What is a mass spectrum? What is a mass spectrum? 1265.6038 100 - - PDF document

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What is a mass spectrum? What is a mass spectrum? 1265.6038 100 - - PDF document

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Mass spectrometry Mass spectrometry in Proteomics Proteomics in Pierre-Alain Binz March 2004 What is a mass spectrum? What is a mass spectrum? 1265.6038 100 MALDI-DE-RE-TOF MS tryptic digest of BSA 80 1394.7169 60 1252.6472 %

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SLIDE 1

Pierre-Alain Binz

March 2004

Mass spectrometry Mass spectrometry in in Proteomics Proteomics

828.0 1263.2 1698.4 2133.6 2568.8 3004.0 Mass (m/z) 20 40 60 80 100 % Intensity 1265.6038 1394.7169 1252.6472 1757.8374 1299.6103 870.4042 1930.0053 1742.8780 1410.7018 1787.7116 2062.0077 2523.2021 950.4584 1083.5082 2848.3 1778.0565 2285.1 2467.1695 1099.5 1859.9261 848.2 2065.0 2266.1 1364.7 1555.7 2501.3228 2734.2 2016.0 2222.2043 * * * * *

MALDI-DE-RE-TOF MS tryptic digest of BSA

What is a mass spectrum? What is a mass spectrum?

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SLIDE 2

Protein Identification using Mass Spectrometry

protein from gel/ PVDF/LC fraction tryptic digestion & peptide extraction PMF identification Mass spectrometry, peptide mass fingerprints

TYGGAAR PSTTGVEMFR EHICLLGK GANK

unmodified and modified peptides 1-DE, 2-DE, LC MS/MS identification Mass spectrometry, peptide MS fragments MS Fragmentation

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SLIDE 3

How are mass spectra produced How are mass spectra produced ? ?

  • Ions are produced in the source and are transferred

into the mass analyser

  • They are separated according to their mass/charge

ratio in the mass analyser (e.g. Quadrupole, Ion Trap, Time of Flight)

  • Ions of the various m/z values exit the analyser and

are ‘counted’ by the detector

Vacuum System Vacuum System

Sample Sample Inlet Inlet Detector Detector Data Data System System Mass Mass Analyser Analyser Ionisation Ionisation Method Method

Atmosphere

Generic description of a mass Generic description of a mass spectrometer spectrometer

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SLIDE 4

Ionization Ionization methods methods

Analytes are ionized to be driven in the mass analyzer Electron impact (EI) Chemical Ionisation (CI) Fast atom bombardment (FAB) Field desorption (FD) Atmospheric Pressure Chemical Ionisation (APCI) ESI Electro-Spray Ionization MALDI Matrix Assisted Laser Desorption Ionization

EI electron impact ionisation: beam of electrons through the gas-phase sample. Produces molecular ions or fragment ions. Typically 70eV. Sample heated. + Reproducible, structural information

  • sample must be volatile and stable, molecular ion often abscent

mass range: < 1000Da CI: chemical ionisation: reagent gaz (methane, isobutane, or ammonia) ionized with electrons. High gaz pressure: (R = reagent, S = sample, e = electron, . = radical electron , H = hydrogen) R + e ---> R+. + 2e R+. + RH ---> RH+ + R. RH+ + S ---> SH+ + R Heated sample. + [M+H]+ often visible, less fragmentation than EI

  • sample must be volatile and stable, less structural info than EI

mass range: < 1000Da DCI: Desorption CI : CI on a heated filament + rapid, simple

  • reproducibility

mass range <1500Da NCI: negative-ion CI: electron capture; use of Methane to slow down electrons + efficient, sensitive; less fragmentation that EI, CI

  • not all molecule compatible, reproducibility

mass range <1000Da

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SLIDE 5

FD: Field Desorption: sample deposited on filament gradually heated by electric field. Sample ionise by electron tunneling. Ions are M+ and [M+Na]+ + simple spectra, almost no background

  • sensitive to alkali, slow, volatile to desorb

mass range <2000-3000Da FI: Field ionisation: sample introduced in gas phase (heaten or not), ionised by electron tunneling near the emitter. + simple spectra, almost no background

