What is a mass spectrum?

EMBnet course 28.1.-1.2.28 Day 1 Mass spectrometry in proteomics Outline Mass spectrometry, general introduction What is a mass spectrum What are the constituents of a mass spectrometer How the instruments look like Pierre-Alain Binz Swiss Institute of Bioinformatics, proteome informatics group Geneva Bioinformatics SA (GeneBio) EMBnet course Basle, 28 Jan, 28 1 What is a mass spectrum? 1265.638 8 1394.7169 MALDI-DE-RE-TOF MS tryptic digest of BSA 6 % Intensity 4 87.442 1252.6472 1299.613 141.718 1757.8374 193.53 1742.878 2 1787.7116 262.77 95.4584 2523.221 183.582 2848.3 1778.565 2285.1 2467.1695 199.5 1859.9261 848.2 265. 2266.1 1364.7 1555.7 251.3228 2734.2 216. 2222.243 828. 1263.2 1698.4 2133.6 2568.8 34. Mass () Protein Identification using Mass Spectrometry Outline 1-DE, 2-DE, LC protein from gel/ PVDF/LC fraction tryptic digestion & peptide extraction Mass spectrometry, peptide mass fingerprints TYGGAAR EHICLLGK GANK PSTTGVEMFR unmodified and modified peptides Mass spectrometry, general introduction What is a mass spectrum What are the constituents of a mass spectrometer PMF identification MS Fragmentation How the instruments look like MS/MS identification Mass spectrometry, peptide MS fragments 1

1 What is a mass spectrum? 1265.638 How does a peptide signal looks like? 8 1394.7169 MALDI-DE-RE-TOF MS tryptic digest of BSA Low resolution 6 % Intensity 4 2 1252.6472 1757.8374 87.442 1299.613 193.53 1742.878 141.718 1787.7116 262.77 95.4584 2523.221 183.582 1778.565 2848.3 2285.1 2467.1695 848.2 199.5 1859.9261 1364.7 1555.7 265. 2266.1 251.3228 216. 2222.243 2734.2 High resolution 828. 1263.2 1698.4 2133.6 2568.8 34. Mass () Isotopic distribution Isotopic distribution Mass resolution.1% vs. 1 ppm Symbol Mass Abund. Symbol Mass Abund ------ ---------- ------ ------ ----------- ------- C(12) 12. 98.9 C(13) 13.3355 1.1 N(14) 14.374 99.63 N(15) 15.19.37 O(16) 15.994915 99.76 O(17) 16.999131.38 H(1) 1.7825 99.99 H(2) 2.1412.15 S(32) 31.97272 95.2 S(33) 32.971459.75 Mass resolution Mass resolution 2 1. FWHM.7 FWHM.5 FWHM Full width 1.3 FWHM.2 FWHM.1 FWHM Half mass 2

1 524.3 1 262.6 95 95 9 9 85 85 Relative Abundance 8 75 7 65 6 55 5 45 4 Singly charged Ion: Distance between Peak and Isotop 1 amu = 1. amu Relative Abundance 8 75 7 65 6 55 5 45 4 Doubly charged Ion: Distance between Peak and Isotop.5 amu =.5 amu 35 35 3 25 2 15 525.3 = 1. amu 3 25 2 15 263.1 =.5 amu 1 5 526.2 1 5 263.6 52 521 522 523 524 525 526 527 528 529 258 259 26 261 262 263 264 265 266 267 Resolution: Example Peptide Mw 2129.64, Ion 4 M ultiply charged myoglobin ions from ES I Intens. x1 5 4 2 Intens. x1 5 533.46 531 532 533 534 1..5 532.62 532.85 533.9 533.33 533.61. 531 532 533 534 Resolution.6 Resolution.2 1 9 8 7 6 5 4 3 2 1 616.2 738.1 77.3 771.5 893.3 848.6 88.2 998.2 942.9 16.5 M 1 1131.1 1211.9 M 2 135. (M 2-1.8) /M 1 -M 2 = Z 1 (Z 1 M 1 )-(Z1.8) = Mwt 1413.5 1541.9 1696. 131.9 1884.2 1428.7 1563. 182.8 1888.9 6 8 1 12 14 16 18 2 Deconvoluted myoglobin spectrum 16951. 1 9 8 MALDI-DE-RE-TOF MS tryptic digest of BSA 1 8 1265.638 1394.7169 % Intensity 1 9 8 7 6 5 4 3 9.9E3 7 6 2 6 5 4 3 2 1 15931. 1614. 16392. 16582. 16784. 1788. 1728. 17562. 1783. 17995. 16 162 164 166 168 17 172 174 176 178 18 mass % Intensity 4 2 1252.6472 1 1757.8374 87.442 1299.613 191. 1918.8 1927.6 1936.4 1945.2 1954. Mass () 193.53 1742.878 141.718 1787.7116 262.77 95.4584 2523.221 183.582 2848.3 1778.565 2285.1 2467.1695 848.2 199.5 1859.9261 1364.7 1555.7 265. 2266.1 251.3228 216. 2222.243 2734.2 828. 1263.2 1698.4 2133.6 2568.8 34. Mass () 3

