C7895 Spectrometry of Biomolecules Schedule of lectures

For schedule, please see a separate file with the course outline. Jan Preisler Consulting The last lecture. Please contact me in advance to make an appointment. Chemistry Dept. 312A14, tel.: 54949 6629, [email protected]

This material is just an outline; students are advised to print this outline and write down notes durin the lectures. The material will be updated during The course is focused on of biomolecules, i.e. the semester. techniques MALDI and ESI, modern mass analyzers, such as time-of-flight MS or traps and bioanalytical applications. However, the course covers much broader area, including inorganic ionization techniques, virtually all Supporting study material: types of mass analyzers and hardware in mass spectrometry. • J. Gross, Mass Spectrometry, 3rd ed. Springer-Verlag, 2017 • J. Greaves, J. Roboz: Mass Spectrometry for the Novice, CRC Press, 2013 • Edmond de Hoffmann, Vincent Stroobant: Mass Spectrometry: Principles and Applications, 3rd Edition, John Wiley & Sons, 2007

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Content

I. Introduction 1

II. Ionization methods and sample introduction

III. Mass analyzers

IV. Biological applications of MS

V. Example problems

Introduction to Mass spectrometry. Brief History of MS. A Survey of Methods and Instrumentation. Basic Concepts in MS: Resolution, Sensitivity. Isotope patterns of organic molecules. Ionization Techniques and Sample Introductin. Electron Impact Mass spectrometry of biomolecules 2018 3 Ionization (EI). (CI) 3 4

I. Introduction Study Material

• Information sources about mass spectrometry Lecture notes • Brief history of mass spectrometry, a survey of methods and Advice: please take notes, but do not copy the slides; the English slides will instrumentation be provided at the end of the semester. The Czech slides can be found on the internet: http:\\bart.chemi.muni.cz • Basic concepts of mass spectrometry Additional literature • Isotope patterns of organic molecules. • Robert J. Cotter: Time-of-Flight Mass Spectrometry - Instrumentation and Applications in Biological Research, American Chemical Society, 1997. • Richard B. Cole et al.: Mass Spectrometry - Fundamentals, Instrumentation & Applications, John Wiley & Sons, Inc., 1997.

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1 Additional Sources of Information Ionization Techniques and Sample Introduction

Internet (GD) Textbook http://www.ms-textbook.com/ Electron impact ionization (EI) Comprehensive information source www.spectroscopynow.com Chemical ionization (CI) Laboratories, e.g. www.mpi-muelheim.mpg.de/stoecki/mass_server.html Field ionization (FI) Inductively coupled plasma (ICP) Protein Prospector prospector.ucsf.edu (FAB) Proteometrics - PROWL www.proteometrics.com Secondary ion mass spectrometry (SIMS) Etc etc. (TSI) Ionspray (IS) Specialized journals Elektrospray (ESI) International Journal of Mass Spectrometry Plasma Desorption (PD) Journal of Mass Spectrometry Laser Desorption (LD) Journal of the American Society for Mass Spectrometry Matrix-assisted laser desorption/ionization (MALDI) Mass Spectrometry Reviews Coupling of separation and mass spectrometry (on-line, off-line, microdevices) Rapid Communications in Mass Spectrometry

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Mass MS Applications

Ion optics. Simulation of ion movement, Simion Analysis of biological compounds: Energy analyzers • Proteins, peptide mapping, protein databases, new methods (ICAT) Magnetic sectors • Peptide analysis (disulfide bonds, post-translational modifications) Quadrupole filter • Nucleic acids Ion cyclotron with Fourier transformation (ICR-FT-MS) • Saccharides (IT), linear trap (LT) Time-of-flight mass (TOFMS) New mass spectrometers: , TOF-TOF, LIFT-TOF Analysis of synthetic polymers (MS/MS, MSn) Collision induced dissociation (CID) and more… Surface induced dissociation (SID) In source and post source fragmentation (ISF and PSD) Principles of vacuum instrumentation Detectors a detection electronics Chromatography - MS (on-line, off-line, in-line, microdevices)

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Brief History of Mass Spectrometry Glow Discharge Ionization

1803 Dalton atomic theory 1880’s Crookes: glow discharge “mass consists of atoms; all atoms o of a kind have the same mass” glowvrstv dischargea iontu … not really: isotopes…

U ~ 700 V DC Proof of existence of isotopes: - + + - viditvisibleelný n edischargegativní výboj + - I ~ 1 mA - optical spectroscopy: a slight shift - + - p ~ 0.2 Pa + of spectral lines ... requires a very high quality instrument + + * - MS: easy determination H2 + H2 → (H2 ) + H2 * (H2) → H2 + 4 h

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2 The First Mass Spectrometer The First Mass Spectrometer

1911 J. J. Thompson: Parabola MS (Phil Mag. 1911, 21, 225) Photographic plate as a detector: 20Ne and 22Ne lines “Rays of positive electricity” 1913

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vvacuumakuová pumppumpa 22Ne+ 20Ne+ Glow discharge in Ne at 1 Torr, hollow cathode, magnet

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Magnetic Sector with Energy Analyzer But the History of MS Goes on ...

1919 F. W. Aston: Mass Spectrograph (Phil. Mag. 1919, 38, 209) 1940 C. Berry: Electron impact ionization (EI) for ionization of organic Magnetic sector with electrostatic energy analyzer compounds

1950-70 MS applied mostly in structural analysis of organic compounds (-) sekineticlekce kienergynetické anmassalýza (+) eanalysisnergie r = f(m/z) hanalysismotnosti 1980+ Analysis of heavy molecules due to new ionization techniques: (+) FAB, PD, ESI a MALDI + + + detection detekce Today MS for qualitative,structural and quantitative analysis (-) + + + Wide scale of commercial mass spectrometers available ziondroj B source štèrbina + + + MS necessary for analysis of organic and biological molecules extrakce slit extraction, Biospectrometry a ffocusingokusace Abundance of most natural isotopes determined by 1930 In Nobel prize ceremony lecture, 1934: “MS is dead, everything's done …”

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Basic Concepts of Mass Spectrometry

Mass spectrometer Ion signal vs. m/z instrument, in which are formed from analytes and their mass-to- charge ratio is analyzed Ion signal charge, current, often converted to voltage, arbitrary units Components of mass spectrometer signal normalization: intensity of the dominant peak = 100% 1. chamber (contains device for sample introduction, ion optics) 2. Mass analyzer (ion optics, electrodes, magnets, detector) normalization of ion signal 3. Vacuum pumps (rough, high and ultrahigh vacuum) %Int. 100 4. Control and data processing unit, software 90

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10 0 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500 Mass/Charge Mass spectrometry of biomolecules 2018 17 Mass spectrometry of biomolecules 2018 18 17 18

3 Mass Spectrum Selected Powers in Mass Spectrometry

Mass, m Mass resolution A measure of separation of two adjacent peaks. a.m.u., u, Da (Dalton), molecular weight number of atom mass units numerically equivalent to molar weight Two definitions: m/z ... mass-to-charge ratio, Th (Thomson) 1. FWHM (full width at half maximum), R = m/m 2. Max. mass (m/z), at which two adjacent peaks with a unit mass difference may be resolved. Number of charges, z: Ion energy, W number of elemental charges of an ion Instead of Joule (J) use electronVolts (eV) and atoms or ions rather than usually ±1 moles exceptions, e.g. electrospray-generated ions: |z| >> 1 1 eV = 1.6 x 10-19 J simplicity: acceleration voltage = 100 V, charge = 1 ... W = 100 eV Pozitive and negative ions, not cations and anions. can be easily compared with , bond energy, photon energy...

Mass spectrometry, not spectroscopy. Pressure, p 1 atm = 760 Torr = 101 325 Pa = 1.01325 bar = 14.70 PSI

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Abbreviations Abbreviations

m mass of ion, atom, molecule (u, a.m.u., Da) n numerical concentration (m-3) z number of charges; (-) I current (A), flux (m-2), intensity (-) m/z mass-to-charge ratio (Th, Thomson) T absolute temperature (K) -19 e elemental charge (1.6x10 C) p pressure (Pa, Torr) U voltage (V) R resolution (-) E intensity of electric field (V/m, N/C) f frequency (Hz) W energy, labor (eV, J) -1 v ion velocity (m/s) w angular frequency (rad/s, s ) r curvature radius (m) a direct proportion L path (m) LD detection limit, also LOD, limit of detection (mol, g, M) l mean free path of a molecule S/N signal-to-noise ratio t time (s) RSD relative standard deviation s collision diameter (m) s2 collision cross-section (m2) m reduced mass (a.m.u., Da, kg)

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Isotope Patterns of Organic Molecules Isotope Patterns of Organic Molecules

Carbon isotopes: 99% 12C, 1% 13C Relative abundance of molecules (%): 12 C60: C60 100 12 13 Pattern as a function of number of carbon atoms in the molecule: C59 C 66 12 13 12 13 C: 99% C 1% C C58 C2 21 12 12 12 13 13 13 12 13 C2: 98% C C 2% C C 0.01% C C C57 C3 4.6 12 12 12 12 12 13 12 13 13 -4 13 13 13 12 C3: 97% C C C 3% C C C 0.04% C C C 10 % C C C C100: C100 100 12 13 C99 C 110 12 13 Binomic formula C98 C2 60 12 13 Relative abundance of the light isotope, a C97 C3 22 Relative abundance of the heavy isotope, b (normalized with respect to the monoisotopic molecule /only 12C/ = 100 %) Number of atoms, n E.g. for n = 2: (a+b)2 = a2 + 2ab + b2 With increasing number of carbon atoms, n, the relative intensity of the monoisotopic form decreases, the monoisotopic peak is not dominant any Monoisotopic molecule contains given atoms in form of a single isotope. more and intensities of other isotopic forms are comparable (wide In the case of biomolecules usually carbon atoms in the form of 12C. envelope) ... see examples below.

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4 Practical Impact of Isotope Abundance Molecular formula: C30H45N6O6S Resolution: 20000 at 50% %Int. - Decrease of sensitivity 617.3 - High resolution R necessary at high m/z for correct determination of m/z 100

+ Use of isotopic internal standards. The best internal standards. 80

Example: 60 5 peptides/proteins with relative abundance of elements C : H : N : O : S = 30 : 45 : 6 : 6 : 1 40 618.3 R = 20 000

C H N O S C H N O S 20 30 45 6 6 60 90 12 12 2 619.3 C90H135N18O18S3 C180H270N36O36S6 (also shown for more R) 0 620.3 621.3 622.3 623.3 C270H405N54O54S9 C360H540N272 O272S12 616 617 618 619 620 621 622 623 624 625 C900H1350N180O180S30 C1800H2700N360O360S60 Mass/Charge

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Molecular formula: C60H90N12O12S2 Resolution: 20000 at 50% Molecular formula: C90H135N18O18S3 Resolution: 20000 at 50% %Int. %Int. 1234.6 1852.9 100 100 1851.9

80 1235.6 80 1853.9

60 60

40 1236.6 40 1854.9

20 1237.6 20 1855.9

0 1239.6 0 1857.9 1234 1236 1238 1240 1852 1854 1856 1858 Mass/Charge Mass/Charge

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Molecular formula: C180H270N36O36S6 Resolution: 20000 at 50% Molecular formula: C270H405N54O54S9 Resolution: 20000 at 50% %Int. %Int. 3705.9 5559.8 100 100 5560.8 3704.9 80 80 5557.8 3707.9 5561.8 60 60

3708.9 5556.8 5562.8 40 3703.9 40

3709.9 5563.8 20 20 3710.9 5555.8 5564.8 5566.8 0 3712.9 0 5569.8 3704 3706 3708 3710 3712 3714 5555 5560 5565 5570 Mass/Charge Mass/Charge

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5 Molecular formula: C360H540N272O272S12 Resolution: 20000 at 50% Molecular formula: C900H1350N180O180S30 Resolution: 20000 at 50% %Int. %Int. 13414.4 18533.4 100 100 13412.4 18530.4 18535.4 80 13416.4 80 18536.4 13411.3 18529.4 60 13417.4 60 18537.5 18528.3 13410.3 13418.4 18538.5 40 40 18527.3 18539.5 13419.4 13409.3 18526.3 20 13420.4 20 18541.5 13408.3 13422.4 18524.2 0 0 18544.6 13405 13410 13415 13420 13425 18520 18530 18540 Mass/Charge Mass/Charge

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Molecular formula: C1800H2700N360O360S60 Resolution: 20000 at 50% Molecular formula: C180H270N36O36S6 Resolution: 500 at 50% %Int. %Int. 37066.0 3706.6 100 100

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m/z ~ 12 m/z ~ 9 40 40

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0 0 37050 37060 37070 37080 3695 3700 3705 3710 3715 3720 Mass/Charge Mass/Charge

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Molecular formula: C180H270N36O36S6 Resolution: 2500 at 50% Molecular formula: C180H270N36O36S6 Resolution: 5000 at 50% %Int. %Int. 3706.2 3705.9 100 100

80 80 3704.8 3707.9 60 60

m/z ~ 4.5 3708.9 40 40 3703.8

3709.9 20 20 3711.0 0 0 3714.0 3718.0 3695 3700 3705 3710 3715 3720 3695 3700 3705 3710 3715 3720 Mass/Charge Mass/Charge

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6 Multiply-charged ions Molecular formula: C180H270N36O36S6 Resolution: 10000 at 50%

%Int. z+ 3705.9 • Typical example: multiply-charged ions [M+zH] by electrospray 100 Bell-shaped envelope • The gaps between peaks are not equidistant (contrary to isotope pattern). 80 3704.9 3707.9 60 S 3708.9 40 3703.9 m/z 3709.9 Example: 20 A small protein with M.W. = 10 000 Da 3710.9 z 4 5 6 7 8 9 10 0 3713.9 3717.9 m/z 2501 2001 1668 1429 1251 1112 1001 3695 3700 3705 3710 3715 3720 Mass/Charge

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What Can Be Said about This Spectrum? II. Ionization Techniques and Sample Introduction

sample (atm. pressure) → sample (vacuum) Note: This is a portion of mass spectrum of a single organic compound. m/z values were determined with accuracy ~ 0.1. Unwanted phenomena • pressure increase in the ion source • cooling and freezing of solvent due to solvent evaporation • adsorption of compound (e.g. water) on the walls of the ion source chamber S 1000.7 1000.3 Classification of samples acc. to the state 1000.0 1001.0 • Fluid samples gaseous liquid (liquid analyte, dissolved analyte) • Solid samples volatile (usually light compounds) m/z nonvolatile (polar, heavy, polymer compounds)

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Sample Introduction Electron Impact Ionization, EI

Off-line vent atmospheric pressure Classical ionization technique. ion source On-line (vacuum) Electrons emitted from a heated filament are accelerated using a medium voltage. c Electron energy, W(e-) = acceleration potential x charge (1). probe sample probe Typical energy: 70 eV. oc vent seal +15 V to pump chamber ABC+ Number of introduced samples × • 1 sample e- • more samples inlet of gaseous sample ABC heated • in a queue, sample series (discrete samples or continual flow) orthogonally oriented to filament the electron beam (×) • in paralel -55 V

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7 Mechanism of EI Mechanism of EI

Interaction of electron with analyte molecule ABC: Appearance energy (AE), at which the fragments AB+ appears, does not e- (fast) + ABC → ABC+ + 2 e- (slow) have to be higher than H(ABC)! Total equation, ABC → ABC+ + e- Ionization energy ABC, H(ABC) ABC → ABC+ + e- ABC → AB+ + C + e- Threshold energy AB+, AE(AB+) is characterized by ionization energy ABC, H(ABC). AE(AB+) = H(ABC) + D(ABC+) Dissociation energy ABC+, D(ABC+) Ions ABC+ with energy excess can undergo fragmentation: (ABC+)* → AB+ + C, A + CB+ etc Absorption of electron during travel through the analyte Reduction of electron flux, dI during the travel through infinitesimally thin Fragmentation extent depends on electron energy, E(e-) and on the analyte analyte layer: structure: dI = -acIdx, a) W(e-) ~ ionization potential  production of molecular ions. after integration: Ionization potential of simple organic molecules ~10 – 12 eV. -acx I = Io e . b) W(e-) >> ionization potential  fragmentation. I electron flux (A) Type of fragmentation depends on analyte structure; compounds of similar c concentration of ABC, (cm-3) (c = p/RT) structure have similar fragmentation spectra. x layer thickness (cm) Interpretation of spectra. Spectral libraries (> 100 000 spekter). a cross-section (cm2) … analogy of e coefficient in the Lambert-Beerově law Mass spectrometry of biomolecules 2018 43 Mass spectrometry of biomolecules 2018 44 43 44

Chemical Ionization (CI) Mechanism of Ion Formation at CI (cont.)