  • sample must be volatile

mass range <1000Da FAB: fast atom bombardment: analyte in a liquid matrix (glycerol, etc.). Bombardment with fast atom beam (xenon at 6keV). Desorbtion of molecular ions, fragments and matrix clusters sample introduced liquid, or LC/MS + rapid, simple, good for variety of compounds, strong currents, high resolution

  • background, sample must be soluble in matrix

mass range ~300-6000Da SIMS: soft ionisation: similar to FAB but with ion beam as gas (Ce+), allowing higher acceleration (energy) + idem FAB

  • idem FAB, target can get hotter, more maintenance

mass range 300-13000Da ESI: electrospray ionisation: The sample solution is sprayed across a high potential difference (a few kilovolts) from a needle into an orifice in the interface. Heat and gas flows are used to desolvate the ions existing in the sample solution. ESI often produces multiply charged ions with the number of charges tending to increase as the molecular weight increases. High to low flow rates 1 ml/min to nl/min. + good for charged, polar or basic compounds, m/z ok for most MS, best for multiply charged ions, low background, controlled fragmentation, MS/MS compatible

  • complementary to APCI: not good for uncharged, non-basic, low-polarity compounds,

low ion currents mass range <200’000Da APCI: atmospheric pressure CI: as in ESI, sample introduced in a high potential difference field. Uses a corona discharge for better ionisation of less polar molecules than in ESI. APCI and ESI are complementary MALDI: Matrix-Assisted Laser Desorption Ionization: analyte co-crystallised in matrix. The matrix chromophore absorbs and distribute the energy of a laser, produced a plasma, vaporates and ionize the sample. + rapid, convenient for molecular weight (singly charged ions mostly)

  • MS/MS difficult, almost not compatible with LC coupling

<500’000Da

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SLIDE 6

S

+ + + + + +

S S S S

+ +

++ + + + + +

S MH SH+

+ +

SnH

+ +

  • Modif. From Alex Scherl

Electrospray Ionization (ESI) Electrospray Ionization (ESI)

MH

+

MH2

2+

S S S SH

+

S MH

+

MH2

2+

droplet Smaller droplet Coulomb explosion: Clusters and ionic species Ions pump

UV or IR laser sample target

Matrix Analytes Membrane, gel or metal

grid

Matrix Assisted Laser Matrix Assisted Laser Desorption Desorption/Ionization /Ionization MALDI MALDI

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SLIDE 7

Matrix Assisted Laser Matrix Assisted Laser Desorption Desorption/Ionization /Ionization MALDI MALDI

Mass Mass Analyzers Analyzers

Mass Spectrometers separate ions according to their mass-to- charge (m/z) ratios – Magnetic Sector – Quadrupole – Ion Trap – Time-of-flight – Hybrid- Sector/trap, Quad/TOF, etc.

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SLIDE 8

+ + + +

  • The ion is transmitted along the

quadrupole in a stable trajectory Rf field. The ion does not have a stable trajectory and is ejected from the quadrupole. RF + DC The quadrupole consists of two pairs of parallel rods with applied DC and RF voltages. Ions are scanned by varying the DC/Rf quadrupole voltages.

Quadrupole mass analyzer Quadrupole mass analyzer Ion Trap mass analyzer Ion Trap mass analyzer

  • Consists of ring electrode and two

end caps

  • Principle very similar to

quadrupole

  • Ions stored by RF & DC fields
  • Scanning field can eject ions of

specific m/z

  • Advantages
  • MS/MS/MS…..
  • High sensitivity full scan

MS/MS

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SLIDE 9

Ion source Detector High vacuum flight tube

time 1 time 2 time 3

Small ions are faster than heavy, and reach detector first

Time of Flight (TOF) mass analyzer Time of Flight (TOF) mass analyzer

Ion source Detector Reflectron High vacuum flight tube

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SLIDE 10

FTMS Ions moving at their cyclotron frequency can absorb RF energy at this same frequency. A pulse of RF excites the ions in the magnetic

  • field. The ions re-emit the radiation, which is picked up by the

reciever plates. The decay produces a free-induction decay signal that can be Fourier transformed to produce the emitted frequencies, and therefore the masses of the ions present.