Ion fragmentation with Mass Spectrometry Tandem MS or MS/MS One set of ions (one value) is selected from a mixture of ions; These ions are fragmented; the fragments are measured. Int. x1 7 4 2 Ab. 1 5 HPLC-ESI-autoMS/MS 4. 5. Time [min] MS, Time=4.42min 545 634 1 2 3 4 5 6 Ab. 1 MS/MS(634), Time=4.458min 373 545 5 249 376 TIC 563 1 2 3 4 5 6 H I O O I H N O HO 634 MS/MS H O O HO 563 I OH I Peptide fragmentation with MS/MS Outline Mass spectrometry, general introduction What is a mass spectrum 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 What are the constituents of a mass spectrometer How the instruments look like How are mass spectra produced? Ions are produced in the source and are transferred into the mass analyser Generic description of a mass spectrometer Atmosphere Vacuum System They are separated according to their mass/charge ratio in the mass analyser (e.g. Quadrupole, Ion Trap, Time of Flight) Sample Inlet Ionisation Method Mass Analyser Detector Data System Ions of the various values exit the analyser and are counted by the detector 4

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 7eV. Sample heated. Reproducible, structural information - sample must be volatile and stable, molecular ion often abscent mass range: < 1Da 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: < 1Da DCI: Desorption CI : CI on a heated filament rapid, simple - reproducibility mass range <15Da 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 <1Da 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 <2-3Da 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 <1Da 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 ~3-6Da 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 3-13Da 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, 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 <2 Da 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 <5 Da Electrospray Ionization (ESI) Matrix Assisted Laser Desorption/Ionization MALDI UV or IR laser S S S droplet S SH MH S S Smaller droplet SnH S S S MH SH 2 S MH2 Coulomb explosion: Clusters and ionic species pump MH 2 MH2 Ions sample target grid Membrane, gel or metal Matrix Analytes Modif. From Alex Scherl 5

Matrix Assisted Laser Desorption/Ionization MALDI Mass Analyzers Mass Spectrometers separate ions according to their mass-tocharge () ratios Magnetic Sector Quadrupole Ion Trap Time-of-flight Hybrid- Sector/trap, Quad/TOF, etc. Time of Flight (TOF) mass analyzer Ion source High vacuum flight tube Ion source High vacuum flight tube Detector Detector time 1 time 2 time 3 Small ions are faster than heavy, and reach detector first Reflectron Quadrupole mass analyzer Ion Trap 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. 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 Advantages - MS/MS/MS.. - High sensitivity full scan MS/MS 6