20th of the 20th century A. J. Dempster 2b) proton transfer (more common) RH+ → R + H+ PA(R) proton affinity + + The same source as in the case of the EI plus an inlet for reagent gas ABC + H → ABCH - PA(ABC) RH+ + ABC → R + ABCH+ E = PA(R) - PA(ABC) Reagent gas (RH) E < 0: exothermic, preferred reaction CH4, butane, H2, NH3 etc. p(ABC) < 10-4 Pa E << 0: energy excess at ABCH+  fragmentation of ABCH+ p(R) ~ 0.1 Pa (l ~ 0.05 mm, many collisions in the source) structural analysis

Mechanism of ion formation E < 0, E → 0  ABC+ a ABCH+ dominate 1) production of RH+ + quantitative analysis e- (fast) + RH → RH+ + 2 e- (slow) + determination of molecular weight of ABC 2a) charge transfer + high ionization efficiency (ABC) RH+ + ABC → RH + ABC+ - no structural information

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Collisions during CI Negative Chemical Ionization

Mean free path The same ion source as in the case of EI plus an inlet for reagent gas Mean path, which a particle travels between 2 collisions l = (2ps2n )-1 Mechanism l(cm) = 0.66/p(Pa) (only the 1st approximation)

Number of collisions 1. Production of thermal (slow) electrons z = ps2(8kT/(pm))1/2 e- (fast) + RH → RH+ + 2 e- (slow) -1 -1 -1 m reduced mass, m = (m1 + m2 ) W(slow e-) ~ 3/2 kT s collision diameter T ~ 400 K  E ~ 0.1 eV s2 collision cross-section CI: 1015-16 collisions  high ionization efficiency (ABC) 2. Electron capture Comparison of CI vs. EI ABC + e- → ABC- + stronger signal Preferred by compounds with electronegative groups (PCB, NO - etc.) - higher noise 3 LD ~ pg + overall S/N higher (LOD of organic compounds ~ pg)

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8 Sample Introduction for EI/CI Glow Discharge. Inductively-Coupled Plasma (ICP). Field 2 Ionization/Desorption. Fast Atom Bombardment (FAB). 1. Gas/volatile liquid Secondary Ion Mass Spectrometry (SIMS). Photoionization (PI). Plasma Desorption (PD) Residual gas analysis open ion source in chamber with analyzed gas: p(chamber) = p(gas)

Eluent from a separation column (GC-MS) Problem: High flow rate of the carrier gas from classical GC columns a) fraction collection or split flow (splitter may mean reduction of sensitivity) b) continual interface (working without interruption) i) particle beam separator for analyte (ABC) enrichment column MS in the carrier gas requires M(ABC) >> M(carrier) ... use He as the carrier gas vacuum pump Mass spectrometry of biomolecules 2018 49 Mass spectrometry of biomolecules 2018 50 49 50

Sample Introduction for EI/CI (cont.) Sample Introduction for EI/CI (cont.)

3. Nonvolatile compounds ii) membrane interface - sample introduction through a membrane, - Large molecules separation of the carrier gas using a gas-permeable membrane - Molecules with many polar groups … many interesting compounds (proteins, DNA, saccharides) c) direct introduction from a capillary GC column lower gas load; lower flow rate of the carrier gas (He) a) Generation of volatile derivates and consecutive standard ionization (EI, CI). Useful for molecules with M < 1000 Da.

Example: esterification, RCOOH + CH3OH → RCOOCH3

2. Volatile, thermally stable solid sample b) Application of classical ionization in desorption arrangement. Sample Direct sample introduction on a probe (glass, ceramics, steel). After deposited on a probe is inserted into an ion source, in which electrons introduction of the probe, the sample begins to evaporate and undergoes interact directly with the sample in condensed state. ionization in the gaseous state. c) Other ionization techniques “Soft” ionization: production of molecular ions without their thermal decomposition: FAB, electrospray, laser desorption techniques

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Inorganic Ion Sources Glow Discharge

Glow discharge The first ion source (J. J. Thompson) Analysis of solid samples, usually conductive. Precise and relatively sensitive analysis: RSD ~1%, LOD ~1 ppb. Discharge in Ar (p ~100 Pa): Ar+ ions sputter metal atoms (M) from a Inductively coupled plasma sample plate and ionize them later ... M+ ions are formed.

Ar Other techniques, e.g. laser desorption – also for organics, will be 100 Pa discussed later do MS, (-) 0.1 Pa cathode, (-) entrance of a sample mass analyzer plate (+) anode

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9 Thermal Ionization (TI) Thermal Ionization

filament M(g) Saha-Langmuir equation heated 0.1 n(M+)/n(M)  exp[(W - IE)/(kT)] filament Pa - to MS e Working function of metal (filament), W ~ 4 eV

M+(g)

Metal K Ca Fe Zn (+) (-) IE (eV) 4.6 6.0 7.8 9.4 (W – IE) (eV) -0.5 -1.5 -3.9 -5.4 Sample deposited on a filament; the filament is resistively heated. efficient weak Evaporation, atomization and ionization: MX (s) → MX (g) → M (g) + X (g) • Three filaments (with different temperature): Substitution of a single M (g) → M+ (g) + e- (filament) filament, which evaporates and ionizes sample too fast. - Generation of more stable ion flow (RSD only ~ 0.1 % !) - Useful for determination of isotopic abundance.

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Inductively Coupled Plasma, ICP MS ICP

plasma coil 101 kPa 100 Pa 0.1 Pa 101 kPa 100 Pa torch sample ++ + ++ aerosol ++ + Plasma + Ar, 1L/min + + ions + ++ radial flow, Ar, series of apertures + + atoms + 10 L/min (differential pumping) +

1. Desolvation of MX (aq, aerosol) 2. Evaporation of MX (s) • Usually 3 vacuum stages (differential pumping). + 3. Atomization of MX (g): dissociation to M(g) and X (g) • Very efficient ionization, n(M )/n(Mtotal) = 90 – 100%. 4. Ionization to M+ (g) • Ions with a single charge prevails. • Non-equilibrium system.

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Differential Pumping ICP

Supersonic jet Hot plasma (5000 K) streams via an aperture (slit) into a chamber and 101 kPa 1k Pa 100 Pa 1 Pa expands at supersonic velocity. Random movement of atoms at the atmospheric side is characterized by wide kinetic energy distribution (5000 K) and relatively low translational velocity. The atoms move at supersonic ion MS velocity with a very narrow kinetic energy dispersion … supersonic cooling source (~300 K). Distribution is later ruined by collisions with molecules of background gas (barrel shock, Mach disc).

Efficient ionization pump 1 pump 2 pump 3 90 - 100 % elements are ionized (very uniform ionization). Applicable for determination of isotopic abundance (low systematic deviation), elemental composition.

- frequently used concept in mass spectrometry Disadvantages - used for atmospheric ionization methods • not useful for structural characterization of analytes • interferences

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10 ICP Spectral Interferences in ICP

Detection limits Formation of molecular ions in ICP 1 ppt (quadrupole) 1. Plasma gas and reaction products (Ar+, Ar2+, ArH+, ArO+, ArC+, ArN+ etc.) 10 ppq (magnetic sector) 2. Sample or solvent (hydride ions, OH+, ClO+, NO+, CaO+, LaO+ etc.) for comparison: LD of ICP-AES and AAS ~ ppm - ppb + + + 3. Chemical ionization of background gas (H2O , H3O , CxHy etc.) ppm ppb ppt ppq Elimination of spectral interference million 106 billion 109 trillion 1012 quadrillion 1015 1. Mathematic corrections (e.g. using isotope distribution)

Interferences 2. Desolvation of aerosol (e.g. by freezing in liquid N2) 1. Non-spectral 3. Cold plasma (relative shifts of ionization degree) Shifts of ionization equlibria as a result of suppression by matrix, acids or 4. Collision cell (thermalization of ions, shifts of reaction equillibria) easily ionizable elements etc. 5. Mass spectrometer with high resolution

2. Spectral Isotopic ions Izobaric molecular ions

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Spectral Interference in ICP Field Ionization, FI

Examples of isobaric ions and required resolution Very high electric field intensity between sharp spires, E > 109 V/m Electron removal due to inner tunnel effect. Isotope Interfering ion Resolution 39K 38Ar1H+ 5690 40Ca 40Ar+ 71700 41K 40Ar1H+ 4890 44Ca 14N14N16O+ 970 12C16O16O+ 1280 52Cr 40Ar12C+ 2380 56Fe 40Ar16O+ 2500 75As 40Ar35Cl+ 7770 80Se 40Ar40Ar+ 9690

Note: higher resolution often means lower sensitivity.

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Desorption Ionization Techniques , FD

LDI Laser Desorption/Ionization H. D. Beckey, Int. J. Mass Spectrom. Ion Phys., 1969, 2, 500-503 1963 R. Honig FD Field Desorption Sample is blown (g) or deposited (l, g) on an emitter, a heated metal 1969 H. D. Beckey filament with a specially modified surface PD Plasma Desorption 1974 R. D. MacFarlane Analytes are formed in very high electric field between sharp spires, E > 109 V/m; electrons are removed by inner tunnel effect. FAB Fast Atom Bombradment 1981 M. Barber Often more ionization mechanisms: SIMS Secondary Ion Mass Spectrometry 1976 A. Benninghoven - thermal ionization - electrospray ... during deposition of liquid samples MALDI Matrix-Assisted Laser Desorption/Ionization 1988 M. Karas & F. Hillenkamp, K. Tanaka Applicable to analysis of organic analytes with M.W. < 2 kDa

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11 Secondary Ion Mass Spectrometry, SIMS Field Ionization. Field Desorption (Fast Ion Bombardment, FIB) … Ionization by ions primary ion beam W ~ 6 keV

to MS analyzer

probe with a sample, +3 kV

probe for liquid injection, FD • Primary ion beam may be scanned; the result is MS image of elements/compounds, analyte topography. Imaging resolution is higher than in the case of optical microscopy since the primary ion beam can be focused tighter, ~nm). • Products … mostly atoms and neutral molecules, also ions. Postionization helpful, e.g. photoionization using a laser. • Inorganic and organic analysis, also lighter biopolymers at the presence of matrix - surface of emitter ”matrix-assisted SIMS”. Source: Bernhard Linden Mass spectrometry of biomolecules 2018 67 Mass spectrometry of biomolecules 2018 68 67 68

Fast Atom Bombardment, FAB Generation of Fast Xe atoms for FAB

(+) 1. Generation of fast Xe+ ions

Fast Xe atoms 2. Conversion of Xe+ to Xe: W ~ 5 keV Xe+ (fast) + Xe (slow) → Xe (fast) + Xe+ (slow) Probe with a layer of viscous solvent and 3. Elimination (deflection) of Xe+ analyte Xe source (glycerol + ABC) (-) Xe source

(+) fast Ionization similar to CI (organic molecules, small peptides …). fast Xe Xe, Xe+ Xe+ Setup: off-line and on-line (flow probe; continuous flow FAB, CF-FAB). Max. m/z ~ 10 000 Da. LOD ~ 10 pmol or even ~ 1 pmol (CF-FAB) Xe+ (-) Usually z =  1. + + - + 5 kV Formed ions: ABCH , [ABC+K] , [ABC+H] , [ABC+N2] , fragments.

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CF-FAB MS/MS of Human Hemoglobin, chain A

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CF CF mod

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norm of a peptide peptide of a in

HK

HK MS FAB

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12 Photoionization, PI Photoionization Schemes

M + n h → M+ + e- Necessary condition: nh > IE(M) e- e- e- Photoionization types 1. Single photon ionization, SPI; n = 1 h h 2. Multiple photon ionization, MPI; n > 1 IE(M) • non-selective analysis useful for inorganic analytes h h h 3. Resonance multiphoton ionization (REMPI) n > 1 • if h = W (energy of electron transfer of M) SPI MPI REMPI • very selective and very sensitive determination • aromatic molecules, dyes, drugs

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Photoionization Plasma Desorption (PD)

Example of arrangement: LD-PI 1974 R. D. Macfarlane PI as a postionization for pro laser desorption (R. D. Macfarlane, R. P. Skowronski, D. F. Torgerson, Biochem. Biophys. Res. Commun. 1974, 60, 616.; R. D. Macfarlane, D. F. Torgerson 1. Desorption laser pulse 2. Desorption of plume (mostly neutrals) Science 1976, 191, 920.)

The first technique capable of ionization of heavy compounds (e.g. proteins).

+ - + - Radioactive californium 252Cf source. Energy of fission fragments ~MeV.

3. Ionization laser pulse 4. Ion extraction Impact of a fission fragment from the radioactive source on sample deposited on polyester foil may ionize analyte molecule.

Other fragment from decay of the same 252Cf flies in the opposite direction and triggers signal recording device. + - + -

Mass spectrometry of biomolecules 2018 75 Mass spectrometry of biomolecules 2018 76 75 76

Experimental Setup of Plasma Desorption Example of Plasma Desorption

Positive PD mass spectrum of ribonuclease A from bovine pancreas

(D. M. Bunk, R. D. Macfarlane Proc. Natl. Acad. Sci. USA 1992, 89, 6215-6219)

(R. D. Macfarlane, R. P. Skowronski, D. F. Torgerson, "New approach to the mass spectrometry of nonvolatile compounds", Biochem. Biophys. Res. Commun. 1974, 60, 616-621.) Mass spectrometry of biomolecules 2018 77 Mass spectrometry of biomolecules 2018 78 77 78

13 Plasma Desorption Characteristics Laser Desorption/Ionization (LDI). 3 Matrix-Assisted Laser Desorption/Ionization, (MALDI). Ionization of relatively large organic molecules (~ thousands Da , e.g. Spray : Thermospray (TS). Ionspray (IS). insulin, 5 735 Da). Electrospray (ESI). Desorption electrospray (DESI).

Formation of molecular ions, ion clusters and multiply-charged ions.

Disadvanatges - radioactive source. - time-consuming signal accumulation.

MALDI and ESI preferred for ionization of heavy analytes nowadays.

Mass spectrometry of biomolecules 2018 79 Mass spectrometry of biomolecules 2018 80 79 80

Laser Desorption Matrix-Assisted Laser Desorption/Ionization

Laser Desorption/Ionization, LDI After laser discovery in 6th decade of 20th century Initially for solid samples: R. Honig, Appl. Phys. Lett. 1963, 2, 138-139. MALDI Later for Organic compounds: M. A. Posthumus, P. G. Kistemaker, H. L. C. Meuzelaar, M. C. ten Neuver de Brauw, Anal. Chem 1978, 50, ESI 985.

Matrix-Assisted Laser Desorption/Ionization, MALDI Karas, M; Bachmann, D.; Bahr, U.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Proc. 1987, 78, 53-68. Tanaka, K.; Waki, H.; Ido, Y; Akita, S.; Yoshida, Y.; Yoshida, T. Rapid Commun. Mass Spectrom. 1988, 2, 151. Source: www.nobel.se October 9, 2002

Mass spectrometry of biomolecules 2018 81 Mass spectrometry of biomolecules 2018 82 81 82

MALDI Schematic (detail) 1987 Karas & Hillenkamp melitin nicotinic acid MALDI Gaseous m/z = 2845 phase

Laser pulse

1988 Tanaka Analyte lysozyme Matrix Co/glycerol m/z = 100 872 (heptamer) Sample layer

Mass spectrometry of biomolecules 2018 83 Mass spectrometry of biomolecules 2018 84 83 84

14 MALDI Schematic Principle of MALDI

1. Very short laser pulse, typical t ~ ns, max. ms. Molecules vaporize before they decompose.

Collisional cooling: conversion of Evib to Etrans .

laser pulse 2. Energy is absorbed mostly by matrix (M), not by analyte. e (matrix) >> e (analyte), c(matrix) >> c(analyte) Matrix → MH+, M+, M*, fragments, fragment ions. target with MS, usually Analyte, originally “dissolved” in matrix, vaporizes together with matrix. a thin layer TOFMS of sample 3. Matrix actively participates on analyte (ABC) ionization. Matrix is excited after absorption of one or more photons. U U 1 2 Dominant ionization mechanism is proton transfer: (U1 > U2 > 0) for + MH+ + ABC → M + ABCH+.

Mass spectrometry of biomolecules 2018 85 Mass spectrometry of biomolecules 2018 86 85 86

Requirements on Matrix (MALDI) Common Matrices for MALDI

1. Absorbtion at the wavelength of the used laser (UV, IR). sinapinic acid (SA) gentisic acid (DHB) 2. Formation of proper crystals with analyte (empiric rule). (3,5-dimethoxy-4-hydroxycinnamic acid) (2,5-dihydroxybenzoic acid) 3. Usually acid (efficient proton transfer to ionize na analyte). 4. Stability. Low volatility. Inert (no reaction with analyte).

Types of matrix aromatic acids (Karas & Hillenkamp) a-cyano-4-hydroxycinnamic acid (CHCA) 3-hydroxypicolinic acid (HPA) glycerol with addition of ultra fine cobalt powder (Tanaka) modified surface, e.g. Si - DIOS (Siuzdak), SELDI

Mass spectrometry of biomolecules 2018 87 Mass spectrometry of biomolecules 2018 88 87 88

Common Matrices for MALDI Common Matrices for MALDI

ferulic acid dithranol (DIT) nicotinic acid-N-oxide 2,-(4-hydroxy-phenlyazo)-benzoic acid (4-hydroxy-3-methoxycinnamic acid) (HABA)

trans-3-indoleacrylic acid picolinic acid (PA) 2',4',6'-trihydroxyacetophenone 2'-6'-dihydroxyacetophenone (THAP) (2-pyridine carboxylic acid)

Mass spectrometry of biomolecules 2018 89 Mass spectrometry of biomolecules 2018 90 89 90

15 Matrices - Applications Properties of Matrices

peptides < 10 000 CHCA, DHB CHCA: „hot“ matrix peptides, proteins > 10 000 SA, DHB for peptides with M < 10 000 Da useful for PSD (structure analysis) oligonucleotides < 3 kDa THAP nucleic acids > 3 kDa HPA synthetic polymers DHB, DIT, IAA DHB: „cold“ matrix carbohydrates DHB, CHCA, THAP universal use

Addition of “comatrices” (e.g. monosaccharides) may to improvements in crystallization, sample homogeneity, mass resolution, suppression of fragmentation etc.

