419.73609.31

29.79371.73

36.35600.7724.02

632.36 74.37590.32

54.52120.08

63.46687.32

42.79663.42

11.69590.37

70.111044.56

38.06814.50

47.91635.31

1.30407.76 74.92

937.5178.70702.21


NL: 1.22E10TIC MS q1652


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288.20

403.23

502.30185.16

201.12 271.18 516.31158.09 386.20308.17120.08 229.12 446.26360.71 573.35485.27417.25

Examine the Data Collected From 20-‐21 Minutes

38


RT: 20.00 - 21.00 SM: 3B

20.0 20.1 20.2 20.3 20.4 20.5 20.6 20.7 20.8 20.9Time (min)

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20.00466.73 20.09

466.7320.17466.73 20.58

690.3320.23466.73

20.27466.73 20.97

544.7720.92397.70

20.21466.7320.16

482.2420.09143.12

20.08444.29

20.35120.08

20.32120.08

20.99684.38

20.59503.28

20.79563.31

20.54288.20

20.73120.08

20.68520.27

20.84218.15

20.47201.12

20.85186.12

20.38669.29


NL: 8.42E9TIC MS q1652


300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z

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461.24 672.38

512.78

690.33578.25

387.70

331.19 560.80921.46628.41

786.40720.41417.88

1024.55826.45 1120.60 1379.66885.48 932.52 1251.64

Tandem Mass Spectra of m/z 464.76

39


RT: 20.00 - 21.00 SM: 3B

20.0 20.1 20.2 20.3 20.4 20.5 20.6 20.7 20.8 20.9Time (min)

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20.00466.73 20.09

466.7320.17466.73 20.58

690.3320.23466.73

20.27466.73 20.97

544.7720.92397.70

20.21466.7320.16

482.2420.09143.12

20.08444.29

20.35120.08

20.32120.08

20.99684.38

20.59503.28

20.79563.31

20.54288.20

20.73120.08

20.68520.27

20.84218.15

20.47201.12

20.85186.12

20.38669.29


NL: 8.42E9TIC MS q1652


150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950m/z

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760.43

831.47

169.10 443.23 689.39618.35240.13141.10

465.76547.32319.11

434.23311.17175.12 573.35651.31476.24382.21 720.41285.13 920.47

219.15247.14

531.29352.04

604.29 798.38 890.42841.46

685.35

Data that Triggered the Acquisi)on of an MS/MS Spectrum

40


RT: 20.00 - 21.00 SM: 3B

20.0 20.1 20.2 20.3 20.4 20.5 20.6 20.7 20.8 20.9Time (min)

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20.00466.73 20.09

466.7320.17466.73 20.58

690.3320.23466.73

20.27466.73 20.97

544.7720.92397.70

20.56464.76

20.32464.72 20.89

464.7420.70464.78




452 454 456 458 460 462 464 466 468 470 472 474 476m/z

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461.24

461.74

462.24460.77453.23 454.22 458.31 467.25462.74 475.24464.76 468.74455.22 474.30473.25465.75 470.74 476.22

Coun)ng Pep)des to “Quan)fy” Proteins?

41

Indx Accession Protein NumPep totalSpec UP003021 UP003022 UP003023 UP003024 UP0030251 A0AUZ9-1 Isoform 1 of Uncharacterized protein C2orf67 2 2 0 0 0 1 12 A0AV96-1 Isoform 1 of RNA-binding protein 47 1 1 0 0 0 1 03 A0AVG3 t-SNARE domain containing 1 1 2 1 1 0 0 04 A0JNW5-1 Isoform 1 of UHRF1-binding protein 1-like 1 1 0 0 0 1 05 A0N2G3 C-myc protein (Fragment) 1 1 0 0 1 0 06 A1A5D9-2 Isoform 2 of Bicaudal D-related protein 2 1 2 0 0 0 1 17 A1Z1Q3-1 Isoform 1 of MACRO domain-containing protein 2 1 1 0 0 0 0 18 A2A288-1 Isoform 1 of Probable ribonuclease ZC3H12D 1 1 1 0 0 0 09 A2CJ06 Dystrotelin 1 2 0 1 0 1 0