Linear Trap Hybrid Mass Spectrometers 3D Trap Full Scan Sensitivity MS 3 (or greater) Tandem in TIME Linear Trap Tandem in SPACE & TIME QqQ MRM Sensitivity Neutral Loss Precursor Scan Tandem in SPACE Full Scan Sensitivity MS 3 MRM Sensitivity Neutral Loss Precursor Scan Novel Scan Types From K Rose 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 Ion Motion in Orbitrap Only an axial frequency does not depend on initial energy, angle, and position of ions, so it can be used for mass analysis The axial oscillation frequency follows the formula w = k m / z w k = oscillation frequency = instrumental const. =. what we want! A.A. Makarov, Anal. Chem. 2, 72: 1156-1162. A.A. Makarov et al., Anal. Chem. 26, 78: 2113-212. 7

Ions of Different in Orbitrap Large ion capacity - stacking the rings Fourier transform needed to obtain individual frequencies of ions of different How Big Is Orbitrap? Outline Mass spectrometry, general introduction What is a mass spectrum What are the constituents of a mass spectrometer How the instruments look like MS instruments used in Proteomics ESI-Triple quadrupole MS ESI-Q-TOF MS ESI-Ion-trap MS ESI-Q-trap MS ESI-FTICR MS ESI-LTQ-Orbitrap SELDI MS MALDI-TOF MS MALDI-TOF-TOF MS MALDI-Q-TOF MS MALDI-Ion-trap MS MALDI-FTICR MS MALDI-TOF-MS MALDI-TOF MS: illustrated examples MALDI sample plates LASER Voyager DE-PRO Applied Biosystems Voyager STR Applied Biosystems I Autoflex Bruker Reflex III Bruker Micromass 8

(ESI) - Triple quadrupole MS Q2 is Non-Linear Collision Cell A B C Q2 collision cell Product Ion Scan (3Q) Q1 Mass selection Ion C Cgas Products Q3 Full Scan Products Q Q1 Q2 Q3 D ESI Probe ions in source Q1 only transmits ion C Fragment the Ion C Q3 Scans for products Square Rod Ion Transmission to Analytical Quads Hyperbolic, high precision quadrupoles Electron Multiplier, Detection System #1 scan mode used in proteomics as it generates primary structure (sequence) information The observed signal is a result of the mass-analyzed product ions derived from a mass-selected precursor ion: low energy collisions Typical product ion spectrum of a peptide fragmented under low energy conditions 4 8 12 A B D ions in source C Q1 Mass selection Q2 Q3 Selected Ion Monitoring (3Q) Ion C Q1 only transmits ion C Q2 only transmits ion C Ion c Q3 only transmits ion C High sensitivity, due to short mass scanning range (can switch) We know what we are looking for (ion C and standard) For complex samples (plasma) it is common to have multiple peaks Ions A-F Neutral Q1 Pass A-H Loss 2 O Scan Q2 collision Mode cell (3Q) Q3 Pass A Ion A-H 2 O Ion B Ion C Ion D Ion E Ion F Scans across mass range (NB A-bond-HOH, not minus) A-H O A 2 A H 2 O Fragment ions one at a timescans across mass range at 18 amu lower than Q1 (linked scan) A The setup of a TSQ can easily be understood: Q1 and Q2 are scanned with an offset of the neutral loss n to be detected. Thus, Q1 passes (M), M fragments in Q2 by loss of n and Q2 passes (M)-n. One important analytical application of CNL scans is their use in SRM (selected reaction monitoring) A B D C Single/Multiple Reaction Q1 Mass selection Monitoring Q2 collision (3Q) cell Ion C SRM/MRM Cgas C1C2C3 Q3 Mass Selection Ion C2 Ions A-F Q1 Mass selection Q2 collision cell Q3 pass only 79 Precursor Ion Scan Mode (3Q) Ion A-PO 4 Ion B Ion C Ion D Ion E Ion F PO 3-79 Ions in Source Q1 only transmits ion C Fragment Ion C Q3 only transmits ion C2 MRM/SRM is performed by specifying the parent mass(es) of the compound for MS/MS fragmentation and then specifically monitoring for (a single) fragment ion(s) MRM/SRM can generate fragment ions that can be measured and quantified from very complicated mixtures (e.g. plasma) SRM typically contain a single peak: ideal for sensitive and specific quantitation Scans across mass range Fragment ions one at a time Transmits only 79 In the precursor ion scan, the instrument looks for a predefined product ion and associates it back to the precursor ion it originated from Example in a negative ion mode: The MS can transmits of -79 (a negatively charged phosphate ion), and identify which peptide ion lost the phosphate ion, thereby identifying it as a phosphopeptide The mass spectrum reveals all precursor ions that fragment to yield a common product ion 9