FTMS FTMS

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SLIDE 11

828.0 1263.2 1698.4 2133.6 2568.8 3004.0 Mass (m/z) 20 40 60 80 100 % Intensity 1265.6038 1394.7169 1252.6472 1757.8374 1299.6103 870.4042 1930.0053 1742.8780 1410.7018 1787.7116 2062.0077 2523.2021 950.4584 1083.5082 2848.3 1778.0565 2285.1 2467.1695 1099.5 1859.9261 848.2 2065.0 2266.1 1364.7 1555.7 2501.3228 2734.2 2016.0 2222.2043 * * * * *

MALDI-DE-RE-TOF MS tryptic digest of BSA

What is a mass spectrum? What is a mass spectrum?

How does a peptide signal looks like? How does a peptide signal looks like?

Low resolution High resolution

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SLIDE 12

Mass resolution 0.1% vs. 1 ppm Symbol Mass

  • Abund. Symbol Mass

Abund

  • H(1) 1.007825 99.99 H(2) 2.014102 0.015

C(12) 12.000000 98.90 C(13) 13.003355 1.10 N(14) 14.003074 99.63 N(15) 15.000109 0.37 O(16) 15.994915 99.76 O(17) 16.999131 0.038 S(32) 31.972072 95.02 S(33) 32.971459 0.75

Isotopic distribution Isotopic distribution Isotopic distribution Isotopic distribution

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SLIDE 13

10002000Half massFull width

Mass resolution Mass resolution

1.0 FWHM 0.7 FWHM 0.5 FWHM 0.3 FWHM 0.2 FWHM 0.1 FWHM

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SLIDE 14

520 521 522 523 524 525 526 527 528 529 m/z 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance

524.3

525.3 526.2

Singly charged Ion: Singly charged Ion: Distance between Peak Distance between Peak and and Isotop Isotop 1 1 amu amu _ _ = 1.0 amu

= 1.0 amu

_ _ = 1.0 amu

= 1.0 amu

m/z 258 259 260 261 262 263 264 265 266 267 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance 262.6 263.1 263.6

Doubly charged Ion: Doubly charged Ion: Distance between Peak and Distance between Peak and Isotop Isotop 0.5 0.5 amu amu _ _ = 0.5 amu

= 0.5 amu

_ _ = 0.5 amu

= 0.5 amu

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SLIDE 15

Resolution: Example Peptide Mw 2129.64, Ion 4+

531 532 533 534 m/z 2 4 5 x10 Intens.

533.46

531 532 533 534 m/z 0.0 0.5 1.0 5 x10 Intens.

532.62 532.85 533.09 533.33 533.61

Resolution 0.6 m/z Resolution 0.2 m/z

Multiply charged myoglobin ions from ESI

10 20 30 40 50 60 70 80 90 100 1060.5 1131.11211.9 998.2 942.9 1305.0 893.3 1413.5 848.6 808.2 1541.9 771.5 1696.0 616.2 738.1 1884.2 1310.9 707.3 1888.9 1428.7 1820.8 1563.0

M2 M1

600 800 1000 1200 1400 1600 1800 2000 m/z

(M2-1.008) /M1-M2 = Z1 (Z1 * M1)-(Z*1.008) = Mwt

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SLIDE 16

Deconvoluted myoglobin spectrum

mass 16000 16200 16400 16600 16800 17000 17200 17400 17600 17800 18000 10 20 30 40 50 60 70 80 90 100 16951.0 17088.0 16784.0 15931.0 17995.0 17280.0 16392.0 16104.0 17562.0 17830.0 16582.0

828.0 1263.2 1698.4 2133.6 2568.8 3004.0 Mass (m/z) 20 40 60 80 100 % Intensity 1265.6038 1394.7169 1252.6472 1757.8374 1299.6103 870.4042 1930.0053 1742.8780 1410.7018 1787.7116 2062.0077 2523.2021 950.4584 1083.5082 2848.3 1778.0565 2285.1 2467.1695 1099.5 1859.9261 848.2 2065.0 2266.1 1364.7 1555.7 2501.3228 2734.2 2016.0 2222.2043 * * * * *

1910.0 1918.8 1927.6 1936.4 1945.2 1954.0 Mass (m/z) 9.9E+3 10 20 30 40 50 60 70 80 90 100 % Intensity

MALDI-DE-RE-TOF MS tryptic digest of BSA

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SLIDE 17

Ion fragmentation with Mass Spectrometry Tandem MS or MS/MS One set of ions (one m/z value) is selected from a mixture of ions; These ions are fragmented; the fragments are measured.