10 A2RTX5-1 Isoform 1 of Probable threonyl-tRNA synthetase 2, cytoplasmic1 3 0 0 1 1 111 A2VCQ4 PRKCSH protein (Fragment) 4 18 3 4 4 3 412 A2VEC9-1 Isoform 1 of SCO-spondin 1 2 0 1 0 0 113 A3KMH1-1 Isoform 1 of Uncharacterized protein KIAA0564 1 1 1 0 0 0 014 A4D1I1 Similar to ribosomal protein S3a; 40S ribosomal protein S3a; v-fos transformation effector protein 11 1 0 1 0 0 015 A4D1S5-1 Isoform 1 of Ras-related protein Rab-19 2 3 1 1 0 0 116 A4D1W8 Ependymin related protein 1 (Zebrafish), isoform CRA_b1 1 1 0 0 0 017 A4D256 Dual specificity protein phosphatase CDC14C 1 2 0 0 2 0 018 A4D997 Uncharacterized protein C9orf102 1 1 1 0 0 0 019 A4FU69-2 Isoform 2 of EF-hand calcium-binding domain-containing protein 51 1 0 1 0 0 020 A4UGR9-1 Isoform 1 of Xin actin-binding repeat-containing protein 21 1 1 0 0 0 021 A5D8V7 Coiled-coil domain-containing protein 151 1 1 0 1 0 0 022 A6NBZ8 Uncharacterized protein 1 1 1 0 0 0 023 A6NC98-1 Isoform 1 of Coiled-coil domain-containing protein 88B1 1 0 0 0 1 024 A6NDU8 UPF0600 protein C5orf51 1 1 0 0 1 0 025 A6NE01 Protein FAM186A 1 1 0 1 0 0 026 A6NEC2-1 Isoform 1 of Puromycin-sensitive aminopeptidase-like protein3 10 0 5 2 1 227 A6NEL2 Ankyrin repeat domain-containing protein 56 1 2 1 0 0 1 028 A6NH00 Olfactory receptor 2T8 1 1 0 0 1 0 0

MS separa)on of specific proteins?

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43

QuanBficaBon: Pep&de concentra&ons are obtained by mul&plying the [IS] concentra&on by the signal ra&o of the analyte / internal standard

NH 2 COOH

Target Protein

QQQ DetecBon: Selected Reac)on Monitoring provides the high sensi)vity and amino acid selec)vity needed to detect pep)des in neat CSF

18 20 22 24 26 28 30 Time (min) 18 20 22 24 26 28 30 Time (min) 18 20 22 24 26 28 30 Time (min)

70 80 90 100

LC -‐ 18 20 22 24 26 28 30

Time (min) 0 10 20 30 40 50 60 70 80 90

100 0 10 20 30 40 50 60

Rela)ve Ab

undance

SRM

Analyte Signal = 200

Int. Std. Signal = 1000

Analyte Pep)de

AQTDIDSPQNLVTDR

NH 2 COOH

Standard Protein IS Pep)de

AQTDIDSPQNLVTDR*

Electrospray Ion

Source

CID Q1 Q3

521.7 757.6

Proteomic Approach #2 -‐ Selected Reac)on Monitoring Assays

Example Targeted Analysis Results

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Proteomic Technology -‐ High Resolu)on Orbital Trap Mass Analyzers Expand Our “View” of the Proteome

45

Orbital Ion Trap More than 100,000 Observable Pep)de Species

Anne8e Michalski, Juergen Cox, Ma8hias Mann, Journal of Proteome Research, Feb 10, 2011.

C:\Xcalibur\data\114186 1/28/2004 7:58:10 PM PBI_167_01PBI_167_01

114186 # 2649-‐2723 R T : 24.01-‐24.50 A V: 37 NL : 1.02E 4F : ITMS + c NS I F ull ms [ 400.00-‐2000.00]

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

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477.8879

456.1795

737.0342

736.9587

604.1706587.2521503.7772563.7734 607.8439521.9630448.4136 755.9727646.6342 722.0627 826.6938442.6017 666.4675 802.7613

910.4764895.1211839.1017 938.8213

114186 # 2649-‐2723 R T : 24.00-‐24.50 A V: 38 NL : 7.32E 4F : F TMS + p NS I F ull ms [ 300.00-‐2000.00]

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

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737.0102

826.3724721.8766604.0323

563.5188477.5853 756.0177 802.9037607.8279

630.3329455.9778 895.4580549.3063 715.8750 910.9570521.7445646.2925448.2320 684.7227498.8625 980.9453827.8761 938.4314775.0247 866.4683

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FTMS Technology – Observe More Pep)des

LTQ Low Resolution Detection

FTMS High Resolution Detection



600 605 610 615 620 625 630 635 640 645 650m/z

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604.1706604.2482

607.8439

604.0094604.5195603.8953604.6180 646.6342627.7416607.9182 637.8765607.7653 620.4253 646.4821608.1470 628.1782 639.5774627.6029 637.7288618.3669 620.6226608.6675 630.3384623.3792 640.8938633.4860 648.3396603.7137 604.7827 641.3740626.5953613.8937 637.3624608.7394

615.3491 643.0638


600 605 610 615 620 625 630 635 640 645 650m/z

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604.0323

603.7816604.2830

607.8279

630.3329637.8310

608.3295604.5336 627.5958620.2660

646.2925638.3321630.8352627.3451620.7674 646.7942631.8751624.3262 640.9806604.7838 637.6454608.8308 618.2879 632.3765624.8277 641.3144 647.2949601.3377