ESI ESI-Q-TOF MS, MS mode Q q TOF ESI ESI-Q-q-TOF, MS/MS mode Q q TOF I Ion 1 Ion 2 Ion 3 I Fragment 2 Fragment 1 Fragment 3 Mod. From Alex Scherl Mod. From Alex Scherl Esquire-LC Ion Optics Q-TOF MS HPLC inlet Capillary Skimmers Octopole End Caps Q Star XL Hybrid Applied Biosystems BioTOF-q Bruker Ion trap MS qtof-ultima Micromass Nebulizer Lenses Ion Trap Ring Electrode LCQ Deca XP Finnigan Esquire 3 Bruker nanolc-esi ESI-Q-TOF Principe of LC-MS/MS Column C18 75 mm Q-Tof = 957.6 time 27.4 min : peak at = 957.6 QIIEEDAALVEIGPR Q96DH1 HPLC Autosampler/Injector 1

Sample Plate 47 TOF/TOF from Applied Biosystems Laser Reflector Detector Reflector MALDI TOF-TOF: TOF: MS/MS Mode CID Cell TIS intensity Mass () Source 1 Source 2 V 1 V 2 source TOF 1 collision cell TOF 2 TOF 1 TOF 2 Timed ion selector operation Bruker UltraFlex TOF-TOF from ion source TIS Deceleration m 12 3 m 12 3 m 1m m 2 3 m 1 m 2 m 3 m 1 m 2 m 3 m 1 m 1 2 2m3 m m 2 m 2 m 2 m m 2 m 2 m 2 m 2 2 31 m 13 TOF 1 - m 13 m3 m 1 m 3 m 1 to collision cell 5 V TTL Pulse V 95 V Switch down time calculated by low mass gate geometry TIS Single Gate V -95 V Switch up time calculated by high mass gate geometry Timed ion selector operation from ion source TIS TOF CID - LIFT TOF 2 Switch down time calculated by low mass gate geometry Few ns Switch up time calculated by high mass gate geometry TIS CID Cell V 1 V 2 11

MALDI TOF-TOF MS AB 47 Proteomics Analyzer with Auto-loader nanolc-maldi MALDI-TOF-TOF Spotting robot Column C18 75 mm HPLC Autosampler/Injector MALDI plate TOF-TOF from Bruker: the Ultraflex Off-line MALDI MS (MS/MS) Bruker APEXIII ElectroSpray MALDI EI/CI Switchable CF-FAB, CF-SIMS GC Interface LC Interface Pulsed valve for MS/MS IRMPD FTMS can provide very high resolution, 1 6, which its main advantage compared to other mass spectrometers. Mass accuracy <1ppm in MS and MS/MS mode Operating mass range (APEX 7e) of 18-66 Daltons Q Trap (Quadrupole linear trap) The Q-trap MS linear-trap MS Q-trap Applied Biosystems and MDS Sciex LTQ-XL Thermo Fisher Scientific 12

LTQ Orbitrap Operation Principle 1. Ions are stored in the Linear Trap 2.. are axially ejected 3.. and trapped in the C-trap 4.. they are squeezed into a small cloud and injected into the Orbitrap 5.. where they are electrostatically trapped, while rotating around the central electrode and performing axial oscillation LTQ-Orbitrap The oscillating ions induce an image current into the two outer halves of the orbitrap, which can be detected using a differential amplifier Ions of only one mass generate a sine wave signal From Thermo Additional info on MS http://www.spectroscopynow.com/ http://www.ionsource.com/ http://www.asms.org/whatisms/index.html 13