Mass spectrometry of biomolecules 2018 91 Mass spectrometry of biomolecules 2018 92 91 92

Lasers in MALDI Influence of Laser Energy UV-MALDI 337 nm nitrogen laser (inexpensive and the most common) Significant dependence of the quality of MALDI mass spectra on laser 355 nm Nd:YAG (3xf) yttrium-aluminum garnet energy, more exactly on power density, PD (power per area). 266 nm Nd:YAG (4xf) 193 nm ArF ... fragmentation! Ion signal fragmentation Note: YAG lasers are more expensive, but their life expectancy is (ABCH+) much higher. The repetition rate of YAG lasers can reach kHz vs. Hz in the case of the nitrogen lasers. resolution decreases IR MALDI 2.94 mm Er:YAG laser 0 working region 10.6 mm CO2 laser PD

PDT

Mass spectrometry of biomolecules 2018 93 Mass spectrometry of biomolecules 2018 94 93 94

Influence of Laser Energy Accumulation of Spectra

Threshold power density, PDT … minimum value of laser power per area, Usually average of 10 – 1000 desorptions is recorded to increase signal- at which analyte peaks begin to appear in mass spectrum. to-noise ratio and reproducibility

+ n For PD > PDT : signal (ABCH ) = k.PD , kde n = 4 – 6. signal, S  n Small variation of power to a large variation in ion signal of ABCH+. noise, N  √n signal/noise, S/N  √n In practice, operator usually slowly increases energy during MALDI experiment, simultaneously moves target with sample and observes mass spectra from single laser shots. After the threshold power is established, the power is set approximately 10–30% above it and spectra are accumulated for 10-1000 laser pulses while the target is slowly being moved.

Mass spectrometry of biomolecules 2018 95 Mass spectrometry of biomolecules 2018 96 95 96

16 MALDI Mass Spectrum MALDI Characteristics

Model spectrum of 2 peptides, Pep1 a Pep2 with matrix M + one of the most ionization techniques applied in mass spectrometry of biopolymers (together with ESI) matrix ions, fragments Ion + + soft ionization of matrix and Pep1H signal analytes, adducts + Na+ Pep2H + simple spectra, usually z = +1 or z = -1 (for analytes with cluster electronegative groups) K+ ions Pep1Na + pulse ionization (predestined for coupling with TOF mass analyzers) + Pep2Na + 0 500 1000 1500 m/z + detection limits ~ amol (for small peptides - best case) + 2+ + [ABC+H] , [ABC+2H] , dimer [(ABC)2+H] adducts with alkaline metals and matrix [ABC-Na]+, [ABC+K]+, [ABC+MH]+ + fast sample preparation and analysis + fragment ions of matrix a analyte, cluster ions, e.g. [M2+Na] Matrix suppression – ideally only the peaks of analytes.

Mass spectrometry of biomolecules 2018 97 Mass spectrometry of biomolecules 2018 98 97 98

MALDI Characteristics MALDI Sample Preparation - uneasy quantitative analysis (inner standard needed) MALDI sample = analyte + matrix c(analyte) = 0.1-10 mM - search for the right spot on the target c(matrix) = 1-100 mM - not useful for low-mass analytes due to intensive background in target: steel, Al, synthetic polymers the region of low m/z (matrix, fragments and matrix clusters)

- mutual ion signal suppression detail MALDI vzorku

www.srsmaldi.com

Mass spectrometry of biomolecules 2018 99 Mass spectrometry of biomolecules 2018 100 99 100

MALDI: Sample Preparation Rules MALDI Sample Preparation Techniques

Recrystallization of matrix. dry droplet – deposit of mixed solution, dry at room temperature Fresh matrix solution. quick & dirty – mix solutions on target, dry at room temperature Selection of proper solvent (ACN, EtOH, MetOH, acetone, water). vacuum drying – speed up drying using reduced pressure pH(matrix) < 4 (adjustment with e.g. 0.1 % trifluoroacetic acid, TFA). fast evaporation – first deposit matrix layer in a volatile solvent Analyte has to be dissolved. overlayer – matrix layer, then layer of analyte with matrix sandwich – layers: matrix, analyte, matrix Purification of analyte prior to MALDI analysis. crushed crystals – matrix crystals crushed and new sample sol’n deposited Unknown analyte – preparation of a series of solutions with concentration of acetone redeposition – dissolve dried sample in acetone droplet and dry analyte in wide range. spin coating – deposition on rotating target Deposited samples are usually stable and can be stored (archived) slow crystallization – slow growth of crystals Thoroughful cleaning of the target electrospray deposition – electrospray aerosol sprayed on target modified targets – for concentration and more reproducible crystallization often using hydrophilic/hydrophobic interface

Mass spectrometry of biomolecules 2018 101 Mass spectrometry of biomolecules 2018 102 101 102

17 Micro Methods Influence of Salt Content MALDI MS of a peptide sample in the presence of Na and K salts - microtargets, piezoelectric pipetors, deposition from a capillary outlet - size of laser focus  size of sample  maximum sensitivity 0.6 high sample density on target

0.4 Signal Signal 0.2 PepH+ + sample: PepNa PepK+ 1 mm S 502 laser = = 0.25% 0.0 S 10002 laser: sample 0 200 400 600 800 1000 1200 50 mm m/z Sample desalting necessary (not a rule, sometimes ionization based on cationization with Na+, K+, Ag+ ...) Presence of salts  adduct formation , sensitivity 

Mass spectrometry of biomolecules 2018 103 Mass spectrometry of biomolecules 2018 104 103 104

Reduction of the influence of salts MALDI Perspective

• segregation on target ... selection of proper crystal for desorption MS analysis of large series of biological samples

• addition of acid (TFA, HCOOH, HCl), NH4 salts • peptide mapping for identification of proteins • target washing (salts are more soluble in water) (MALDI MS of products enzymatic protein digests) • catex on target • peptides, proteins, oligonucleotides, saccharides

• desalting prior to deposition: separation, dialysis, ZipTip (C18) desalting Micro methods 1. 2. H2O 3. 50% ACN ZipTip Coupling with separation techniques • advantage of sample archiving on MALDI target • recent availability of MS/MS spectrometers for MALDI peptide • complementar to ESI (various ionization efficiency for various (MALDI analytes) sample solution: target) peptide, Na+ Na+ (waste)

Mass spectrometry of biomolecules 2018 105 Mass spectrometry of biomolecules 2018 106 105 106

MALDI MS of Numerous Sample Series MALDI MS of a Digest: Identification of BSA

1439.47 1, 10, 96, 100, 384, 1536 ... 100 1639.51 2.2E+4 samples/target

80

1178.32

60 Signal 40

927.34 1193.37 20 1867.50 1567.36 1750.52 550.60 1682.50 2854.35 764.33 1249.361419.39 555.31 2612.34 1905.46 2358.28 3815.52 0 0 500.0 1200.0 1900.0 2600.0 3300.0 4000.0 384 samples/target m/z

Analyte: 1 pmol of tryptic digest BSA, desalted using ZipTipC18 Matrix: 10 mg/ml aCHCA in ACN/0.1% TFA : 70/30 Sample preparation: dried-droplet. MS: Voyager DE-STR Mass spectrometry of biomolecules 2018 107 Mass spectrometry of biomolecules 2018 108 107 108

18 Micro Methods Comparison of LDI and MALDI

(A) sample LDI MALDI processing and injection Ionization relatively hard soft (B) reactor with immobilized Sample only analyte analyte in excess of enzyme matrix (C) micropipetor (D) nanovials (300 x Max. m (Da) <2 000 106 300 x 20 mm) on MALDI target Typical analyte small organic peptides, (E) automated molecules, small MALDI-TOF MS proteins, peptides, synthetic DNA, polymers saccharides, synthetic polymers (Ekstrom, S., Onnerfjord, P., Nilsson, J., Bengtsson, M., Laurell, T., Marko- Varga, G. Anal. Chem. 2000, 72, 286-93) Mass spectrometry of biomolecules 2018 109 Mass spectrometry of biomolecules 2018 110 109 110

On-Line Ionization Techniques Thermospray, TS Atmospheric Pressure Ionization heated metal (also spray ionization techniques) capillary spray of (~ 200 ºC) droplets eluent from to mechanical LC or CE Common attributes pump (100 Pa) • Analyte: polar, often ionized and dissolved in solution. column • Heated capillaries and other elements of ion source (hundreds ºC) • Additional elements to increase ionization efficiency (electron beam, (-) electric arc) to MS • Differential pumping • Solution boils in the capillary tip, droplets are generated, then dry aerosol • Often after separation  2D separation (MS as the second dimension). and finally MH+ ions. Spectra similar as in the case of CI, molecular ions • Ionization process stages: prevail. Electrodes may be inserted into the source to further increase 1) formation of aerosol droplets ionization efficiency. 2) solvent vaporization • Electronegative compounds may form negative ions. (An electron source - 3) ion analysis can be inserted into the chamber to promote formation of negative ions M .)

• Use of volatile compounds, such as NH4Ac, to prevent capillary tip clogging.

Mass spectrometry of biomolecules 2018 111 Mass spectrometry of biomolecules 2018 112 111 112

Particle Beam, PB Particle Beam

Principle • Derived from the generator of monodisperse aerosol (R. C. Willoughby, R. F. Browner, Anal. Chem., 56, 1984, 2626-2631). • Similar to TSI and heated nebulizer. Additional beam of particles, usually He. Separator of He atoms from ions. • Additional EI source may be used to increase ion production. Results are EI spectra (with higher noise due to presence of ions and molecules of solvent).

Characteristics • Spectra similar as in the case of TS. More fragments. • Less sensitive than TS and ESI. • Useful for thermally stable, nonionic compounds with medium mass. Typical PB setup for GC and LC. (www.micromass.co.uk ) Mass spectrometry of biomolecules 2018 113 Mass spectrometry of biomolecules 2018 114 113 114

19 Heated Nebulizer Ion Spray, IS

heated element heated gas element ions extracted gas LC, CE discharge ions extracted through apertures LC, CE gas through into mass gas analyzer apertures into mass analyzer (+) (-) atmospheric N2 atmospheric pressure (drying gas) pressure N2 (drying gas)

• Spectra similar to CI spectra, usually capture of 1 proton ([M+H]+). • Low fragmentation (soft ionization). • Only positive ions penetrate through electrodes, negative are removed. • Suitable for molecules with medium masses (~2000 Da). • Formation of multiply charged ions [M+H]+, [M+2H]2+, [M+3H]3+, [M+4H]4+... • Structure? ... Additional collision cell needed (MS/MS). • Packed LC columns with a splitter, micro columns, capillary columns.

Mass spectrometry of biomolecules 2018 115 Mass spectrometry of biomolecules 2018 116 115 116

Electrospray (ESI)*, Nanoelectrospray+ ESI Principle

* Yamashita, M; Fenn, J. B. J. Phys. Chem. 1984, 88, 4451. * Yamashita, M; Fenn, J. B. J. Phys. Chem. 1984, 88, 4671. + Wilm, M. S.; Mann, M. Int. J. Mass Spectrom. Ion Processes 1994, 136, 167. + + + + Schematics + + + - + - + + + + + + + + tip (quartz, metallic, counter electrode with an - + + + + + + + + + - ++ + + + quartz, metal) aperture, "nozzle" (0 V) + + + + + + + + + - - + + + + + + + - + + + + + infusion, mLC, CE + - + + + + + k MS + + sheath liquid heated + "skimmer" (optional) 1–5 kV capillary + additional gas – atmospheric 100 Pa vacuum optional pressure + -

Mass spectrometry of biomolecules 2018 117 Mass spectrometry of biomolecules 2018 118 117 118

ESI Principle

• Formation of Taylor cone in electric field. Concentration of positive charge in the cone, destabilization of the meniscus and emission of droplets with excess of positive charge. • Volume reduction and increase of surface charge density of the droplets due to solvent evaporation. • Unsymmetrical fission of charged droplets (Rayleigh stability limit); original droplet loses ~15% of charge, but only 2% of volume. • Droplet size: mm → nm. Number of charges in a droplet: 105 → 10. (Note: Size of a macromolecule ~ nanometers.) • Formation of secondary ions in gaseous phase, secondary reactions in gaseous phase. • Ion transfer into the mass spectrometer. • No discharge; discharge is not desirable.

Mass spectrometry of biomolecules 2018 119 Mass spectrometry of biomolecules 2018 120 119 120

20 ESI Arrangement ESI Arrangement

• Additional (curtain) gas – N2 stream, heated capillary behind the entrance • Arrangement of needle and aperture (nozzle) aperture: better desolvation, reduction of cluster formation. ... separation of ions from ballast - on-axis • Optional coaxial stream of liquid through an additional capillary. - off-axis - diagonal (tilted) and orthogonal • Needle: i.d. < 100 mm, o.d. 100 mm – 1 mm, tip < 100 mm. - Z-spray

• Distance tip – counter electrode: 1 – 3 cm. Flow rate < 10 mL/min. • Voltage connection - through additional liquid (sheath flow) • Nanoelectrospray: smaller dimensions,without additional coax. liquid and - liquid junction forced flow, flow rate < 100 nL/min. - metallic or metallized needle tip (sheathless interface)

• Nanospray = registered commercial name

Mass spectrometry of biomolecules 2018 121 Mass spectrometry of biomolecules 2018 122 121 122

ESI Characteristics Generation of Multiply Charged Ions

• Very soft ionization suitable for biomolecules. + Higher z reduces m/z  mass analyzer with lower m/z limit can be used • Very high mass limit, M.W. ~ 106 (m/z much lower). Ionization of heavy for ions with very high M.W. polymers (also virus particle). + Two adjacent [M+nH]n+ peaks sufficient for determination of m and z: • Generation of multiply charged ions (m/z)n = (m+n)/n Bell-shaped envelope. Typical for ion spray and electrospray. (m/z)n+1 = (m+n+1)/(n+1) The gaps between adjacent peaks are not equidistant (in contrast to + Alteration of number of charges, z ? isotope pattern). • pH (solution): pH  z S pH  z or even negative charge promoted • b- emitter or other e- source (electron capture  z ) Example: - MS of mixture more complex (but can be evaluated). Single compounds m/z M.W. = 10 000 Da are present in several forms and give several peaks in the spectrum ... complex spectra, reduced sensitivity. z 4 5 6 7 8 9 10 - Satellite peaks, e.g adducts [M+Na]+, [M+K]+ etc. m/z 2501 2001 1668 1429 1251 1112 1001

Mass spectrometry of biomolecules 2018 123 Mass spectrometry of biomolecules 2018 124 123 124

ESI Factors Influencing ESI

• Application of electric field (nozzle-skimmer)  fragmentation in source. • Type of analyte • For structure analysis – additional chamber for collision dissociation after • Needle, spray tip (dimensions, arrangement) the first mass analyzer. • ESI signal dependent on analyte concentration, c(analyte); at very low • Voltage between needle and counter electrode concentrations dependent on the amount of analyte (nanoelectrospray). • Solution composition (solvents, additives, salts, ion-pair reagents) • Signal α c(analyte) (10-7 – 10-3 M), it reaches plateau at higher c(analyte). • ESI closely related to other API: • Flow rates of sample, sheath liquid and drying (curtain) gas e.g. APCI (Atmospheric Pressure Chemical Ionization) • Temperature of the entrance capillary (solvent acts as a reagent gas ... ionization similar to CI) - heated needle - zero voltage on needle - additional elements: - electrode for discharge in front of the entrance nozzle - piezoelectric element for better nebulization - nebulizer (ion spray sometimes called pneumatic ESI)

Mass spectrometry of biomolecules 2018 125 Mass spectrometry of biomolecules 2018 126 125 126

21 ESI – Some Rules Most frequent API techniques

• Use volatile buffers: • Most frequently used API techniques in organic analsis: ESI, APCI, APPI - CH COOH 3 • APCI (Atmospheric Pressure Chemical Ionization) - HCOOH - pneumatic nebulizer - TFA (trifluoroacetic acid) - reagent = solvent (mobil phase), additives ... analogy with CI + - NH4 salts of volatile acids - energy for reaction: • Keep salt concentration < 20 mM - electrode for corona discharge in front of the MS inlet • Avoid use of sulfates, phosphates etc. - heated ion source • Orthogonal or Z spray may partially help in the cases the rules mentioned - no voltage on the spray tip (in contrary to ESI) above cannot be applied. • APPI (Atmospheric Pressure Photoionization) • For positive ionization, pK (electrolyte) < pK (analyte) – 2 a a - pneumatic nebulizer • For negative ionization, pK (electrolyte) < pK (analyte) – 2 b b - UV lamp (e.g. krypton) for photoionization • Proper sample preparation = easier analysis - heated ion source, possibly additional elements desalting, removal of surfactant and other contaminants - no voltage on the spray tip (in contrary to ESI) - APLI: a laser is the excitation source

Mass spectrometry of biomolecules 2018 127 Mass spectrometry of biomolecules 2018 128 127 128

Applications of ESI, APCI and APPI DESI

A schematic diagram of applications Desorption electrospray ionization - electrospray in desorption arrangement ESI - interactions with surface - examples of applications: medications in tablets cocaine on banknotes metabolites on skins

weight APPI - a wide group of ambient techniques (DART, MALDESI, LAESI ...)

ecular APCI mol

polarity

Z. Takáts, J. M. Wiseman, B. Gologan, R. G. Cooks Science 306, 471 (2004) Mass spectrometry of biomolecules 2018 129 Mass spectrometry of biomolecules 2018 130 129 130

III. Mass Analyzers Analyzers: Ion Optics. Wein Filter. Energy Analyzer (E). Mass analyzers. Magnetic Sector (B). 4 • Ion optics basics. Simulation of ion movement

• Energy analyzer

• Mass analyzers

• Detection of ions and data acquisition

• Vacuum techniques

• Coupling of separation to MS. Microfabricated devices

• New techniques/instrumentation

Mass spectrometry of biomolecules 2018 131 Mass spectrometry of biomolecules 2018 132 131 132

22 Ion Optics Lorentz and Coulomb Forces

Analogy with light optics Lorentz force at magnetic induction B: slit slit lens lens F = ze ( v x B ) prism, grating W : energy analyzer, deflector mag. kin B m/z: mass analyzer mirror ion mirror Coulomb force at el. field intensity E: optical fiber ion guide

Fel. = zeE F v Total: Differences from light optics F = ze (E + v x B ) wavelength, l kinetic energy, Wkin mass/charge, m/z refraction index (const.) electric or magnetic fields (tunable, can be For simplicity only: altered even during experiment) F = zeE Intensity-independent space charge effects – mutual ion repulsion el. Fmagn. = zevB

Mass spectrometry of biomolecules 2018 133 Mass spectrometry of biomolecules 2018 134 133 134

Elements of ion optics Ion Lens (Electrostatic, Einzel Lens)

Slit  restriction of ion beam  restriction vs. transmission + +

+V

Deflector • analogy of classical lens -V  deflection of ions • focal length may be changed by variation of the voltage V  2 deflectors for x, y scans • higher transmission compared to slit • focal length is not a function of m/z (for ions accelerated in the same ion + source). The lens is ideal only for ions with the same kinetic energy and at reasonable numbers (no space-charge effect) • the schematic above shows only one of the possible arrangements +V

Mass spectrometry of biomolecules 2018 135 Mass spectrometry of biomolecules 2018 136 135 136

Ion Mirror (reflector, reflectron) Construction of Dual Stage Ion Mirror

two grids

R1 R1 R1 R1 R1 R1 R2 R2 R2 R2 R2 R2 R2 R2 R2 R2 + vx 0 +U Kinetic energy vs. potential energy in electric field: 1) mv 2 ion mirror: ion returns with the same velocity as the x  zeU velocity, at which the ion entered the mirror 2

2) energy filter: ions with sufficient velocity (energy) (U2) U3>U1 mv 2 x  zeU penetrate through the second grid … mirror with a partial 2 transmission grids: definition of equipotential planes rings: shielding of the ground potential (vacuum apparatus walls) Ion mirror: with grids or gridless, with one or two stages, nonlinear fields. potential at the rings defined using a series of resistors and U3 Use: e.g. for correction of initial energy dispersion in TOF MS. U1 ... acceleration voltage

Mass spectrometry of biomolecules 2018 137 Mass spectrometry of biomolecules 2018 138 137 138

23 Ion Characteristics Ion Properties

Characteristics of a single ion Example: Initial velocity dispersion at MALDI

(Pv … fraction of ions moving at velocity interval ) m/z mass/charge v velocity (kinetic energy, W = mv2/2) t time Pv x, y, z coordinates protein a angle (... direction)

t0 time of ion formation (index0 … ion formation) matrix Characteristics of ion groups

(usually for the ions of the same type, i.e. the same m/z)

… described by dispersions of v (W), x, y, z, t0, a 0 1000 2000 v0 (m/s)

Mass spectrometry of biomolecules 2018 139 Mass spectrometry of biomolecules 2018 140 139 140

Wein Velocity Filter Wein Velocity Filter

For ion flying on axis of the filter: +V/2 V zeE = zevB (E = ) v L detector E + v = L B + B

-V/2 ... only ions with specific speed pass through the filter.

