Mass Spectrometry in Proteomics

Mass spectrometry in Proteomics

Pierre-Alain Binz

March 2004

WhatWhat isis aa massmass spectrum?spectrum? 1265.6038 100

MALDI-DE-RE-TOF MS 80 1394.7169 tryptic digest of BSA

1252.6472 1757.8374

870.4042 1299.6103 * % Intensity 40 * 1930.0053 1742.8780 * * 1410.7018

1787.7116 2062.0077 950.4584 2523.2021 20 * 1083.5082 2848.3 1778.0565 2285.1 2467.1695 848.2 1099.5 1859.9261 2065.0 2266.1 1364.7 1555.7 2501.3228 2016.0 2222.2043 2734.2

0 828.0 1263.2 1698.4 2133.6 2568.8 3004.0

Mass (m/z) Protein Identification using Mass Spectrometry

protein from gel/ tryptic digestion & 1-DE, 2-DE, PVDF/LC fraction peptide extraction LC TYGGAAR EHICLLGK GANK PSTTGVEMFR unmodified and Mass spectrometry, modified peptides peptide mass fingerprints

PMF identification MS Fragmentation

Mass spectrometry, MS/MS identification peptide MS fragments HowHow areare massmass spectraspectra producedproduced ??

• 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 Vacuum System

Sample Ionisation Mass Data Detector Inlet Method Analyser System IonizationIonization methodsmethods 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 <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 ElectrosprayElectrospray IonizationIonization (ESI)(ESI)

pump

S S + + S S SH MH + + + + + + + S + + + + S MH SH MH +++ + 2+ + + S + + 2+ S + S SnH MH2 MH2 S + Smaller Coulomb explosion: Ions droplet droplet Clusters and ionic species Modif. From Alex Scherl

Matrix Assisted Laser Desorption/Ionization MALDI

UV or IR laser

sample target Membrane, gel or metal grid Matrix Analytes Matrix Assisted Laser Desorption/Ionization MALDI

MassMass AnalyzersAnalyzers

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. QuadrupoleQuadrupole massmass analyzeranalyzer

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

IonIon TrapTrap massmass analyzeranalyzer

• 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

High vacuum Ion source flight tube

Detector time 1 Small ions are faster than heavy, and reach detector time 2 first time 3

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.

FTMSFTMS WhatWhat isis aa massmass spectrum?spectrum? 1265.6038 100

MALDI-DE-RE-TOF MS 80 1394.7169 tryptic digest of BSA

1252.6472 1757.8374

870.4042 1299.6103 * % Intensity 40 * 1930.0053 1742.8780 * * 1410.7018

1787.7116 2062.0077 950.4584 2523.2021 20 * 1083.5082 2848.3 1778.0565 2285.1 2467.1695 848.2 1099.5 1859.9261 2065.0 2266.1 1364.7 1555.7 2501.3228 2016.0 2222.2043 2734.2

0 828.0 1263.2 1698.4 2133.6 2568.8 3004.0

Mass (m/z)

How does a peptide signal looks like?

Low resolution

High resolution Isotopic distribution

Mass resolution 0.1% vs. 1 ppm

Symbol Mass Abund. Symbol Mass Abund ------

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 H(1) 1.007825 99.99 H(2) 2.014102 0.015 S(32) 31.972072 95.02 S(33) 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 524.3 100

95 90 85 80 75 Singly charged Ion: 70 65 Distance between Peak 60 and Isotop 1 amu 55

Abundance 50 45 = 1.0 amu Relative 40 _

35 30 525.3 25 _ = 1.0 amu 20

15 10 526.2 5 0 520 521 522 523 524 525 526 527 528 529 m/z

262.6 100

95 90

85 80 75 Doubly charged Ion: 70 65 Distance between Peak and 60 Isotop 0.5 amu 55

Abundance 50 45 = 0.5 amu Relative 40 _

35 30

20 263.1

15 _ = 0.5 amu 10 263.6 5

0 258 259 260 261 262 263 264 265 266 267

m/z Resolution: Example Peptide Mw 2129.64, Ion 4+

Intens. x105 533.46 Resolution 4 0.6 m/z 2 0 531 532 533 534 m/z Intens. x105 532.62 532.85 533.09 Resolution 1.0 533.33 0.2 m/z 0.5 533.61 0.0 531 532 533 534 m/z