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617 618 619 620 621 622 623 624 625 626 627 628 629 630m/z

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627.7416

620.4253628.1782

627.6029618.3669

620.6226617.9780 623.3792618.4467

629.2044624.2963622.3300 628.2169

620.2675 623.3145618.5809 626.5953619.9922 626.0928625.2161 628.4862621.6592 627.4194623.2839

619.9135 629.2421624.3618621.1584619.6698

617.5070 629.8379


617 618 619 620 621 622 623 624 625 626 627 628 629 630m/z

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627.5958

620.2660

627.8463627.3451

620.7674

624.3262

623.3261618.2879 628.0974623.5773 628.3008623.0754 624.8277618.0365 618.5384 620.8176 626.2913

621.3194 625.9589622.3470 628.6346626.6250618.7888 625.3291 628.8247

624.3002

621.8208 622.8491




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620.0 620.5 621.0 621.5 622.0 622.5 623.0 623.5 624.0 624.5 625.0m/z

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620.4253

620.6226623.3792

624.2963622.3300 623.3290623.8901623.6011620.2675 623.3145

621.6592 623.2839620.6414 622.9736622.6492620.9719 624.3618624.0942621.5428 623.2045622.7194 624.9265

621.7531 622.2361

621.8773


620.0 620.5 621.0 621.5 622.0 622.5 623.0 623.5 624.0 624.5 625.0m/z

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620.2660

620.7674

624.3262

623.3261

623.5773623.0754 624.8277620.8176

621.2696621.3194 622.3470

623.8274623.7992

624.0492

624.3002

621.8208 622.8491621.6639

Mass Spectrometry – Detec)on and Separa)on of Ions


FTMS Detect attomole quantities of peptides Resolving power in excess of 200,000 Mass measurement error below 5 ppm

50

Sky and Telescope Ground-‐Based Image Archive Credit & Copyright: Image by Jay GaBany, San Jose, CA Observed with a 12-‐inch f/9 telescope and CCD camera 12 hour exposure from July 1st to 7th 2005.

“How” you look can define “What” you discover M51 – The Famous Whirlpool Galaxy

Supernova 2005cs

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Differen)al Mass Spectrometry (dMS)

Key Benefits •  supports clinically accessible samples •  no pooling or chemical labeling •  individual sample integrity retained •  supports complex mul)-‐factor experiments

•  no need for an)body reagents •  quickly translates from animals to humans •  )me matched with pharmaceu)cal research

An efficient MS based strategy for comparing complex biological systems. Un-‐biased analysis of all ions detected by full scan MS, not just ions that have corresponding MS/MS spectra and pep1de sequences.

Differen)al MS Workflow

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Randomized Sample Set

Sample Processing prior to MS Analysis (e.g. Immunodeple)on or Ultrafiltra)on)

Desalt & run LC-‐MS

AD Control

Archived Human CSF (400 uL)

R.K*QLATK*AAR.-‐ Pep)de AA Sequence

dMS Feature

)me

m/z

CTRL

AD

Differen1al MS (dMS)

High Res MS

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158360 # 2685-‐2809 RT: 24.81-‐25.34 AV: 3 NL: 5.29E5 F: FTMS + p NSI Full ms [ 300.00-‐2000.00]

300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 m/z

0 10 20 30 40 50 60 70 80 90

100

Rela)ve Ab

undance

544.298

913.487 722.371

787.882 969.482 1087.593 738.707

646.322 1094.057 867.410

552.295 457.245 959.447 521.786 670.337 639.631 592.338 1024.499 391.286

1107.560 452.238 481.238 881.943 695.631

828.440 776.410 919.446 342.265 982.878

The analysis of 1 CSF sample generates ~ 2000 high resolu)on spectra and that contain the m/z and intensity of more than > 100,000 pep)de ions.

Differen)al Mass Spectrometry (dMS) Reveals Features of Interest in High Resolu)on MS Data

dMS dMS

Summary

•  Fundamentals •  ESI •  High and Low Resolu1on (Isotopes) •  Tandem Mass Spectrometry •  LC-‐MS and LC-‐MS/MS analysis •  Data types

•  Base peak chromatogram •  Selected ion chromatogram •  Mass spectra •  Tandem mass spectra

•  Proteomics •  Shotgun proteomics •  Targeted and global analysis of proteins •  Trace analysis by selected reac1on monitoring •  Differen1al mass spectrometry

Focus: Introduction to Mass Spectrometry and Methods for the Structural an Quantitative Analysis of Biomolecules.

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Intro to MS Yates - University of Pittsburgh · 365.23 22.27 444.74 20.58 690.33 21.06 436.76 21.80...

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Transcript of Intro to MS Yates - University of Pittsburgh · 365.23 22.27 444.74 20.58 690.33 21.06 436.76 21.80...