In case all ions were accelerated by voltage U: • Transverse electric field between two flat electrodes mv 2 = zeU • Transverse magnetic field 2 • Electric intensity field vector E is perpendicular to the vector of magnetic m eB2 induction B = 2U z E 2 • Ions are drawn by electric and magnetic forces with opposite direction … Wein filter can be used for mass analysis (m/z).

Mass spectrometry of biomolecules 2018 141 Mass spectrometry of biomolecules 2018 142 141 142

Energy Analyzer; Electrostatic Analyzer (ESA, E) Energy Analyzer mv 2 2V Acceleration: = zeE , where E = . r L +V ESA mv 2 entrance slit Energy: = zeU L 2 Radius of curvature: 2U exit slit r = ion -V E source + • Radius of curvature is directly proportional to ion kinetic energy. r • Not a function of m/z. • Even thick ion beam (parallel ion trajectories) are focused. +U • Using electrodes curved also axially, ions can be focused in two dimensions. • Sector – shape of a slice, a portion of a circle

Mass spectrometry of biomolecules 2018 143 Mass spectrometry of biomolecules 2018 144 143 144

24 Mass Analyzers Magnetic Sector (MAG, B)

Magnetic sector (MAG, B) magnetic sector Quadrupole analyzer (Q, q) B + + + + entrance m /z > m /z slit Ion cyclotron (ICR-FT-MS) 1 1 2 2 + + m1 r m ion Ion trap (IT), Linear trap (LT) 2 + detector source (+) Time-of-flight (TOF)

Vector of magnetic induction B is perpendicular to the velocity vector of ions streaming from the ion source into the sector.

Mass spectrometry of biomolecules 2018 145 Mass spectrometry of biomolecules 2018 146 145 146

Principle of Magnetic Sector Principle of Magnetic Sector

1.) Lorentz force = centripetal force: m eB2r 2 mv 2 = Bzev = z 2U r m eBr = Scan types: z v • Scan U, const. B. Problems with low extraction efficiency at low values of U. 2.) Kinetic energy = acceleration energy: • Scan B, const. U. Difficult originally, prevailing nowadays. • Move the exit slit, r. Const. U, B. Usually not used. mv 2 = zeU 2 Peak switching Stepwise change of one of the variables (B, U). Suitable for monitoring of m eB2r 2 = limited number of ion types. z 2U

Mass spectrometry of biomolecules 2018 147 Mass spectrometry of biomolecules 2018 148 147 148

Tandem Electrostatic Analyzer – Magnetic Sector Tandem EB (ESA-MAG)

Double focusing mass spectrometer • Dispersion of ion characteristics (v, x, a) and instability of fields (B, U) reduce quality of spectra. • Placing ESA prior to MAG solves problem with velocity (kinetic energy, Forward-geometry, ESA-MAG, EB Wkin) dispersion of ions entering into mass sector. 1. energy analyzer: selections of ions with a certain kinetic energy 2. magnetic sector: mass analysis Practical EB geometries: 1. Nier-Johnson +U 90o ESA + 60o MAG ion exit focused for given radius, r + source B suitable for scanning spectrometers -V . . . . 2. Mattauch-Herzog 31.8o ESA + 90o MAG . . single focal plane for ions with different m/z detector ESA suitable for planar detectors (photographic plate, array) 3. Matsuda +V MAG suitable for compact instruments

Mass spectrometry of biomolecules 2018 149 Mass spectrometry of biomolecules 2018 150 149 150

25 Mattauch-Herzog Geometry Nier-Johnson Geometry

Mass spectrometry of biomolecules 2018 151 Mass spectrometry of biomolecules 2018 152 151 152

Other Combinations of E and B Quadrupole Filter (Q). Ion Trap (IT). Linear Trap (LT). Fourier Transform Ion Cyclotron Resonance Mass Spectrometer Reverse-geometry, BE 5 (FT-ICR-MS). Orbitrap. Electrostatic Trap. 1. magnetic sector: mass filter 2. energy analyzer: sorting of ions according to kinetic energy

MIKES (Mass-Analyzed Ion Kinetic Energy Spectrometry) the first MS/MS technique, tandem mass spectrometry (1973) 1. Magnetic sector allows passing of only ions with certain m/z. 2. Metastable ions may undergo decay in the region between magnetic and energy analyzer. 3. Daughter ions from the decay are sorted according to kinetic energy in the energy analyzer.

A variety of hybrid instruments based on E and B: EBE, BEB, EBEB, BEBE ...

Mass spectrometry of biomolecules 2018 153 Mass spectrometry of biomolecules 2018 154 153 154

Quadrupole Filter (Q, q) Quadrupole Filter

r xz plane: heavy ions pass yz plane: light ions pass -U-Vcos(wt)

y + ... only ions with a specific m/z will pass through the quadrupole filter, all other ions are deflected.

z Note: important exception is m/z-unselective RF quadrupole

x U+Vcos(wt)

4 rods with hyperbolic (also round, square or other) cross-section U ... DC voltage component V ... AC (RF) voltage component w … angular frequency, w = 2pf, f = 1 - 3 MHz, phase shift 180o

Mass spectrometry of biomolecules 2018 155 Mass spectrometry of biomolecules 2018 156 155 156

26 Ion Trajectories in Quadrupole Filter

xz xz

yz yz

Mass spectrometry of biomolecules 2018 157 Mass spectrometry of biomolecules 2018 158 157 158

Ion Trajectories in Quadrupole Filter Stability Diagram

xz ... graphic representation of the solution of the Mathieu equations, which describe trajectories of ions in the quadrupole filters.

xz const. a/q (const. U/V) a 8 eU m+1 m-1 a = m w 2 r 2 m ( z) yz

yz 4eV transmission q = m w 2 r 2 region ( z) q

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Resolution Properties of Quadrupole Filter … is determined by the value (a/q) of the slope of the scan line MS scan r a f usually const.; U a V scanned simultaneously at const. U/V.

Upper m/z limit: 2 000 - 4 000.

0.316V max(m )~ [V, cm, MHz] z r 2f 2

To increase the upper m/z limit: V, r, f.

Resolving power on the order of ~ 103 similar to all quadrupole analyzers

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27 Properties of Quadrupole Filter Quadrupole Filter - Notes

To increase resolution: (Resolution, R < 10 000, usually R < 2 000.) External source

• For acceleration voltage, Uacc (towards the quadrupole): • fringe field, especially at the entrance - responsible for unstable trajectories; significant fraction of ions does not m 2eU l = acc v = get inside z v 2 t - mass discrimination of heavy ions, which spend longer period in the fringe field, are more influenced m l 2 • solution t = z - entrance electrostatic lens 2eU acc - entrance RF quadrupole (q, RF only), hexapole or octopole ... only RF component: a = 0, U = 0  unselective filter, through For given quadrupole length, l, the voltage Uacc must be low enough to enable sufficient number of ion oscillations, tens to hundreds at the given which ions of all m/z can pass frequency, w, during the time of flight, t, through the filter. Triple quadrupole (QqQ) • Better manufacturing, very low mechanical tolerances. • popular tandem mass spectrometer for collisional dissociation • middle quadrupole (q, RF only) serves as a collision chamber

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Quadrupole Ion Trap (Ion trap, IT)

1953 Wolfgang Paul (Paul trap) – almost unnoticed 1983 Finnigan – commercial instrument (one of best selling MS’s)

Schematics of ion trap ring

lids incoming z ions r detector

DC AC DC DC: constant voltage or ground Bruker, Model ESQUIRE-LC AC: constant + alternate (RF) component, U + Vcos(wt)

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Stability Diagram (Ion Trap)

az instability region: ions leave trap through apertures in lids

m3 m2 m1 stability region:

qz ions oscillates inside trap, they are trapped (storage mode)

m3 > m2 > m1 (z = 1) 16eU az = q  V/(m/z) for movement along z axis (a = 0, U = 0) m w 2 r 2 z ( z) Scan: by increasing V. First lighter, then heavier ions 8eV qz = are expelled from the trap. (Also resonant ejection m w 2 r 2 ( z) helps to improve resolution.)

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28 Ion Trap • Experiment stages: ionization, accumulation, scan a detection. • Accumulation: ions are being trapped even during ionization pulse – the result is very low detection limits. The total time of experiment depends on following requirements: Sensitivity ... Longer accumulation means higher sensitivity. Scan time ... Wider m/z range, higher m means longer scan (see next page). Resolution ... Longer scan means slower (and longer) scan. • Resonance ejection of ions of specific m/z by application of RF voltage on trap lids. A hole in the stability diagram is created. It is used to increase resolution even in regular scan mode. (1983, G. Stafford: “mass selective instability mode“) • Buffer gas (low mass gas: He, 1 mTorr) Much better results than with analyte itself. Collisions of analyte with buffer gas molecules lead to better distribution of energy among analyte ions and keeps ions in the center of the trap. Advantageous in GC-MS.

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Ion Trap Ion Trap

• MS/MS – trap can replace tandem of two mass spectrometers • Ion transmission only half ions may be detected CID, CAD: collision-induced (activated) dissociation • Physical restrictions • High R … < 10 000, usually ~ 5 000 dimensions, voltage and frequency limited.

6 • Upper mass limit, m/zmax ~ 70 000 (resonance ejection) • Maximum dynamic range (10 for single ion type) limited: - minimum by sensitivity (usually 10; even 1 ion may be detected) • Miniaturization: ion micro trap, trap on a chip (also quadrupole filter) - maximum by space charge effect: for number of trapped ions higher + total size only 1 cm than 106 the trap does not function properly – due to mutual ion + useful in space exploration repulsion. Note that the charge limit involves sum of charges of all ion types! - much lower mechanical tolerances, demanding manufacturing

- low parameters (R, m/zmax )

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Linear Trap Linear Trap with Radial Ejection

Linear trap or “2D trap” is based on quadrupole ion filter/ion guide. The + previously described ion trap is sometimes called “3D trap”.

Ions are injected into the quadrupole and trapped by elevating potential on lids. (Ions oscillate in a potential well of RF-only quadrupole.) Later the - ions can be gradually ejected and detected through the rods or lids. detector I detector II

Two approaches of ion ejection from LT: +

A. Schwartz, J.C., Senko, M.W., Syka, J.E.P., J. Am. Soc. Mass Spectrom. 13 , 2002, 659. Ions are ejected from LT in radial direction.

B. Hager, J.W., Rapid Comm. Mass Spec. 16, 2000, 512. (From: Schwartz, J.C., Senko, M.W., Syka, J.E.P., J. Am. Soc. Mass Spectrom. 13 , 2002, 659.)

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29 Linear Trap with Axial Ejection Advantages of 2D Ion Traps

Capture Efficiency: 50-75% (3-D: 5 % - 20%, and depends on mass)

Extraction Efficiency: 25-50% (3-D : 20%)

Sensitivity increased by a factor of 5-10

Ion Capacity: 20 - 30 times larger than 3-D

Linear range increased more than 2 orders, up to 106

Ions are ejected from the LT (Q3) along quadrupole axis. Resolution: ~10 000 at scan rate 300 a.m.u./sec

(From: J. C. Y. Le Blanc et al. Proteomics 3, 2004, 859-869.) Wider variety of ion handling (accumulation, scans...)

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Differences b/w Quadrupole Filter and Traps Comparison of Quadrupole Analyzers

Quadrupole filter QIT QQQ LQIT - no ion storage - only ion with certain m/z can pass through a filter at a time Sensitivity ++ - ++

... scanning instrument, loss of ions and hence lower sensitivity Dynamic range - ++ + (except in single ion monitoring) Mass range - + + ... no space charge effects, hence higher dynamic range MS3 ++ - ++

Neutral loss scan/Precursor scan - + +

m/z accuracy + - +

Resolving power + - +

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Ion Cyclotron (FT-ICR-MS) FT-ICR-MS (Fourier Transform - Ion Cyclotron Resonance - Mass Spectrometer) …another type of ion trap, geometry: cylindric, cubic, hyperbolic Ions are introduced into the trap along z-axis (parallel to vector B), trapped in a potential well (by increasing potential on the lids), excited by electric Trap consists of two pairs of electrodes and two lids field perpendicular to vector B and detected inductively

B mv 2 B Bzev = + + v + r ++ Ion movement in magnetic field: + + + r x + + + z + + + B y

excitation excitation v Be w = = r m Angular (cyclotron) frequency: ( z) detection detection

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30 FT-ICR-MS Characteristics Fourier Transform

- (superconducting) magnet with very high magnetic induction B ~ 10 Tesla ... transformation of signal from time domain to frequency (m/z) domain - very low pressure, p < 10-7 Pa (UHV, ultra high vacuum) Ideal signal of ions with single m/z ... high cost ~ $700 000 St S - limited dynamic range ~ 103, 100 – 105 ions FT t + very high precision and accuracy, m/z, ~ ppm m/z + very high resolution, R ~ 106 Real FT-ICR-MS signal + multiplex (Fellgett) advantage of FT … ion signal is acquired for all m/z S S during entire acquisition period  improvement of S/N 103x, reduction of t acquisition period ~106x (FT-ICR vs. ICR) FT + suitable for MSn, commonly n = 2 – 3, even n = 10 demonstrated t m/z • Collisions with background molecules and inhomogeneity of magnetic Other geometries of cell: cylindric, hyperbolic (Penning) field result in signal decrease. Higher p  more collisions  lower R. • Lorentz peak shape. Mass spectrometry of biomolecules 2018 181 Mass spectrometry of biomolecules 2018 182 181 182

FT-ICR-MS Principle FT-ICR-MS Principle Ionization for FT MS Detection 1. internal: analyzer = source (e.g. EI, LDI) 1. nondestructive (inductive) – ions attract electrons in the detection plates 2. external: ionization in an external source and introduction to the cell during passage nearby: non-destructive detection (FT-ICR) - ion guides, quadrupoles, electrostatic lens 2. destructive – ions strike the detection plates (ICR) - differentially pumped chambers (ions are formed at the first cell at higher pressure and then introduced into analyzer cell through an aperture along z-axis) Data acquisition • Higher sampling frequency  higher upper m/z limit. Excitation Nyqist criterion: the highest achievable frequency = sampling frequency/2 1. Pulse ... high amplitude, extremely short duration • Longer acquisition period, t  higher resolution and lower m/z limit. 2. Chirp ... fast scan through frequencies in required interval • Result ... necessity of storage of many data points 3. SWIFT ... Stored Waveform Inverse Fourier Transform - heterodyne frequency mixer (shift of signal frequency down allows to use lower sampling frequency) profile of the excitation waveform generated by iFT of required m/z profile (iFT = inverse FT, transformation from frequency (m/z) to time domain

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FT-ICR-MS Principle FT-ICR-MS

Z-trapping Resolution m Bt a m m • DC voltage ~1-5 V on the lids to keep ions inside (and not to leave in z-axis z direction. Upper m/z limit. From the mean quadratic speed of thermal movement, 2kT v = • Presence of an additional electric field causes magnetron oscillations (~10 m , Hz) of ions in addition to cyclotron oscillation (~ MHz). The result of combined movement are more complex calibration, peak shift and higher which is responsible for pre-excitation of ions, it can be expressed: loss of heavy ions. mv 2mkT r = = zeB zeB . In a trap characterized by B and r, m of ions that can be stored is lower than (zeBr )2 m = 2kT .

