Mass spectrometry in Proteomics

Pierre-Alain Binz
March 2004

What is a mass spectrum?

100 1265.6038 80 1394.7169

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

60 % Intensity 1252.6472 1299.6103 * * 1410.7018 950.4584 1083.5082 848.2 1099.5 1364.7 1555.7 1742.8780 1930.0053 * 2062.0077 2285.1 2065.0 2266.1 2222.2043 2016.0 828.0 1263.2 1698.4 Mass (m/z) 2133.6 1757.8374 *

870.4042 40

1787.7116 20 * 1778.0565 1859.9261

2523.2021 2848.3 2467.1695 2501.3228 2734.2 0 3004.0

2568.8

Protein Identification using Mass Spectrometry

1-DE, 2-DE, LC

protein from gel/ PVDF/LC fraction

tryptic digestion & peptide extraction

TYGGAAR EHICLLGK GANK PSTTGVEMFR

Mass spectrometry, peptide mass fingerprints

unmodified and modified peptides

PMF identification MS Fragmentation

MS/MS identification

Mass spectrometry, peptide MS fragments

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

Generic description of a mass spectrometer

Atmosphere
Sample Inlet Ionisation Method

Vacuum System
Mass Analyser Data System

Detector

Ionization 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

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 <200000Da 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 <500000Da

Electrospray Ionization (ESI)

S S

+++ + + + ++ S
droplet

S S

pump

SH S

+
++ + + +++ + +

MH

+ +
S

S MH2

S nH

2+

MH

S S SH

MH2

2+

MH

Smaller droplet

Coulomb explosion: Clusters and ionic species

Ions

Modif. From Alex Scherl

Matrix Assisted Laser Desorption/Ionization MALDI

UV or IR laser

sample target grid

Membrane, gel or metal Matrix Analytes

Matrix Assisted Laser Desorption/Ionization MALDI

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

Quadrupole mass analyzer

+
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. 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.

+ +

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

Time of Flight (TOF) mass analyzer

Ion source High vacuum flight tube

Detector

time 1 time 2 time 3

Small ions are faster than heavy, and reach detector first

Ion source

High vacuum flight tube

Detector Reflectron

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

What is a mass spectrum?

100 1265.6038 80 1394.7169

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

60 % Intensity 1252.6472 1299.6103 * * 1410.7018 950.4584 1083.5082 848.2 1099.5 1364.7 1555.7 1742.8780 1930.0053 * 2062.0077 2285.1 2065.0 2266.1 2222.2043 2016.0 828.0 1263.2 1698.4 Mass (m/z) 2133.6 1757.8374 *

870.4042 40

1787.7116 20 * 1778.0565 1859.9261

2523.2021 2848.3 2467.1695 2501.3228 2734.2 0 3004.0

2568.8

How does a peptide signal looks like?

Low resolution

High resolution

Isotopic distribution
Mass resolution 0.1% vs. 1 Symbol -----C(12) N(14) O(16) H(1) S(32) Mass ---------ppm Abund . Symbol ----------Mass ----------Abund ------1.10

12.000000 98.90 C(13) 14.003074 99.63 N(15) 15.994915 99.76 O(17) 1.007825 99.99 H(2) 31.972072 95.02 S(33)

13.003355 15.000109 0.37 16.999131 0.038 2.014102 0.015 32.971459 0.75

Isotopic distribution

Mass resolution
10002000Half massFull width

Mass resolution
1.0 FWHM 0.7 FWHM 0.5 FWHM

0.3 FWHM

0.2 FWHM

0.1 FWHM

100 95 90 85 80 75 70 65 Relative Abundance 60 55 50 45 40 35 30 25 20 15 10 5 0 520 521 522 523

524.3

Singly charged Ion: Distance between Peak and Isotop 1 amu _ = 1.0 amu
525.3

_ = 1.0 amu
526.2

524 m/z

525

526

527

528

529

100 95 90 85 80 75 70 65 Relative Abundance 60 55 50 45 40 35 30 25 20 15 10 5 0 258 259 260 261 262