Multiply chargedmyoglobin ions from ESI

(M 2-1.008) /M1-M2 = Z1 1060.5 100 M1 (Z 1 * M1)-(Z*1.008) = Mwt 90 1131.1 998.2 1211.9 M2 942.9 80 1305.0

70 893.3 60 848.6 1413.5

40 808.2 1541.9 30 771.5 20 1696.0 616.2 738.1 10 707.3 1310.9 1884.2 1428.7 1563.0 1820.8 1888.9 0 600 800 1000 1200 1400 1600 1800 2000 m/z Deconvoluted myoglobin spectrum

16951.0 100

10 15931.0 16104.0 16392.0 16582.0 16784.0 17088.017280.0 17562.0 17830.0 17995.0 0 16000 16200 16400 16600 16800 17000 17200 17400 17600 17800 18000 mass

9.9E+3 MALDI-DE-RE-TOF MS 100 90 tryptic digest of BSA 80 1265.6038 100 70

50 80 1394.7169 % Intensity 40

20 60

1252.6472 10 1757.8374

0 0 870.4042 1299.6103 1910.0 * 1918.8 1927.6 1936.4 1945.2 1954.0 % Intensity 40 * Mass (m/z) 1930.0053 1742.8780 * * 1410.7018

1787.7116 2062.0077 950.4584 2523.2021 20 * 1083.5082 2848.3 1778.0565 2285.1 2467.1695 848.2 1099.5 1859.9261 2065.0 2266.1 1364.7 1555.7 2501.3228 2016.0 2222.2043 2734.2

0 828.0 1263.2 1698.4 2133.6 2568.8 3004.0 Mass (m/z) 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. H I 7 x10 TIC O O 4

H 2 I N O 0 4.0 5.0 Time [min] HO Ab. MS, Time=4.420min 100 634 m/z 634

50 MS/MS 545 0 100 200 300 400 500 600 m/z H I O Ab. MS/MS(634), Time=4.458min 545 OH 100 373

I 50 249 376 563 O HO 0 m/z 563 100 200 300 400 500 600 m/z Peptide fragmentation with MS/MS

K C S C N P D M MAPNCSCK 2+ y3 [M+2H] MAPNCSC K y1 MAPNCS CK y7 y4 y5 MAPNC SCK y2 y6 MAPN CSCK 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 MALDI-TOF-MS

LASER

m/z

MALDI-TOF MS: illustrated examples MALDI sample plates

Voyager STR Voyager DE-PRO Applied Biosystems Applied Biosystems

Autoflex Reflex III Bruker Bruker

Micromass (ESI) - Triple quadrupole MS

Q2 is Non-Linear Collision Cell

Q0 Q1 Q2 Q3

ESI Probe

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

ESI-Q-TOFESI-Q-TOF MSMS

Q q TOF ESI

I Ion 2 ! Ion 1 Ion 3

m/z

Mod. From Alex Scherl ESI-Q-q-TOFESI-Q-q-TOF

Q q TOF ESI

Fragment 2 I Fragment 1 ! Fragment 3

m/z

Mod. From Alex Scherl

Esquire-LC Ion Optics

HPLC inlet Skimmers Octopole End Caps

Capillary

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

Ion Trap Nebulizer Lenses Ring Electrode Q-TOF MS

Q Star XL Hybrid BioTOF-q qTOF-Ultima Applied Biosystems Bruker Micromass Ion trap MS

LCQ Deca XP Esquire 3000 Finnigan Bruker

nanoLCnanoLC-ESI-Q-TOF-ESI-Q-TOF

Q-Tof

Column C18 75 µm

HPLC Autosampler/Injector PrincipePrincipe ofof LC-MS/MSLC-MS/MS

time 27.4 min : peak at m/z = 957.6 m/z = 957.6 QIIEEDAALVEIGPR

Q96DH1

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

TOF 1 TOF 2 collision source cell MALDI TOF-TOF MS

AB 4700 Proteomics Analyzer with Auto-loader

TOF-TOF from Bruker: the Ultraflex

nanoLCnanoLC-MALDI-TOF-TOF-MALDI-TOF-TOF

Spotting robot

Column C18 75 µm

MALDI plate HPLC 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

ElectroSpray MALDI EI/CI Switchable CF-FAB, CF-SIMS GC Interface LC Interface Pulsed valve for MS/MS IRMPD Bruker APEXIII

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