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31 New Mass Spectrometers Electrostatic Orbital Trap, ”Orbitrap“

Mass spectrometer of future? A. Makarov, HD Technologies Ltd., USA, ASMS Conference 1999 ... not a single solution rings (+) electrode (-) + + ion injection Trends: + ring (+) - • Withdrawal of magnetic sector instruments. • Hybrid spectrometers + + electrode (-) Modern spectrometers and their hybrids gain popularity (ion traps, detection orthogonal TOF analyzers). Principle • New spectrometers? • Injected ions are trapped in radial electrostatic field between central Linear quadrupole traps (discussed earlier) electrode and rings, they circulate around the central electrode. Electrostatic trap ”Orbitrap“ • Ion also oscillate along the electrode axis and are inductively detected. Linear electrostatic trap • Mass spectra are obtained after Fourier transform of signal acquired in time, f = f(m/z).

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Orbitrap Properties Linear Electrostatic Trap

+ Very high resolution – close to FT-ICR-MS (R = 500 000). Detection tube in potential well Trap electrodes + Very high precision and accuracy of m/z… close to FT-ICR-MS. (ion mirrors)

+ No magnet necessary.

+ No RF generators necessary. electrons are attracted and repelled, AC current is produced - Ultrahigh vacuum needed as in the case of FT-ICR-MS.

Detected frequency = f(m/z) - Difficult injection of ions into orbitrap

(Benner, Anal. Chem. 1997, 69, 4162-4168) Very wide spreading instrument nowadays, there are several hybrid versions of O. It redefines the field of MS.

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Time-of-flight mass analyzer

6 TOF = time-of-flight ... m/z calculated from the time of ion travel

1. Pulse formation of ions

2. Acceleration of ions The same acceleration energy, W(el.) for ions with the same z. Transformation W(el.) = W(kin.)

3. Drift (flight) of ions: Separation of ions according to m/z; W(kin.) = mv2/2

4. Impact of ions on detector Acquisition of signal in time, I(t)

5. Determination of m/z from the time of flight Time-of-Flight Mass Spectrometer (TOFMS). Techniques for Transformation I(t)  I(m/z) Enhanced Resolution in TOFMS (Reflector, Delayed Extraction and Orthogonal Extraction). Simion: Examples. Animations. Mass spectrometry of biomolecules 2018 191 Mass spectrometry of biomolecules 2018 192 191 192

32 TOFMS History TOFMS Geometry

1946 principle of TOF Linear geometry W. E. Stephens, Phys. Rev., 1946, 69, 691 ... the simplest arrangement 1948 1st TOF spectrometer (ion velocitron) ... ions are not focused optimally on the detector A. E. Cameron, D. F. Eggers, Rev. Sci. Instrum. 1948, 19, 605 1955 two-stage ion source Wiley, W. C.; McLaren, I. H.; Rev. Sci. Instrum., 1955, 26, 1150 ionization beam: 1973 Ion mirror e.g. pulse e- beam (B. A. Mamyrin, V. I. Karataev, D. V. Schmikk, and V. A. Zagulin, Sov. Phys. JETP 1973, 37, 45) (+) 1995 Delayed (pulse) extraction (time-lag-focusing) repeller, drift zone, E = 0 detector (Whittal, R. M.; Li, L., Anal. Chem. 1995, 67, 1950-1954; ~20 kV Brown, R. S.; Lennon, J. J., Anal Chem. 1995, 67, 1998-2003; Vestal, M. L.; Juhasz, P.; Martin, S. A, Rapid Commun. Mass Spectrom. entrance and exit grids 1995, 9,1044-1050)

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TOFMS Geometry TOFMS Geometry

Wiley-McLaren linear geometry (1955) TOF MS with ion mirror (reflector) ... adjustment of the extraction electric field does not depend on the value of ... for higher resolution the total acceleration voltage ... for structural analysis (PSD) ... allows focusing of ions on the detector at the end of the flight tube

ionization beam: e.g. pulse e- beam or laser pulse ionization beam: ion mirror, e.g. laser pulse (+) detector >20 kV (+) repeller, (+) extraction grid, ~17 kV ~20 kV drift zone (-) (E = 0) repeller, drift zone, E = 0 detector ~20 kV (+) source ion grids and grids deflector acceleration exit grid, electrodes and gate (+) grid, 0 V 0 V of reflector

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ion source TOFMS Principle flight tube detector 1. Pulse ionization (pulse width ~ ns): fast generation of ion plume (LDI, laser MALDI, PD, EI …) 2. Extraction and acceleration of ions in electric field 3. Separation of ions in drift zone at E = 0 (field-free region, flight tube) 4. Detection of ions, signal acquisition and transformation to m/z domain

mv 2 diffusion digital oscilloscope acceleration energy: z eU = kinetic energy pump 2 L length of drift zone v = flight time delay generator t m t 2 S = 2eU mechanical control unit z L2 pump vacuum controller (m/z)1<(m/z)2

high voltage source MALDI TOFMS TOF Mass spectrometry of biomolecules 2018 197 Mass spectrometry of biomolecules 2018 198 197 198

33 TOFMS Principle TOFMS Calibration

Exact relation includes also the time spent in in the ion source and the m/z=(2eU/L2)t2 detector regions:  m/z=k (t-t )2 voltage: U 0 0 U 1 0 s d  1/2 t=c0+c1(m/z) distances: s L d 1/2 t=c0+c1(m/z) +c2(m/z) -1/2 1/2 t=c0+ c-1(m/z) +c1(m/z) +c2(m/z) tTOF = ts + tL + td

2ms 2 mL2 d m correction of data acquisition trigger t s = t L = td = ( Us + Us + 4Ud ) zeU s 2zeU s U d 2ze correction of the original ion velocity before extraction pulse correction of non-ideal extraction pulse shape 1/2 Note: 1) ts, tL, td and hence tTOF are proportional to (m/z)

2) for rough estimate tTOF: tTOF ~ tL (L >> s, d)

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Principal Advantages of TOFMS Ion Sources for TOFMS

No theoretical upper m/z limit 1. Ion source for gaseous samples e-, ions, laser … Ideal for pulse ionization + Fellgett advantage: for each of pulses, entire spectrum recorded, no need (+) (-) for scanning 2. Ion source for condensed samples (desorption) Very short duration of acquisition of a spectrum (~10-4 s) laser, ions, atoms … High ion transmission ... a prerequisite of high sensitivity

Simplicity (+) (-)

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Ideal Ion Source for TOFMS Real Ion Source for TOFMS

All ions are created: Ions are characterized by initial dispersions of

time (time of creation t0),

• in time t0 (with period of formation, t = 0) space (position of creation x0, y0, z0),

• at distance x0 (x … flight axis of TOFMS) velocity v0 (energy Wkin0) and

(preferably in a single point [x0, y0, z0]) angle a (deviation of the flight x-axis).

• with the same velocity vx0 (, which does not have to be zero,) along x- axis. Result: drop of resolution, R

(preferably vy0 = 0 a vz0 = 0) Wiley-McLaren ion source for gaseous samples: y Adjustment of voltage on the first grid allows to find a compromise between z contribution of v0 a x0 dispersions to reach optimal resolution (ion focusing into detector plane) + x

source detector

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34 Ion Source (detail) Ion Properties

Example: Dispersion of initial ion velocity in MALDI

(Pv … probability of occurrence of molecules with velocity in )

Pv analyte

matrix laser

0 1000 2000 v0 (m/s)

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MALDI-TOF MS Coupling? Pulse Generation of Ions

Desorption Methods (MALDI, LDI) – a special case: 1. Extraction using constant electric field (DC)

t0~ns negligible (very short laser puls) pulse ionization beam + constant extraction field

x0~mm negligible (tthin sample layer, small sample crystals)

z0,y0~ negligible (focused laser beam: 50 mm) 2a. Pulsed extraction, PE (delayed extraction, DE) pulse ionization beam + pulse extraction field

v0 >100 m/s the major reason of low resolving power 2b. Orthogonal extraction 0 a0 ~10 contributes to additional broadening of velocity distribution continuous generation (introduction) of ions + pulse extraction field

Due to energy (velocity) distribution (v ~ 700 m/s, v > 500 m/s), ions of the same m/z hit detector at different times.

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Enhancement of Resolution in TOFMS 1. High Acceleration Voltage

1. High acceleration voltage

mv 2 m v 2 2zeU 2. Ion mirror. = 0 + zeU  v = v 2 + 2 2 0 m 3. Pulse extraction contribution contribution of desorption of electric field ... important decision when buying TOFMS Increase of acceleration voltage will minimize contribution of initial velocity dispersion of analyte.

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35 Influence of Acceleration Voltage 2. Ion Mirror (reflector, reflectron)

Př.: MALDI TOFMS of a peptide, m = 2000 Da, z = 1, v0 = 750 m/s, v0(FWHM) = 500 m/s, L = 1 m. (2 ions: v01 = 500 m/s, v02 = 1000 m/s.) 1 source detector II 2 detector I (-) Pv + 100% (+) U1 deflector U2 U3>U1 500 50% ion mirror m/s

• Ions 1 and 2 with same m/z and velocities v1 a v2: faster ion penetrates deeper into the ion mirror and its trajectory is longer. 0 500 1000 v0 (m/s) • If adjusted properly, two ions will hit the detector II simultaneously.

U = 1 kV: t1 = 101.673 ms, t2 = 101.284 ms R ~ 130 (B. A. Mamyrin, V. I. Karataev, D. V. Schmikk, and V. A. Zagulin, Sov. Phys. JETP 1973, 37, 45; U = 10 kV: t1 = 32.190 ms, t2 = 32.177 ms R ~ 1300 Mamyrin, B. A.; Shmikk, D. V.; Sov. Phys. 1979, 49, 762)

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Construction of Ion Mirror Construction of Ion Mirror

Linear ion mirror ... E is constant along the ion mirror axis Single stage ... intensity of electric field, E is constant in the entire ion R R R R R R R R R R R R R R R R mirror (Alikhnov) 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2

Dual stage ... ion mirror is divided in two regions with different E (B. A. Mamyrin, V. I. Karataev, D. V. Schmikk, V. A. Zagulin, Sov. Phys. JETP 1973, 37, 45)

Nonlinear ion mirror ... E varies along the ion mirror axis

Quadratic field (D. R. Jardine, J. Morgan, D. S. Alderdice, P. J. Derrick, (U2) U3>U1 Org. Mass Spectrom. 1992, 27, 1077) Curved field (T. J. Cornish, R.J.Cotter, Rapid Commun. Mass Spectrom. grids: definition of equipotential plane 1993, 7, 1037-1040) rings: electric shielding of vacuum apparatus walls + additional patents (Japan, Soviet Union) voltage on the rings is defined by series of resistors

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Construction of Ion Mirror Single Stage Ion Mirror

detector L L (-) 2 m ion mirror

s L1 (+) Us Ur

tTOF = ts + tL + tr ... sum of periods ion spends in source, tube and mirror 2 v 1 2s  L 2a  v 2 1 2s  3L 2a  v 2  0   0   0  tTOF = t 0 − + 1 − +  − 1 − +   + ... a 2 a  2s ar  2as 8 a  2s ar  2as  2s  L 2a    t 0 = 1 + +  L = L1 + L2 ... drift zone, E=0 a  2s ar  a ... acceleration in source, a=qEs /m

ar ... acceleration in mirror, a=qEr /m (Moskovets, E. Rapid Commun. Mass Spectrom. 2000, 14, 150-155) Mass spectrometry of biomolecules 2018 215 Mass spectrometry of biomolecules 2018 216 215 216

36 Dual Stage Ion Mirror Dual Stage Ion Mirror

Two-stage ion mirror ... ion mirror is divided in 2 stages with different E

For positive ions:

• positive voltage on the middle grid, 0 > U2 > U3 +

- a variety of arrangements, various ratio of stage lengths, Wkin

e.g. ~1:2, ~2:3 U2 U3 - short 1st stage with higher E allows shortening of the entire mirror length tTOF = f(Wkin, U2, U3) energy dispersion ... the main reason of peak broadening and low R • negative voltage on the middle grid: - space focusing in the 1st stage Mamyrin: 2 - energy focusing in the 2nd stage   Solution f (W ,U ,U ) = 0 and f (Wkin ,U 2 ,U 3 ) = 0 kin 2 3 W 2 Wkin kin

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Dual Stage Ion Mirror Nonlinear Ion Mirror

+ Applicable to energy dispersion below ~20%. Aim: tTOF independent on their Wkin for ions of all m/z E.g. for energy dispersion < 5% theoretical resolution ~100 000 k In quadratic field U (z ) = (z − a)2 + C 2 - Only ions that penetrate deep into the ion mirror (85-100%) are focused. d 2z q 2 = − k (z − a) - Mass spectra of ions with wider energy dispersion have to be recorded at dt m several values of the potential on the mirror. The resulting spectrum is ion reflection  f(Wkin0) stitched from these several segments. z ... axis of potential change a ... parabola minimum k a C ... constants f ... frequency of oscillations (A. A. Makarov, E. N. Raptakis, and P. J. Derrick, Int. J. Mass Spectrom. Ion Processes 1995, 146/147, 165.)

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Nonlinear Ion Mirror Nonlinear Ion Mirror

Other practical fields: axially symmetric hyperbolic potential planar hyperbolic potential axially symmetric hyperlogarithmic potential

nonlinear (curved, quadratic) field

linear field

(T. J. Cornish, R. J. Cotter “Non-linear field reflector“, US Patent 5 464 985)

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37 Nonlinear Ion Mirror 3. Pulsed Extraction (Delayed Extraction Time-Lag Focusing) Usually quadratic field or its approximation Extraction field is turned on after a delay: 1. Laser pulse at t = 0 2. Expansion of ions at zero extraction field for a period t (delay) 3. Extraction field is turned on at t = t, with fast rise time Creation of nonlinear field V using unequal resistors in series 15 kV V(repeller) using unequal spacers between rings and/or grids 12 kV V(grid)

+ simultaneous focusing of ions with wide dispersion of Wkin , e.g. products of post-source fragmentation, for entire m/z range - more complex calibration 0 0 t t - ideal only for ions moving on the axis of the reflector ... only limited R can be achieved in practice (Brown, R. S.; Lennon, J. J.; Anal. Chem. 1995, 67, 1998)

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Pulsed Extraction Orthogonal Extraction

repeller grids drift zone detector analyte ion beam from a continuous ion source 1. + (+) t = 0: + repeller, drift zone, E = 0 E = 0 pulse detector e.g. 12 kV 12 kV 0 kV voltage, U1 acceleration exit grid, 2. grid, 0 V 0 V t = t : + extraction grid, U + 2 E > 0 3. e.g. 15 kV 12 kV 0 kV Pulse extraction for continual ion sources t = tof: Analyte stream is extracted by pulse extraction field orthogonal to the entering ion beam. + + Usually with ion mirror for higher resolution.

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Other Factors Influencing Resolution in TOFMS Importance of Alignment in TOFMS

• non-ideal ion source Strict requirement on parallel arrangement L • ion optics • tube length dilatation • properties of detector + drift zone • sampling frequency of A/D converter + • sample preparation MCP • acceleration voltage fluctuations ion source detector

E.g. acceleration voltage fluctuations: MCP with narrow channels

2 −1 dm  dU dt  m = const. Ut  R = = const . + 2  L m  U t  + drift zone 10o dt given by max. frequency of AD converter, detector speed and time + dispersion of ion generation ion source dU given by high voltage power supply (drifts and noise) 3 mm MCP (detail) Mass spectrometry of biomolecules 2018 227 Mass spectrometry of biomolecules 2018 228 227 228

38 Importance of Alignment in TOFMS Influence of Grids

Resolution: m Grids ... electrodes transparent for ions R = ... in ion source, ion mirror and in front of detectors m m t 2 =2eU t z L2 R = MS with grids 2t + precise definition of potential t = L/v L - ion losses (impact, deflection, reaction) R = - secondary ion formation on grids, sputtering of grid material 2L - field penetration E.g. for R = 15 000 and L = 3 m: Gridless MS L  100 mm + higher resolution without losses on grids - more complex design (extra curvature of ion trajectories, lens effect) Note: MCP with channel angle 10o and diameter 10 mm: L = 10 mm/tan(10o) = 57 mm For details, see: T. Bergmann, T. P. Martin, H. Schaber Rev. Sci. Instrum. 1989, 60, 347.

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Additional TOF MS parameters MALDI MS of Numerous Sample Series

Target (MALDI): 384 spots on target sized as microtitration well plate, 1, 10, 96, 100, 384, 1536 ... sufficient area for collection of eluent from a separation column samples/target Axial sample irradiation: higher resolving power

Collision chamber in ion source: increase of fragmentation degree

Length of the drift zone (flight tube): typically 1-2 m, but even 10 cm or 5 m

Ion guide – it may be placed in the tube to increase ion transmission

Detectors: microchannel plate (MCP), usually double MCP hybrid detector s(scintilating layer + photomultiplier) 384 samples/target

Detection electronics: AD converter (8 bits, 0.5 - 4 GS/s) TD converter + segmented detector

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Comparison of TOFMS Systems TOFMS Characteristics

1. Unlimited theoretical upper m/z limit. Practical limit: ionization and detection efficiency, metastable ion decay.

system geometry extraction R 2. Entire spectrum recorded at once.

TOFMS linear DC 500 3. High speed of data acquisition (1 spectrum in ~10-4 s), e.g. insulin (m/z DE-TOFMS linear pulse 5 000 = 5735) accelerated by 15 kV overcomes 1-m drift zone in ~ 50 ms.

rTOFMS reflector DC 10 000 4. High ion transmission, especially in linear mode (> 50 %).