HPLC-ESI-autoMS/MS

4.0 5.0 Time [min] 2 4 7 x10 Int. 50 100 Ab. 100 200 300 400 500 600 m/z

545 634

MS, Time=4.420min

50 100 Ab. 100 200 300 400 500 600 m/z

249 373 376 545 563

MS/MS(634), Time=4.458min

TIC MS/MS m/z 634 m/z 563

O O I H H N O HO I

O O OH I H I HO

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SLIDE 18

Peptide fragmentation with MS/MS MAPNCSCK MAPNCSC K MAPNCS CK MAPNC SCK MAPN CSCK ...

[M+2H]2+

y1 y2 y3

K C S C N P D M

y4 y5 y6 y7 y8

MS instruments used in Proteomics

ESI-Triple quadrupole MS ESI-Q-TOF MS ESI-Ion-trap MS ESI-Q-trap MS ESI-FTICR MS SELDI MS MALDI-TOF MS MALDI-TOF-TOF MS MALDI-Q-TOF MS MALDI-Ion-trap MS MALDI-FTICR MS

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SLIDE 19

LASER

m/z

I

MALDI-TOF-MS

Voyager DE-PRO Applied Biosystems Voyager STR Applied Biosystems Reflex III Bruker MALDI sample plates

MALDI-TOF MS: illustrated examples

Autoflex Bruker Micromass

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SLIDE 20

ESI Probe

Q0 Q1 Q2 Q3

Hyperbolic, high precision quadrupoles Square Rod Ion Transmission to Analytical Quads Electron Multiplier, Detection System

Q2 is Non-Linear Collision Cell

(ESI) - Triple quadrupole MS

ESI-Q-TOF MS ESI-Q-TOF MS

  • m/z

I

Q q TOF ESI

Ion 1 Ion 2 Ion 3

  • Mod. From Alex Scherl
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SLIDE 21

ESI-Q-q-TOF ESI-Q-q-TOF

  • m/z

I

Q q TOF ESI

Fragment 1 Fragment 2 Fragment 3

  • Mod. From Alex Scherl

Capillary Skimmers Octopole Lenses Ion Trap HPLC inlet Nebulizer

+ + + + + + + + + + + + + +

End Caps Ring Electrode

Esquire-LC Ion Optics Esquire-LC Ion Optics

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SLIDE 22

Q-TOF MS Ion trap MS

LCQ Deca XP Finnigan Esquire 3000 Bruker Q Star XL Hybrid Applied Biosystems BioTOF-q Bruker qTOF-Ultima Micromass HPLC Autosampler/Injector Column C18 75 µm Q-Tof

nanoLC nanoLC-ESI-Q-TOF

  • ESI-Q-TOF
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SLIDE 23

Principe of LC-MS/MS Principe of LC-MS/MS

time 27.4 min : peak at m/z = 957.6

m/z = 957.6

QIIEEDAALVEIGPR

Q96DH1

MALDI TOF-TOF: MALDI TOF-TOF:

MS/MS Mode MS/MS Mode

source TOF 1 collision cell TOF 2

Mass (m/z) intensity

TIS

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SLIDE 24

AB 4700 Proteomics Analyzer with Auto-loader TOF-TOF from Bruker: the Ultraflex MALDI TOF-TOF MS

HPLC Autosampler/Injector Column C18 75 µm

nanoLC nanoLC-MALDI-TOF-TOF

  • MALDI-TOF-TOF

Spotting robot MALDI plate Off-line MALDI MS (MS/MS)

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SLIDE 25

FTMS can provide very high resolution, 106, which its main advantage compared to other mass spectrometers. Mass accuracy <1ppm in MS and MS/MS mode Bruker APEXIII ElectroSpray MALDI EI/CI Switchable CF-FAB, CF-SIMS GC Interface LC Interface Pulsed valve for MS/MS IRMPD Operating mass range (APEX 70e) of 18 - 66000 Daltons

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SLIDE 26

The Q-trap MS Q Trap (Quadrupole – linear trap)

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SLIDE 27

Q-TRAP MS

Q-trap Applied Biosystems and MDS Sciex

Additional info on MS

http://www.spectroscopynow.com/ http://www.ionsource.com/ http://www.asms.org/whatisms/index.html