262.6

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

263.1

_ = 0.5 amu
263.6

263

264

265

266

267

m/z

Resolution: Example Peptide Mw 2129.64, Ion 4+

Intens. x105 4 2 0 531
Intens. x105 1.0 0.5 0.0 531 532 533

533.46

Resolution 0.6 m/z

532 533 534 m/z

532.62

532.85 533.09 533.33 533.61

534 m/z

Resolution 0.2 m/z

Multiply charged myoglobinions from ESI

(M 2-1.008) /M1 -M2 = Z1
100 90 80 70 60 50 40 30 771.5 20 616.2 738.1 10 0 600 800 1000 1200 m/z 1400 1600 1800 2000 707.3 1310.9 1428.7 1563.0 1884.2 1820.8 1888.9 1696.0 848.6 893.3 1413.5 1060.5

M1 M2
1305.0

(Z 1 * M 1)-(Z*1.008) = Mwt

998.2 942.9

1131.11211.9

808.2

1541.9

Deconvoluted myoglobin spectrum

100 90 80 70 60 50 40 30 20 10 0 16000 16200 16400 16600 16800 17000 mass 17200 17400 17600 17800 18000 16951.0

15931.0 16104.0

16392.0 16582.0

16784.0

17088.017280.0

17562.0

17830.0 17995.0

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

100 1265.6038
% Intensity

100 90 80 70 60 50 40 30

9.9E+3

80

1394.7169

60 % Intensity 1252.6472 1299.6103 * * 1410.7018 950.4584 1083.5082 848.2 1099.5 1364.7

20 10 0 1910.0

1757.8374 *
1918.8 1927.6 1936.4 Mass (m/z) 1945.2 0 1954.0

870.4042 40

1742.8780

1930.0053 * 2062.0077 2285.1 2065.0 2266.1 2222.2043 2016.0

1787.7116 20 * 1778.0565 1555.7 1859.9261

2523.2021 2848.3 2467.1695 2501.3228 2734.2 0 3004.0

828.0

1263.2

1698.4 Mass (m/z)

2133.6

2568.8

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
Int. x107 4 2 0 Ab. 100 50 0 100 200 300 Ab. MS/MS(634), Time=4.458min 100 400 500
O I H I O H

TIC

4.0
MS, Time=4.420min

5.0

Time [min]

HO

634 545
600 m/z

m/z 634 MS/MS

H O OH I I

373 376

545

50 0 100

249
200 300

563
500 600 m/z

O HO

400

m/z 563

Peptide fragmentation with MS/MS

MAPNCSCK MAPNCSC K MAPNCS CK MAPNC SCK MAPN CSCK ...

C
y1

S
y3

[M+2H]2+
y7 y4 y5 y6 y8

y2

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

MALDI-TOF-MS
LASER

I
m/z

MALDI-TOF MS: illustrated examples

MALDI sample plates

Voyager DE-PRO Applied Biosystems

Voyager STR Applied Biosystems

Autoflex Bruker Micromass

Reflex III Bruker

(ESI) - Triple quadrupole MS

Q2 is Non-Linear Collision Cell

Q0

Q1

Q2

Q3

ESI Probe

Square Rod Ion Transmission to Analytical Quads

Hyperbolic, high precision quadrupoles

Electron Multiplier, Detection System

ESI-Q-TOF MS
Q ESI q TOF

I
! Ion 1

Ion 2 Ion 3

m/z

Mod. From Alex Scherl

ESI-Q-q-TOF
Q ESI q TOF

I
!

Fragment 2 Fragment 1 Fragment 3

m/z
Mod. From Alex Scherl

Esquire-LC Ion Optics

HPLC inlet Skimmers Octopole

End Caps

Capillary

+ ++ + + ++

+ +

+ +

+ +

Nebulizer

Ion Trap Lenses

Ring Electrode

Q-TOF MS

Q Star XL Hybrid Applied Biosystems

BioTOF-q Bruker

qTOF-Ultima Micromass

Ion trap MS

LCQ Deca XP Finnigan

Esquire 3000 Bruker

nanoLC-ESI-Q-TOF
Q-Tof

Column C18 75 m HPLC

Autosampler/Injector

Principe of LC-MS/MS
time 27.4 min : peak at m/z = 957.6
m/z = 957.6

QIIEEDAALVEIGPR
Q96DH1

MALDI TOF-TOF:
MS/MS Mode
TIS
intensity Mass (m/z)

TOF 1 source

collision cell

TOF 2

MALDI TOF-TOF MS AB 4700 Proteomics Analyzer with Auto-loader

TOF-TOF from Bruker: the Ultraflex

nanoLC-MALDI-TOF-TOF
Spotting robot

Column C18 75 m

HPLC

MALDI plate Autosampler/Injector

Off-line MALDI MS (MS/MS)

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

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

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