DE-rTOFMS reflector pulse 20 000 5. High resolving power (R > 10 000).

oTOFMS reflector pulse 10 000 6. Simplicity and relatively low cost. 7. MS/MS techniques available R ~ 100 000 in commercially available instruments

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39 TOFMS Perspective Comparison of Common Mass Spectrometers

Advantages of TOFMS upper m/z limit, speed and high sample throughput, high sensitivity and MS max. m/z [Da] R m/z accuracy [Da] cost resolution, relatively lost cost Q 103 103 0,1 ☺☺ IT, LIT 103 103 0,1 ☺ Huge spread of TOFMS in the last decades B 104 104 0,01  MALDI + PE TOF, rTOF MS TOF 105 104 0,01 ☺ API, AP MALDI + oTOF MS O 104 105 0,001  FT-ICR 104 106 0,0001  New techniques for MS/MS (TOF-TOF, LIFT, QTOF) Note: Competing technologies The values in the table are approximate, as they describe average LT, FT-ICR and hybrid MS instruments, the parameters of research-grade systems might be significantly higher (differences in order of magnitudes).

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Simulation of Ion Movement Simion

• Exact calculation of ion movement complex even for relatively simple Example: Simulation of electrostatic lens using program Simion. electric and magnetic fields. Lens: 3 segments • Simulation of ion movement: program Simion diameter, d = 36 mm

length of segments, y1 = 28 mm, y2 = 26 mm, y3 = 32 mm Development of ion optics using the program Simion: gaps between segments, l = 2 mm 1. Input of geometry (schematics of electrodes). ion origin in xyz [0, 0, 0] mm potentials, U = 0; U = tunable; U = 0 2. Input electrode potential, define magnetic field. 1 2 3 Ions: number, n = 5 3. Definition of ions (number n, v , x , y , z , a , f ). 0 0 0 0 0 0 mass, m = 100 4. Movement simulation  result (graphical representation, text). kinetic energy, W0 = 100 eV coordinates xyz [0, -30, 0] mm o a0(i) = (-4 + 2i ) , where i = 0 … n – 1

Aim: Verify function of the lens for voltage of the middle ring, U2 = 0, 85, 100, 120 a 133 V. Solution: Presented in the lecture

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Collision-Induced Dissociation (CI). Tandem Mass Scan: a mass spectrum acquisition Spectrometry (MS/MS). Additiona Dissociation Options (ECD, 7 ETD, IRMPD, In-Source Decay (ISD), Post-Source Decay S(m/z) = f(X) = f(t) (PSD). Techniques TOF-TOF, LIFT. Scanning instruments Scanned power Scan period magnet sector (B) B, U ~ 1 s quadrupole filter (Q) U+V, f ~ 0.1 s ion traps (IT, LT) U, V, f ~ 0.1 s

Not-scanning instruents (direct spectrum acquisition in time): time-of-flight (TOF) ~ 100 ms ion cyclotron (FT-ICR) ~ 1 s orbitrap (O) ~ 1 s

Other not-scanning instruments may use array detectors.

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40 Simple mass spectrometry Simple mass spectrometry

Acquisition of a „whole” spectrum Selected (single) ion monitoring (SIM)

… „kompletní“ informace www.jimkellyjr.com/2008_07_01_archive.html Peak switching: acquisition of selected ion signals Acquisition of a spectrum interval (zoom scan)

Techniques 2, 3 and 4: … vybraný interval m/z … data reduction, higher frequency (spectra/s) … important in chromatography with MS detection

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Tandem mass spectrometry Basic MS/MS modes

Ion selection → fragmentation → ion analysis 1. Product ion scan, daughter ion scan, fragment ion scan Set MS1, scan MS2

Q1 q2 Q3 2. Precursor ion scan, parent ion scan MS1 CID MS2 Scan MS1, set MS2

precursor ions product ions 3. Neutral loss scan Scan MS1 and MS2 simultaneously, m/z = const. mother ions daughter ions parent ions 4. Selected reaction monitoring/multiple reaction monitoring Set MS1 and MS2 – select precursor and product its ion. Classical instrument for low-energy CID: triple quadrupole (triple quad, TQ, QQQ, QqQ, Q3)

Q1: 1. analyzer (MS1)

q2: collision chambre (RF-only Q)

Q3: 2. analyzer (MS2)

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Product ion scan Example: Product ion scan

MS–MS Fragmentation Patterns of Set MS1: transmit ions with selected m/z Cholesterol Oxidation Products Scan MS2: spectrum of product ions of the selected precursor B. Rossmann, K. Thurner, W. Luf Monatsh Chem 138, 437–444 (2007) … useful for structural elucidation ... fingerprint of the analyzed molecule APCI QqQ … simplified spectrum ... signal-to-noise ratio (S/N) increase toxicologically relevant products of cholesterol oxidation … attention: MS1 does not guarantee isolation of a single precursor ion

MSn ... mass spectrometry to the n-th stage

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41 Example: Product ion scan Precursor ion scan

Scan MS1: spectrum of precursor ions Set MS2: detection of product ions of a given m/z

… for identification of a compound class, similar compounds with the same functional group or structural motif … simplified spectrum ... S/N increase

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Example: Precursor ion scan Example: Precursor ion scan

Parent ion scans of unseparated peptide Parent ion scans of unseparated mixtures peptide mixtures M. Wilm, G. Neubauer, M. Mann Anal. Chem. M. Wilm, G. Neubauer, M. Mann Anal. 68, 527-33 (1996) Chem. 68, 527-33 (1996)

(A) MS of peptide GLHINDYALNITKSFLLDK (A) Pozitive MS of phosphopeptide Mr,avg = 2175,5; c = 10 fmol/mL EPQYPEEIPIYL,

(B) Precursor ion scan for immonium ions of Mr,mono = 1471,6; c = 1 fmol/mL. isoleucine and leucine (m/z = 86 Da) (B) Precursor ion scan for (C) Product ion scan - [M+3H]3+ phosphogroup (m/z = 79 Da) in negative mode at the same sample concentration

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Neutral loss scan Example: Neutral loss scan

Constant neutral loss scanning for the Simoultaneous scan of MS1 and MS2 characterization of bacterial Constant m/z difference of MS1 and MS2 phospholipids desorbed by fast atom bombardment D. N. Heller, C. M. Murphy, R. J. Cotter, … loss of the same neutral porticle C. Fenselau, O. M. Uy ... useful for compounds with a similar functional group Anal Chem 60, 2787 - 2791 (1988) … simplified spectrum ... S/N increase phosphoethanolamine: m/z = 141 phosphoglycerol: m/z = 172 methylphosphoethanolamine: m/z = 155

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42 Example: Neutral loss scan Example: Neutral loss scan

MS MS

MS/MS NLS, m/z = 141 phosphoethanolamine MS/MS MS/MS NLS, m/z = 172 NLS, m/z = 155 phosphoglycerol methylphosphoethanolamine

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Coupling of MS with chromatographic techniques Monitoring of selected reaction(s) (MS chromatogram, ion chromatogram) … MS/MS analogy of SIM and peak switching … record of ion intensity/intensities during separation Selected/Single reaction monitoring, SRM Set MS1: precursor ion of a given m/z Total ion current (TIC) Set MS2: product ion of a given m/z … monitorg of a wide m/z interval (hundreds Da) … very specific proof of analyte via its reaction … useful overall separation picture, all peaks included … not useful for separation of complex mixtures Multiple reaction monitoring Set MS1: peak switching – precursor ions of given m/z values Selected ion monitoring, SIM Set MS2: peak switching – product ions of given m/z values … detection at a given m/z … very specific proof of analyte via their reactions … monitor a selected compound … for more ions - peak switching High S/N, employed in combination with chromatographic techniques Selected reaction monitoring (SRM) … a more specific analogy to SIM using tandem MS … for more reactions - multiple reaction monitoring, MRM

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Coupling of MS with chromatographic techniques TIC: MS

Complete spectra are acquired during multidimensional analyses analýzách (LC- MS, GC-MS, CE-MS) during separation. TIC or intensity of a selected ion(s) can be acquired from the data TIC: MS/MS

Extracted ion chromatogram, XIC, EIC Reconstructed ion chromatogram, RIC XIC: MS @ 701 Quality of a „reconstructed “ chromatogram might be lower than „true “ SIM or MRM

recorded with Q or QqQ , , Bruker Ultra HCT IT

Base peak intensity chromatogram, BPC ... intensity of dominantn peaks in given retention times MS @ 19,8 min

... can be more simple than TIC due to ignoring of a sum of minor peaks

: Zbyněk : Zbyněk Zdráhal

redit C

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43 Example: Multiple reaction monitoring Example: HPLC – APCI – SRM

B. Rossmann, K. Thurner, W. Luf Monatsh Chem 138, 437- 444 (2007) SRM for dominant reactions from the previous table

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Example: HPLC – APCI – MRM MRM: TIC and XIC

(A) TIC for selected alcoholdehydrogenase peptides (B) XIC of a specific ackoholdehydrogenase peptide, precurzor: z = 3 (C) XIC of a specific ackoholdehydrogenase peptide, precurzor: z = 2 (with standard for quantitation)

A multiple reaction monitoring method for absolute quantification of the human liver alcohol dehydrogenase ADH1C1 isoenzyme D. J. Janecki, K. G. Bemis, T. J. Tegeler, P. C. Sanghani, L. Zhaib, T. D. Hurley, W. F. Bosron, M. Wanga, Anal.Biochem. 369, 18-26 (2007) Mass spectrometry of biomolecules 2018 261 Mass spectrometry of biomolecules 2018 262 261 262

Why MS/MS? Mass spectrometers for MS/MS

Structural elucidation of organic compounds 1. Triple quadrupole filter (Triple quad, QQQ, TQ, Q3)

Analysis of analyte mixtures a substitute for separation coupled to 2. Ion traps (IT, LIT, FT-ICR) MS? • All analytes are ionized at the same time in the case of MS/MS, a 3. BE (magnetic sector – electrostatic analyzer) with reverse geometry single precursor ion is isolated at a time followed by another one (MS1) and they are identified after fragmentation (MS2). 4. TOF/TOF MS • Attention! Mutual suppression in the case of complex mixture alalysis

5. Hybrid spectrometers, such as QTOF, IT-TOF, EBQ etc. Increase of S/N • Increase of selectivity  decrease of noise • Simpler spectra Classification of tandem mass spectrometry • All MS/MS scans and SRM/MRM 1. in space – 1, 3, 4, 5 2. in time – 2, 5 (IT-TOF)

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44 Other Mass Spectrometers for MS/MS Other Mass Spectrometers for MS/MS

Tandem magnetic and electrostatic sector with reverse geometry (BE) Tandem IT - TOFMS (IT-TOFMS) 1. Isolation of precursor ABC+ in B. IT: accumulation and MSn option in the 1st stage. 2. Fragmentation in collision chamber. TOF: sensitive detector with high mass resolution&accuracy as the 2nd stage. 3. Kinetic energy analysis in E. Daughter ions are characterized with W; W = f(m/z). Ion traps: IT, FT-ICR-MS or O More sophisticated combinations: EBE, EBEB. • MSn option • the same spectrometer as for MS, only software upgrade needed EBqQ • the same price EB: precise precursor selection • Procedure:

q1: RF-only (collision cell) 1. isolation of precursor (after accumulation of all ions)

Q2: selection/analysis of products 2. excitation of precursor (amplitude boost) for a longer period 3. product scan (or back to the 1st step for MSn, n >2) Tandem quadrupole filter - oTOFMS (QqTOFMS) Tandem LT – FT-ICR-MS or O TOF-TOF plenty of scan types and operational modes

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Dissociation types Dissociation types

Dissociation ... monomolecular reaction, t(induction) << t(dissociation) 1. collisions with molecules of gas in the collision chamber at elevated pressure, p ~100 Pa Fragmentation due to: CID/CAD collisionally induced/activated dissociation 1. Collision with atom or molecule 2. dissociations with surface: SID, surface induced dissociation collision-induced dissociation (CID) - surface: a layer of an organic compound (polymer or monolayer of 2. Collision with surface small organic molecules, e.g. alkanthiols) on a suitable substrate (Au) surface-induced dissociation (SID) - dissociations as a result of acceleration in electric field, e.g.: voltage 3. Photon – photodissociation (PD) nozzle-skimmer e.g., infrared multiphoton dissociation (IRMPD) 3. photodissociation 4. Electron RMPI, resonant multiphoton ionization electron capture dissociation (ECD) 4. capture/transfer of electron electron transfer dissociation (ETD) ECD, Electron capture dissociation ETD, Electron transfer dissociation Note: Determination of the main reason of fragmentation may not be simple, 5. excitation during ionization (e.g., high energy at LDI and MALDI) e.g. fragmentation during MALDI (ISD and PSD) may be induced ISF, in-source fragmentation … v TOF MS byphotons as well as collisions. PSD, post-source decay … v TOF MS Mass spectrometry of biomolecules 2018 267 Mass spectrometry of biomolecules 2018 268 267 268

Ion Stability Ion Collisions

1. Stable: lifetime, t > 10-6 s Fragmentation Ion flights through entire MS without decomposition. • intended fragmentation aimed at elucidation of analyte structure • usually with atoms of rare gases 2. Metastable: lifetime, t ~ 10-7 - 10-6 s Ion is decays during flight in the m/z analyzer. Elastic collisions Kinetic energy is conserved. 3. Unstable: lifetime, t < 10-7 s Ion fragments in the ion source. Inelastic collisions Part of kinetic energy is converted into inner ion energy:

Note: This is historical classification according to the time ions spend in Ein <= E(Mt/(Mt + Mi)) magnetic sector. t = target, i = ion Use heavy target to transfer more energy on ion. Reactions: unimolecular, bimolecular 1 eV/ion ~ 100 kJ/mol (100 kJ/5 g tatranky)

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45 Low-Energy Collisions High-Energy Collisions

Collision Energy: 1 – 100 eV Collision energy: keV vibrational excitation, t (ion-target interaction) ~ 10-14 s electron nature of excitation, t (ion- target interaction) ~ 10-15 s converted energy ~ 1 – 3 eV Collision efficiency usually sufficient due to many collisions in collision cell Collision efficiency He – reduces angular scatter of products Instrumentation (scatter has negative impact on m/z analysis) triple quadrupole filter, ion traps, hybrid MS Ar, Xe – enables more efficient energy conversion

Instrumentation The most widespread technique nowadays. hybrid sector instruments, TOF/TOF

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Example: McLafferty Rearrangement Mechanism of McLafferty Rearrangement

Electron impact-induced breakage of carbonyl compounds with hydrogen in g position and formation of enolic fragment and olefin:

F. W. McLafferty, Anal. Chem. 31, 82 (1959).

D. G. I. Kingston et al., Chem. Rev. 74, 215 (1974); K. Biemann, Mass Spectrometry (New York, 1962) p 119; Djerassi et al., J. Am. Chem. Soc. 87, 817 (1965); 91, 2069 (1969); 94, 473 (1972) M. J. Lacey et al., Org. Mass Spectrom. 5, 1391 (1971); G. Eadon, J. Am. Chem. Soc. 94, 8938 (1972); F. Turecek, V. Hanus, Org. Mass Spectrom. 15, 8 (1980).

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ECD, ETD ECD

Electron capture dissociation • interaction of ions with thermal electrons (low E) • application: peptide fragmentation (ions c, z ), no fragmentation of side chain • FT-ICR-MS

Electron transfer dissociation • transfer of electron from a reagent • the same character as fragmentation as ECD • quadrupole ion traps

(Syka J.E.P,. Coon J.J., Schroeder M.J., Shabanowitz J., Hunt D.F. PNAS 101, 9528-9533, 2004)

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46 ETD In-Source Decay (ISD)

• Also called In-Source Fragmentation (ISF)

• Technique for study of molecular structure

• Elevated laser power at MALDI leads to excessive “heating" (vibrations) of molecules/ions of analyte and fragmentation of analyte in the source

• Intensity of fragments << intensity of parent ion ([M+H]+)

• Pulse extraction necessary to: • reach sufficient resolution and sensitivity • prolong ion stay in the ion source (more collisions)

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ISD Characteristics

+ Ion mirror not necessary. - Pulse extraction needed. - Preparation of clean analyte required (no option of precursor selection). - May require special sample preparation (e.g. higher salt concentration)

Use: MALDI TOFMS of peptides, saccharides

ISD. Analyte: 20 pmol KGF analogue. Matrix: sinapinic acid. Laser: N2. (V. Katta, D. T. Chow, M. F. Rohde Anal. Chem., 70, 4410-4416, 1998.) Mass spectrometry of biomolecules 2018 279 Mass spectrometry of biomolecules 2018 280 279 280

Post-Source Decay (PSD)

• Ion selector (ion gate): selection of precursor (m/z interval 1 - 20 Da).

• Analysis of product ions in ion mirror: - Fragment ions of analyte are formed during flight through the drift zone (as a result of excessive excitation), e.g.: - ABC+ → AB+ + C - ABC+ → A+ + BC etc. - Kinetic energy conservation for the first equation: 2 2 2 - mABCv /2 = mABv /2 + mCv /2.

