Mass spectrometry in Proteomics Pierre-Alain Binz March 2004 100 What is a mass spectrum? 1265.6038 80 1394.7169 MALDI-DE-RE-TOF MS tryptic digest of BSA 60 % Intensity 40 870.4042 1252.6472 1299.6103 1410.7018 1757.8374 1930.0053 1742.8780 20 1787.7116 2062.0077 950.4584 2523.2021 1083.5082 2848.3 1778.0565 2285.1 2467.1695 848.2 1099.5 1859.9261 1364.7 1555.7 2065.0 2266.1 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 1-DE, 2-DE, LC protein from gel/ PVDF/LC fraction tryptic digestion & peptide extraction Mass spectrometry, peptide mass fingerprints TYGGAAR GANK EHICLLGK PSTTGVEMFR 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 Vacuum System Sample Inlet Ionisation Method Mass Analyser Detector Data System
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. [MH] 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 [MNa] 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
Electrospray Ionization (ESI) S S S droplet S S SH MH S SnH Smaller droplet S S S MH SH 2 S MH2 Coulomb explosion: Clusters and ionic species pump MH 2 MH2 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 Small ions are faster than heavy, and reach detector 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. FTMS
100 What is a mass spectrum? 1265.6038 80 1394.7169 MALDI-DE-RE-TOF MS tryptic digest of BSA 60 % Intensity 40 870.4042 1252.6472 1299.6103 1410.7018 1757.8374 1930.0053 1742.8780 20 1787.7116 2062.0077 950.4584 2523.2021 1083.5082 2848.3 1778.0565 2285.1 2467.1695 848.2 1099.5 1859.9261 1364.7 1555.7 2065.0 2266.1 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
100 524.3 Relative Abundance 95 90 85 80 75 70 65 60 55 50 45 40 35 Singly charged Ion: Distance between Peak and Isotop 1 amu _ = 1.0 amu 30 25 20 15 525.3 _ = 1.0 amu 10 5 0 520 521 522 523 524 525 526 527 528 529 m/z 526.2 100 262.6 Relative Abundance 95 90 85 80 75 70 65 60 55 50 45 40 35 30 Doubly charged Ion: Distance between Peak and Isotop 0.5 amu _ = 0.5 amu 25 20 15 10 5 0 258 259 260 261 262 263 264 265 266 267 263.1 263.6 _ = 0.5 amu m/z
Resolution: Example Peptide Mw 2129.64, Ion 4 Intens. x10 5 4 2 533.46 Resolution 0.6 m/z 0 531 532 533 534 m/z Intens. x10 5 1.0 0.5 532.62 532.85 533.09 533.33 533.61 0.0 531 532 533 534 m/z Resolution 0.2 m/z Multiply charged myoglobin ions from ESI (M 2-1.008) /M 1 -M 2 = Z 1 100 90 80 942.9 1060.5 1131.1 998.2 1211.9 M 1 M 2 1305.0 (Z 1 M 1 )-(Z1.008) = Mwt 70 60 893.3 848.6 1413.5 50 40 30 20 10 0 616.2 738.1 707.3 771.5 808.2 1541.9 1696.0 1310.9 1884.2 1428.7 1563.0 1820.8 1888.9 600 800 1000 1200 1400 1600 1800 2000 m/z
Deconvoluted myoglobinspectrum 100 16951.0 90 80 70 60 50 40 30 20 10 0 15931.0 16104.0 16392.0 16582.0 16784.0 17088.017280.0 17562.0 17830.0 17995.0 16000 16200 16400 16600 16800 17000 17200 17400 17600 17800 18000 mass MALDI-DE-RE-TOF MS tryptic digest of BSA 100 1265.6038 100 90 80 70 9.9E3 80 1394.7169 % Intensity 60 50 40 30 60 20 % Intensity 40 870.4042 1252.6472 1299.6103 1410.7018 10 1757.8374 0 1910.0 1918.8 1927.6 1936.4 1945.2 0 1954.0 Mass (m/z) 1930.0053 1742.8780 20 1787.7116 2062.0077 950.4584 2523.2021 1083.5082 2848.3 1778.0565 2285.1 2467.1695 848.2 1099.5 1859.9261 1364.7 1555.7 2065.0 2266.1 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. x10 7 4 TIC H O I O 2 0 Ab. 100 4.0 5.0 Time [min] MS, Time=4.420min 634 O I HO m/z 634 N H 50 545 0 100 200 300 400 500 600 m/z Ab. MS/MS(634), Time=4.458min 545 100 373 O H MS/MS I OH 50 249 376 563 0 100 200 300 400 500 600 m/z O HO m/z 563 I
Peptide fragmentation with MS/MS MAPNCSCK MAPNCSC K MAPNCS CK MAPNC SCK MAPN CSCK... K C S C N P D M y3 [M2H] 2 y1 y7 y4 y5 y2 y6 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 I m/z MALDI-TOF MS: illustrated examples MALDI sample plates Voyager DE-PRO Applied Biosystems Voyager STR Applied Biosystems Autoflex Bruker Reflex III Bruker Micromass
(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 ESI Q q TOF! I Ion 1 Ion 2 Ion 3 m/z Mod. From Alex Scherl
ESI-Q-q-TOF ESI Q q TOF! I Fragment 1 Fragment 2 Fragment 3 m/z Mod. From Alex Scherl Esquire-LC Ion Optics HPLC inlet Capillary Skimmers Octopole End Caps Nebulizer Lenses Ion Trap Ring Electrode
Q-TOF MS Q Star XL Hybrid Applied Biosystems BioTOF-q Bruker Ion trap MS qtof-ultima Micromass LCQ Deca XP Finnigan Esquire 3000 Bruker nanolc-esi-q-tof Q-Tof Column C18 75 µm HPLC Autosampler/Injector
Principe of LC-MS/MS m/z = 957.6 time 27.4 min : peak at m/z = 957.6 QIIEEDAALVEIGPR Q96DH1 MALDI TOF-TOF: MS/MS Mode TIS intensity Mass (m/z) source TOF 1 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 µm HPLC MALDI plate Autosampler/Injector Off-line MALDI MS (MS/MS)
FTMS can provide very high resolution, 10 6, 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