ISD • The heavier the fragment ion is, the higher kinetic energy it has and the Analyte: oxidized bovine insulin, chain B deeper it penetrates into the ion mirror  longer flight time. Matrix: thiourine Laser: Er:YAG (l = 2936 nm)

(http://medweb.uni-muenster.de/institute/impb/research/hillenkamp/publicat/abs-cm99.html)

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47 Ion Mirror as an Energy Analyzer in PSD TOFMS Precursor ABC+ m v 2 m v 2 m v 2 ABC+  AB+  A+ 2 2 2 + v detector 2 CH + ABC+ ABH ABCH+ source detector 1 (-) v + for neutrals Fragmentation 1 AB+ (+) (BC, C etc.) +U1 deflector/selector 0 +U2 , (U2 > U1) v C ion mirror

Clasical ion mirror allows focusing of only product ions with < 20 % Fragmentation 2 v energy dispersion and hence in < 20 % m/z interval. Total PSD A+ spectrum has to be put together from few PSD spectrum increments obtained at different reflector potential. v Quadratic ion mirror allows focusing of products ions in wide m/z interval. BC

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MALDI PSD of a Derivatized Peptide YLYELAR MALDI PSD of a Peptide

[Abs. Int. * 1000] a Y y R A 3.6 a .i. 3.4 3.2 3.0 2.8 20 0 0 2.6 2.4 2.2 2.0 1.8 15 0 0 1.6 1.4 1.2 1.0 0.8 10 0 0 0.6 0.4 0.2 0.0 100 200 300 400 500 600 700 800 900 1000 1100 m/z 50 0

Spectrum composed from spectral increments recorded at different ion

mirror potential. Only ions that penetrate deep into the mirror were 0 focused. Dual stage ion mirror used. (Source: Z. Zdráhal) 100 200 300 400 500 600 700 800 900 m /z MALDI PSD: Dual stage ion mirror. (Source: Z. Zdráhal) Mass spectrometry of biomolecules 2018 285 Mass spectrometry of biomolecules 2018 286 285 286

Tandem Mass Spectrometer QTOF MALDI PSD of a Peptide [M+H]+ 80 b • the most typical oTOF API, AP MALDI... b 3 b B 2 5 b 7 60 • useful for low-energy CID b laser (MALDI) 1 y 5 a 4 40 • MS/MSIon beam is wide after Q1: precursor selection a y a 3 6 y 7 exit from Q3 (space dispersion 7 y a b 8 Intensity [%] Intensity 2 y b - x ), but ions have negligible 20 y 4 4 a 6 0 a 3 6 Q2: CID 1 energy dispersion - v0 in the direction of flight in TOF 0 Q3: ion guide & focusing

200 400 600 800 1000 1200 1400 1600 ion optics Mass/Charge

pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 detector ion (+) MALDI PSD, a quadratic-field reflector used. mirror (O. Šedo et al. Anal. Chim. Acta 2004, 515, 261-269)

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48 CID in TOFMS? QTOFMS: hybrid TOF MS

CID (in QqQ, IT...)  ISD or PSD Taking benefit of both analyzers: Fragmentation results of collision of precursor ion with gas molecule Q: 1st MS, collision chamber and ion beam shapingg TOF: better parameters in 2nd MS (compared to Q) Collision chamber - additional collision chamber in TOF ion source (negligible effect) Exchangeable ion source decoupling of ionization from mass analyzer useful for continuous, quazicontinuous and pulsed ionization techniques Two ways of CID in TOF Example: QqTOF: API (continuous ion source) 1. hybrid instrument (e.g. QqTOF) AP MALDI (pulsed, quazicontinuous ion source) 2. TOF/TOF

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Hybrid QTOF MS for API and MALDI Hybrid QTOF MS for API and MALDI

Source: Micromass/Waters Source: Micromass/Waters

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TOF-TOF LIFT TOF MS

22 kV

source

6 kV LIFT cell ion mirror 15 kV (pulse)

tube LIFT cell: with grids entrance exit grid grid TOF-based MS/MS (Medzihradszky, K. F.; Campbell, J. M.; Baldwin, M. A.; Falick, A. M.; 15 kV (pulse) Juhasz, P.; Vestal, M. L.; Burlingame, A. L. Anal. Chem. 2000, 72, 552).

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49 Ion Detection

Faraday cup Electron multiplier Channeltron Microchannel plate, MCP Induction (FT-ICR, O) “Daly” detector Array detectors Photographic plate Hybrid detectors, e.g. MCP-diode array

Detectors. Data Acquisition. Vacuum: Principles & Techniques. 8 Coupling of Separation and MS (on-line, off-line, chips) Mass spectrometry of biomolecules 2018 295 Mass spectrometry of biomolecules 2018 296 295 296

Faraday Cup Electron Multiplier

conversion dynode, -2 kV dynodes -10 V + etc. + collector, 0 V -100 V

• Analogy of photomultiplier; conversion dynode (instead of photocathode) - Very small currents followed by a series of dynodes with decreasing potential and a collector. E.g.: 100 ions/s  current = 1.6 x 10-17 A Electron multiplier is not encapsulated in a glass bulb. • Conversion of ions to electrons on the first dynode (e.g. Cu-Be alloy). Multiplications of electrons on dynodes. Electron capture on collector, generation of current. • Gain ~ 106

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Channeltron Microchannel Plate (MCP)

2 MCP’s w/microchannels 0 V Dimensions of MCP -100 V • thickness ~1 mm + - • diameter 1 - 10 cm. collector + Microchannels + • slightly tilted, 0 V -3 kV + • diameter ~ 3 - 20 mm. + • Glass tube with a layer of semiconducting PbO inside and applied voltage • covered with a PbO layer, on the ends. Current flowing through the PbO layer forms potential ... formation of gradient along gradient along the tube (rather than dynodes kept at discrete potential microchannels (electron -2.2 kV -200 V values). Electron multiplication similar to electron multiplier. -1.2 kV multiplication on continuous dynodes) • Chevron structure in sets of more MCP’s. • Conversion dynode necessary for detection of positive ions. Array detector with large sensitive area useful in TOFMS Gain of single MCP ~103, dual MCP ~106 or higher. • Gain ~ 106

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50 “Daly” Detector Array Multichannel Detectors

polished metal … e.g. for magnetic sector electrode, -30 kV Photographic plate + - historical detector, time-consuming processing thin metal film phosphorus layer Combination of MCP and diode array phosphorus photomultiplier + diode + array 2 MCP’s optical fiber Ion → electron(s) → 4 photons (phosphorus) bunch + Very low noise. + Very high gain (photomultiplier usually works in photon counting mode). Incorporation of optical fiber bunch for higher positional resolution. - Light interferes. - Very low pressure (p < 10-5 Pa) necessary. Other detectors ... combination of the detectors discussed before E.g. MCP and phosphorus scintillator … isolation of high voltage

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Data Acquisition in MS Mass Spectrum Acquisition

ions charge, q I(m/z) = f(X) = f(t) conversion (conversion efficiency,amplification) electrons Scanning instruments Scanned X Scan period I = q/t (t ... time) MAG magnetic sector B, U ~ 1 s Q quadrupole filter U, V, f ~ 0.1 s current IT, LT ion traps U, V, f ~ 0.1 s

U = IR (R ... impedance) Direct signal acquisition in time: voltage TOF ~ 100 ms FT-ICR ion cyclotron ~ 1 s Different schematic recording device for photographic plate Other non-scanning instruments may use array detectors. and scintillating detectors

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Mass Spectrum Recording Devices

y-axis: current, voltage, charge, ions, counts??? digital converters • A/D converter (analog to digital) a.u. (arbitrary units) or relative intensity • T/D converter (time to digital) photographic plate chart recorder normalization of ion signal analog oscilloscope with a camera

%Int.

100

90

80

70

60

50

40

30

20

10 0 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500 Mass/Charge

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51 A/D Converter (ADC) A/D Converter

Measurement (digitization) of voltage Example: 2-bit converter with input voltage range 0-3V and sampling frequency 10 S/s

Basic parameters level high-bit low-bit number of levels = 22: number of bits (resolution) 0 0 0 sampling frequency, number of samples pre second [Sample/s] 1 0 1 2 1 0 period, T = 1/10 = 0.1 s max. frequency (cut-off frequency) 3 1 1 polarity: unipolar (negative), bipolar input voltage range S (V) S (#) max. input voltage A D 3 number of data point (memory length, buffer size) 2 stability 1 etc. 0 0 0.2 0.4 0.6 t (s) 0 0.2 0.4 0.6 t (s)

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Signal Precision and Accuracy Selection of ADC

10 Precision and (accuracy) of measurement given by number of bits of ADC Solution: 8 • higher detection range Example: for 8-bit ADC, 28 = 256 levels: • converter with higher number of bits 6 deviation might be up to 1/2 of the difference between two adjacent levels • signal accumulation (averaging) -1 -1 min. rel. deviation > (2x(number of levels - 1)) = (1/2x255) ~ 0.2 % 4

Iontovýsignál 250

2 number of bits 1 8 12 16 24 200

0 number of levels 2 256 4 096 65 536 16 777 216 150 100 102 104 100 min. rel. deviation (%) 50 0.2 0.01 8x10-4 3x10-6 m/z Iontovýsignál

Possible reasons: 50 dynamic range - 50 800 13 000 3 000 000 low signal level (rel. deviation < 10%) 0 converter with low number of bits 100 101 102 103 104 105 m/z Mass spectrometry of biomolecules 2018 309 Mass spectrometry of biomolecules 2018 310 309 310

Acquisition Speed ADC Speed

... given by sampling frequency of ADC Example 2: How fast ADC is needed for TOFMS in order to record ion flying 20 ms with resolution 2000, provided the peak should be Factors: scan period characterized by 10 points? scan range Signal required resolution 100% required quality of peaks (number of data points/peak) t = 20 ms, R = 2 000 t 50% 5 ns Example 1: R = m/m = t/(2t) Requirements on a Q: scan period = 0.1 s, unit resolution in m/z range = 200 – 2 000, >10 pts/peak. What is min. sampling frequency? t = t/2R = 20 ms/4000 = 5 ns (FWHM) 20 ms t min. sampling frequency = (2 000-200)x10/0.1 = 180 ksample/s memory length = (2 000-200)x10 = 18 000 samples entire (~10 points) … 10 ns  tsample = 1 ns (1 ns/sample) … for 16-bit converter: 36 kByte (1 data point = 19 bits or 2 bytes)

sampling frequency, f = 1/ tsample = 1 Gsample/s

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52 Influence of Sampling Frequency of ADC ADC: Current State of Art (in TOFMS) High number of bits: 24 bits 25000 25000 • but only at low sampling frequency (1 kHz) 2 Gsample/s 1 Gsample/s 20000 500 Msample/s

18000 High sampling frequency: 20 Gsamples/s 20000 20000 16000 • but only with low-bit ADCs (8 bits)

14000 15000 15000 High performance 12000 • data accumulation (averaging) directly in PC card • on-board compression (algorithms without and with data loss)

Ion (a.u.) signal Ion 10000 10000 10000 • fast data transfer from card to PC 8000 ... PCI card in computer, direct PC memory access

6000 5000 5000 10 ns 10 ns 4000 10 ns Time of flight (ms) Mass spectrometry of biomolecules 2018 313 Mass spectrometry of biomolecules 2018 314 313 314

T/D converter (TDC) SA (V) Analog spectrum 100 % Pulse counters (1 pulse) Impact of ion on detector → pulse → amplifier → discriminator → counter threshold signal level 0 0 20 40 60 t (ms) TDC (time to digital converter) 1-bit A/D converter SD1 (#) 2 levels: 0 a 1 1 Digitized spectrum (1 pulse)

Parameters: 0 0 20 40 60 t (ms) time resolution dead time Accumulation of n spectra SDn (#) number of channels n etc. Accumulated digitized spectrum (n pulses) 0 0 20 40 60 t (ms) Mass spectrometry of biomolecules 2018 315 Mass spectrometry of biomolecules 2018 316 315 316

TDC TDC

Only one of a group of ions is used during one acquisition period (single TDC with multiple channels channel TDC). ... to increase dynamic range ... using a multiple channel TDC in combination with a segmented detector Suitable for acquisition of relatively low signals with high repetition rate. Classical TOF detector Relatively low cost (compared to ADC).

Applications: MCP T/D 1 bit TOFMS with high frequency of extraction pulses (>kHz) ESI - oTOFMS AP-MALDI – oTOFMS TOFMS with low numbers of ions PD – TOFMS

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53 TDC Informatics & Bioinformatics

Segmented TOF detector Hardware: IBM PC platform most popular

T/D 1 bit Software: Windows XP T/D 1 bit 4 bits Seg. MCP T/D 1 bit T/D 1 bit Exponential growth of performance: Moore law

Ions hit randomly four segments with similar probabilities ...total ion fluence might be higher Example: Evaluation of MS/MS spectra using SEQUEST program

SCX - LC - MS/MS of Y2H yeast digest (the next slide)

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PC Cluster for evaluation of SCX-LC-MS/MS

SCX-LC 1x master PC (2 processors P4, 3 GHz) 8 x slave PCs (2 processors P4, 3 GHz) interconnected with 1-Gbit network

MS/MS

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Vacuum Instrumentation Background Gas Flow Classification

Evangelista Torricelli (1608-1647) 1. Turbulent flow (p > 10 kPa) Pressure unit: 1 Torr = 1 mm Hg very short l many collisions between molecules Pressure, p 2 p = force/area [Pa, N/m ] 2. Viscous, continuous flow (p = 1000 - 0.1 Pa, „rough" vacuum) 1 atm = 760 Torr = 101 325 Pa = 101.325 bar = 14.70 psi 1 Torr = 133 Pa l = mm - cm: still much shorter compared to dimension with vacuum apparatus Vacuum more collisions molecule – molecule than molecule – wall gaseous state with p < 101325 Pa 3. Molecular flow (p < 0.01 Pa, “high" or “ultra high” vacuum, UHV) Mean free path of a molecule l > m: Mean path of a molecule (atom, ion, particle) travels between 2 collisions collisions of molecules with walls of vacuum apparatus prevail l = (2ps2n )-1 usual situation in mass spectrometer l(cm) = 0.66/p(Pa) ... only rough estimation for air at 25°C (exceptions: collision cells, CI, ion mobility spectrometry)

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54 Gas Flow Regimes Pumping Speed Gas Flow Rate, Q Q Pumping speed, S S = [S] = L/s [Q] = Pa m3/s, Pa L/s p 1/S = 1/S + 1/C C … conductance of a tube with diameter D and length L chamber pump tot C = f(D, L, p, gas, T), [C] = L/s Appropriate design: Ctot > Spumpa i.e., using expensive efficient pump together with long narrow tubes or At molecular flow regime, conductance is not a function of pressure p: hoses does not make sense C  D3/L (approximation) Notes: At viscous flow, conductance C depends on pressure p: • In molecular flow regime pump does not suck gas. Pump acts as a trap; molecules that come to the pump will not return to chamber. C  pD4/L (approximation) • Pumping speed varies with gas type (drops with gas M.W.). • Pressure is not constant throughout the system, positioning of pressure Design of a vacuum apparatus sensor matters. • Short thick connectors  C  faster pumping. • Outgassing. Time needed for reaching sufficient vacuum is prolonged by • In serial connections, the thinnest tube (bottleneck) limits pumping presence of volatile compounds adsorbed (water, sample, finger prints)

of the entire system: 1/Ctot = S1/Ci and absorbed in the system (gases and water in gaskets, plastics).

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Vacuum Pumps Rotary Oil Pump

Mechanical pumps (rotary oil, membrane, roots) • One, two or even more stages.

Diffusion pump • Rotor immersed in oil - min. pressure limited by oil vaporization - trap for oil vapors needed (to keep apparatus clean - oil contamination!) Turbomolecular pump - trap for oil mist on outlet needed (to keep operator’s lungs clean) - periodic oil changes required Cryotrap, sorption traps, chemisorptions traps • Pressure > 0.1 Pa … “rough" vacuum, pumping speed, S: 1 – 500 L/s (typical)

• The most common rough pump in commercial systems.

• Other mechanical pumps: membrane pump (no contamination, but higher limit p and lower pumping speed)

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Turbomolecular Pump Diffusion Pump

• Series of turbines rotating at extremely high speed (up to 50 000 rpm). • Special non-volatile oil heated with a heater at the base of the pump, Gas molecules are reflected by turbine blades. Perimeter speed of heated oil vapors flow upwards through a central tube, stream as circular turbine blades > speed of gas molecules. jets downwards on cooled walls, condense and flow down to the base. Molecules of pumped gas are drawn by oil jets. • Dependence of inlet and outlet pressure on gas molecular weight: • Often used in combination with a trap cooled with water or liquid nitrogen; ln (p /p )  m this may prevent oil backstreaming or increase pumping speed (solvent out in vapors). • UHV (ultra-high vacuum) pump, limit pressure ~10-6 Pa, pumping speed -8 • UHV pump, limit pressure ~10 Pa up to ~ 10 000 L/s. • Reliable, low maintenance. • Pumping speeds up to ~ 3000 L/s • Slow start, backstreaming.

• Commonly used UHV pump in commercial systems. Cryotrap • Sorbent (e.g. charcoal) cooled down to 4 K (liquid He). • Regeneration necessary.

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55 UHV System Heating Pressure Sensors (Pressure Gauges)

Molecules of background gas (especially water) adsorb on inner walls of Hydrostatic pressure gauge mass spectrometer. Slow desorption of gas molecules continues to elevate • Pressure determined from difference of liquid levels. pressure and prolongs pumping time. Pumping can be fastened by heating • Range down to 1 kPa, or 0.1 Pa for special constructions. of the entire system (e.g. by wrapping the system using a resistively-heated tape), which shift equilibrium from adsorption to desorption. Mechanical gauge • Determination of pressure from deviation of elastic membrane. • Reading of the deviation - mechanical - change of capacitance (range 105 – 10-2 Pa)

Thermocouple gauge (Pirani) • Bimetallic thermocouple and resistively heated filament. • Heat transfer from filament to thermocouple dependent on pressure and type of gas. • Range p: 100 Pa - 0.1 Pa or 100 kPa - 0.1 Pa (convectron).

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Pressure Gauges Coupling Separation to Mass Spectrometry

Ion gauge Why separation? Bayard & Alpert: ion tube with heated cathode. • Three electrodes in a glass bulb: spiral (+), collecting wire (-) in the center ... analysis of very complex samples, mixtures of many analytes, e.g. of spiral and heated filament (-) outside the spiral (heated cathode). common sample in proteomics contains >102 peptides • The same setup as in the case of open ion source: electron from heated filament are extracted towards the spiral, ionize gas, ions and electrons Simple MS or even MS/MS is not powerful enough for several reasons: are captured on the wire and current is measured. p = f(current). Note: • high probability of occurrence of 2 or more analytes with the same m/z current depends on gas composition! • overlap of isotopic envelopes of analytes with close m/z • Pressure range: 10-2 - 10-10 Pa (high vacuum, UHV) • mutual ion suppression • limited dynamic range of mass spectrometer Penning: ion tube with cold cathode. • resolution of the 1st stage at MS/MS often unsatisfactory (R ~ 500) • ions are formed in electric discharge in magnetic field. 1 • removal of contaminants • Pressure range: 1 - 10-4 Pa, some constructions down to 10-10 Pa. • source of additional information about analytes

Note: commercial spectrometers employ thermocouple and ion gauges.

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Ion Suppression in Peptide Mixtures Classification of Interfaces

0.25 Gas → vacuum AIII c(peptides) = 1 mM 0.20 AF 1-7 • jet separator for analyte enrichment in carrier gas (particle beam) AII 0.15 AF 3-8 AI • membrane interface • capillary column or classical column + splitter 0.10 AG 1-14 0.05

0.00 Liquid → vacuum 600 800 1000 1200 1400 1600 1800 2000 • API ionization techniques (ionization directly from liquid at atmospheric 0.25 pressure) AII (off scale) 0.20 • Flow probes (FAB) Ion Intensity (V) Intensity Ion c(peptides) = 1 mM • Sample deposition on a target 0.15 besides c(AII) = 50 mM 0.10 - moving belt (deposition at atmospheric pressure – transport, differential pumping - ionization) 0.05 - Fraction collection 0.00 600 800 1000 1200 1400 1600 1800 2000 m/z

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56 Separation - ESI MS Separation of biomolecules: MALDI or ESI ?

Most common techniques: ESI MS

2D GE - MS + Separation compatible + Commercially available, routinely used • 2-dimensional gel electrophoresis on polyacrylamid gel + ESI-MS/MS well established • band excision and protein processing + High sensitivity • common planar technique for protein separation + Easy automation - No sample archiving only on-line RPHPLC – ESI MS - Minor components not analyzed in MS-MS mode - Quantification vs. MS-MS • liquid chromatography on reverse phase – ESI MS - LC gradient often slow • common column technique for peptide analysis MALDI MS Data dependent scan ... one MS scan followed by few MS/MS scans (discussed again later) + Simpler spectra + Separation decoupled from mass spectrometry + Capability of sample archiving

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MALDI Interface MALDI Interface Off-line Common strategy: Targets with hydrophilic spots (anchorchip). 1. Deposition of liquid sample on target Micromethods using piezoelectric pipetors and microtargets. 2. Sample drying Deposition using electrospray 3. Target insertion into mass spectrometer and analysis

Regime On-line • On-line Flow probe with or without frit • Off-line Nebulizer for aerosol generation • In-line In-line Addition of MALDI matrix ROBIN Interface • Mixing with analyte solution (sheath flow, T, liquid junction) Deposition on target at subatmospheric pressure, moving belt interface • Deposition of analyte solution on target precoated with matrix layer Off-line vs. on-line Collection of eluent • Discrete fractions + Separation decoupled from mass analysis • Continuous streak + Sample archiving

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Sample Archiving (MALDI) Flow Probe (on-line)

Strategy for separation - MALDI MS and MS/MS

1. Deposition of eluent on target 2. MALDI MS analysis (<10% sample consumed) 3. Analysis of MALDI MS data 4. MALDI MS-MS selected peaks (detail analysis)

(The overall procedure may be interrupted anytime after the first step.)

(Whittal, R. M., Russon, L. M., Li, L. J. Chromatogr. A 1998, 794, 367-375. )

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57 Nebulizer for Aerosol Generation (on-line) Moving Belt Interface (in-line)

Interface for continuous introduction of nonvolatile sample in volatile solvent into mass spectrometer

warm N2 LC eluent heated slits IR heating element

ion source

moving polyimide belt to vacuum pumps

(McFadden W. H., Schwartz H. L. and Evans S. J. Chromatogr. 122, 389, (Fei, X., Wei, G., Murray, K. K. Anal. Chem. 1996, 68, 1143-1147. ) 1976.) Mass spectrometry of biomolecules 2018 343 Mass spectrometry of biomolecules 2018 344 343 344

ROBIN (Rotating Ball Inlet), in-line Vacuum or Subatmospheric Deposition (in-line, off-line) 2ND GENERATION VACUUM DEPOSITION INTERFACE

source chamber (p ~ 10-6 Torr) atmospheric pressure deposition support repeller

target repeller center coil (tape guide) probe with capillary source coil rubber propelled wheel shaft interface flange Mylar tape

Orsnes, H., Zenobi, R. Chem. Soc. Rev. 2001, 30, 104-112.

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Automated Fraction Collection (off-line) Microfluidic Devices and Mass Spectrometry Why connect micro and macro? ... possible advantages of mdevices: • integration of more steps, “lab on a chip“ (sample preparation, purification, injection, separation...) • paralell analysis (repeated motif on a single device) • low cost – single use mdevice (serial production) • all chemistry on the mdevice, MS as a detector

Basic types: 1. 2D array • affinity array: first selective preconcentration and/or capture of specific compounds, then MALDI MS directly from the mdevice • array of mvials with tips for ESI: higher reproducibility

2. Fluid mdevices (mdevices with channels) • systems for paralell analysis – for ESI, MALDI ... • systems integrating several steps E.g. commercially available Probot (www.lcpackings.com)

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58 Micromethods Example of mdevice for Paralell Analysis

capillaries for infusion into mass spectrometer

common liquid junction

separation channels

capillaries for sample injection from a microtitration well plate (Laurell, T.; Nilsson, J.; Marko-Varga, G. Trends Anal. Chem. 2001, 20, 225-231. ) (Courtesy of T. Rejtar) Mass spectrometry of biomolecules 2018 349 Mass spectrometry of biomolecules 2018 350 349 350

mdevice vs. Conventional Device Biological applications: Proteins and Peptides. Protein 9 Identification: Peptide Mapping, Sequence Tag, Accurate Mass Tag

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Biological Applications of MS Proteomics

Genome, proteome, metabolome. Proteome analysis much more complex than genome analysis: Small organic molecules, biomolecules, drugs, petrochemical products. - more building blocks (amino acids) Biopolymers (DNA, proteins, carbohydrates). - higher variability – modification of amino acids Synthetic polymers. - no existing amplification method for proteins analogical to PCR - low levels of many proteins (e.g. regulatory proteins) Proteomics

Characterization of peptides and proteins – protein complement of genome. Differential proteomics Nowadays the main and the most perspective application of mass Determination of relative protein expression (presence or absence) in spectrometry. v influenced and healthy organism (organ, tissue, cell).

Genome  proteome. Level of protein expression (gen → protein) varies. Functional proteomics Determination of all interactions (protein-protein, protein-DNA, etc.) in given Genome is static, proteome dynamic: expression depends on type and organism (organ, tissue, cell). function of protein, location in cell, state and health of cell. Function of organism is directly related to proteome.

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59 IUPAC: Amino Acids IUPAC: Amino Acids

Trivial name Symbols Formula Trivial name Symbols Formula

Alanine Ala A CH3-CH(NH2)-COOH Isoleucine Ile I C2H5-CH(CH3)-CH(NH2)-COOH Arginine Arg R H2N-C(=NH)-NH-[CH2]3-CH(NH2)-COOH Leucine Leu L (CH3)2CH-CH2-CH(NH2)-COOH Asparagine Asn H N-CO-CH -CH(NH )-COOH N 2 2 2 Lysine Lys K H2N-[CH2]4-CH(NH2)-COOH Aspartic acid Asp HOOC-CH -CH(NH )-COOH D 2 2 Methionine Met M CH3-S-[CH2]2-CH(NH2)-COOH Cysteine Cys C HS-CH2-CH(NH2)-COOH Phenylalanine Phe F C6H5-CH2-CH(NH2)-COOH Glutamine Gln Q H2N-CO-[CH2]2-CH(NH2)-COOH Glutamic acid Glu HOOC-[CH ] -CH(NH )-COOH Proline Pro P E 2 2 2

Glycine Gly G CH2(NH2)-COOH Serine Ser S HO-CH2-CH(NH2)-COOH

Histidine His H

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Development of Techniques for Ionization of IUPAC: Amino Acids Peptides and Proteins Trivial name Symbols Formula

1963 LDI Laser Desorption/Ionization (R. Honig) Threonine Thr T CH3-CH(OH)-CH(NH2)-COOH 1969 FD Field desorption (H. D. Beckey)

Tryptophan Trp W 1974 PD Plasma desorption (R. D. McFarlane) 1976 SIMS Secondary ion mass spectrometry (A. Benninghoven)

1981 FAB Fast atom bombardment (M. Barber) Tyrosine Tyr Y 1984 ESI Electrospray (J. B. Fenn)

1988 MALDI Matrix-Assisted Laser Desorption/Ionization Valine Val V (CH3)2CH-CH(NH2)-COOH (M. Karas & F. Hillenkamp, K. Tanaka) 1994 nano-ESI Nanoelectrospray (M. S. Wilm, M. Mann)

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Current Ionization Techniques in Proteomics Mass Spectrometers in Proteomics

FAB MALDI MS high sample throughput max. m ~ 10 000 Da TOF LD ~ 20 pmol (classical FAB), < 1 pmol (CF-FAB) ESI MS-MS structure elucidation QqTOF, IT, FT ICR ESI max. m ~ 100 000 Da (z >> 1) LD < 10 fmol (routine) New instrumentation MALDI TOF-TOF, MALDI LIFT TOF LD ~ amol or 10-9 M (selected applications) MALDI QqTOF

MALDI • fast identification in MS mode max. m ~ 106 (practically unlimited – TOF analyzer) • option of later detail analysis in MS/MS mode relatively least vulnerable to contaminants (result-dependent analysis) LD < 10 fmol (routine) LD ~ amol or 10-9 M (selected applications)

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60 Separation Methods in Proteomics Gel Electrophoresis (GE)

GE 2D SDS PAGE gel electrophoresis on polyacrylamide gel 2D: 2-dimensional electrophoresis HPLC 1. dimension: according to pI (isoelectric focusation) high performance liquid chromatography on reverse phase 2. dimension: according to size of SDS after denaturation (charge/size = const.) IEC ion-exchange chromatography PA: polyacrylamide gel as separation medium AC affinity chromatography • Useful for separation of tens of thousands proteins, for very simple mixtures 1D GE sufficient CE • Fast identification and protein characterization on 2D gel is the most capillary electrophoresis common analysis of current proteomics • Approximately 5% proteins and 30% peptides exhibit abnormal migration Centrifugation • PTM’s influence apparent protein m (errors up to 50%) common centrifugation, gradient centrifugation • Problematic protein transport from gel matrix (electroblot on a membrane)

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Example: 2D GE of Human Plasma RP HPLC

• Column: classical, capillary (f ~300 mm) or nano LC (f ~75 mm)

• Packing: C18, particle size: 3 – 10 mm (RP ... reverse phase)

• Gradient elution, e.g.: - organic phase: ACN (acetonitrile) + 0.1% TFA (trifluoroacetic acid) - aqueous phase: 0.1% TFA - start: 10% organic phase + 90% aqueous phase - end: 80% organic phase + 20% aqueous phase

• Organic solvent promotes peptide solubility, enables detection in UV (230 – 240 nm) and evaporates fast, which is convenient for fraction collection, e.g. in MALDI MS.

• Standard technique of column separation for peptides and proteins

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Example: RP HPLC of BSA Digest Enzymatic Protein Digestion enzyme protein peptide mixture

Reasons for moving from high m/z region to low m/z region: 1. Better parameters of mass spectrometers (higher resolving power, precision and accuracy of m/z determination, sensitivity) 2. Narrower isotopic envelope (simpler spectra, higher sensitivity)

Note.: digestion = enzymatic cleavage digest = mixture of protein fragments, products of digestion modifications before digestion: • reduction of -S-S- bridges using, e.g., dithiothreitol (DTT) • alkylation –SH using, e.g., iodoacetamide (IAA)  m: + 57 Da • purpose: „unfolding of protein“ ... better approach for enzyme

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61 Enzymatic Protein Digestion Enzymatic Protein Digestion in 2D Gel 1. Wash the gel slices for at least 1 hr in 500 microliters of 100 mM ammonium bicarbonate. Discard the Most commonly used enzyme is trypsin (modified, e.g. TPCK to suppress wash. 2. Add 150 microliters of 100 mM ammonium bicarbonate and 10 microliters of 45 mM DTT. Incubate at 60 chymotrypsin activity, methylation etc.). degrees centigrade for 30 min. 3. Cool to room temp and add 10 microliters of 100 mM iodoacetamide and incubate for 30 min in the dark at room temperature. Other enzymes and reagents: lysine, chymotrypsin, CNBr etc. 4. Discard the solvent and wash the gel slice in 500 microliters of 50% acetonitrile/100 mM ammonium bicarbonate with shaking for 1 hr. Discardthe wash. Cut the gel into 2-3 pieces and transfer to a 200 microliter eppendorf style PCR tube. 5. Add 50 microliters of acetonitrile to shrink the gel pieces. After 10-15 min remove the solvent and dry the Products of enzymatic cleavage gel slices in a rotatory evaporator. 6. Re-swell the gel pieces with 10 microliters of 25 mM ammonium bicarbonate containing Promega • specific fragments of analyzed protein modified trypsin (sequencing grade) at a concentration such that a substrate to enzyme ratio of 10:1 has been achieved. (If the amount of protein is not known, add 0.1-0.2 microgramsof modified trypsin in 10 E.g. trypsin cleaves on C-terminus of amino acids K or R, if P is not a microliters of 25 mM ammonium bicarbonate).After 10-15 minutes add 10-20 microliters of additional neighbor. (Actual rules more complex): buffer to cover the gel pieces. Gel pieces need to stay wet during the digest. Incubate 4 hrs to overnight at 37 degrees Centigrade.Proceed to step 8 if further extraction of the gel is desired (recommended)- N terminus-X-X-X-X-X-K-Y-Y-Y-Y-Y-Y-C terminus (YP) otherwise continue with step 7. 7. Approximately 0.5 microliters of the supernatant may be removed for MALDI analysis and/or the • non-specific fragments of analyzed protein supernatant acidified by adding 10% TFA to a final concentration of 1% TFA for injection onto a narrow- or microbore reverse phase column. (If necessary the sample's volume may be reduced~1/3 on a • artifacts (modification of amino acids, e.g. oxidative, due to PA gel etc.) rotatory evaporator.) • fragments of enzyme (autolysis) 8. Extraction (Optional)- Save supernatant from step 7 in tube X, and extract peptides from gel twice with 50 microliters of 60%acetonitrile/0.1% TFA for 20 min. Combine all extracts in tube X (using the same • keratin fragments ( ) pipet tip to minimize losses), and speed vac to near dryness.Reconstitute in 20 microliters of appropriate solvent. Proceed with chromatography or MALDI analysis. (http://www.abrf.org/ResearchGroups/ProteinIdentification/EPosters/pirgprotocol.html) Mass spectrometry of biomolecules 2018 367 Mass spectrometry of biomolecules 2018 368 367 368

Strategy of Analysis in Proteomics Strategy of Analysis in Proteomics

Basic Analysis Types Top-down • protein isolation 1. Identification (confirmation of presence of a known protein in sample) • protein processing, enzymatic cleavage 2. Relative quantification in differential proteomics • analysis of peptides (MS, MS/MS) 3. Sequencing of unknown protein/peptide (de novo sequencing)

Sample is usually mixture of many proteins/peptides Bottom-up       • enzymatic cleavage Analysis is based on combination of separation with MS and MS/MS • separation of peptides • MS and MS/MS analysis of peptides

Identification Amino acid sequence of many proteins has already been described and it is not necessary to analyze it again.

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Some Useful Terms Protein Identification

genotype genetic “equipment” of an organism, disposition Information known prior analysis: phenotype actual state of organism, result of interaction • Sample source (organism) with environment • Isoelectric point (pI) from 2D PAGE in vivo in living organism • Molecular weight of protein from 2D PAGE, or possibly from MALDI MS in vitro outside living organism, in artificial environment • N- and/or C- terminal portion of sequence (Edman degradation) in silico in computer ... The information is not sufficient for protein identification

Methods for protein identification A. 2D GE + X + MS (peptide mass fingerprinting, peptide mapping) B. 2D GE + X + RPHPLC + MS and MS/MS (sequence tag, AMT) C. X + 2D LC (e.g. IEC and RHPLC) + MS and MS/MS http://www.meta-library.net/gengloss D. X + AC (afinity chromatography) + MS and MS/MS (IMAC, ICAT) E. Strategy using X and isotopic reagents/standards X ... selective enzymatic cleavage

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62 Protein Identification Peptide Mass Fingerprinting, PMF (Peptide Mapping) • MS is used to obtain information specific for protein Confirmation of protein identity using MS of products of enzymatic cleavage • Comparison of specific characteristics of analyzed protein with a protein 1. Separation (and possibly quantification) of proteins: 2D GE library (containing specific characteristics) of known proteins. Specificity is based on: 2. Band excision, modifications, enzymatic cleavage 1.Analysis of more peptides related to the protein peptide mass fingerprinting (peptide mapping), MS analysis of protein band excision products of enzymatic cleavage of the protein extract isolated protein 2.Detailed analysis of a single peptide of the protein (in gel) a) sequence tag, MS-tag – MS/MS analysis of a portion of the protein cells, (fragmentation of a peptide in gaseous phase) 2D gel tissue chemical modification and b) accurate mass tag, AMT – determination of amino acid composition of electrophoresis a portion of the protein (accurate m of product of enzymatic cleavage) selective cleavage (e.g. trypsin, CNBr) • Requires accurate MS analysis and a quality database • MS-based identification is much faster and more sensitive than chemical methods, such as Edman degradation) • Negative result of database search may mean new protein discovery!!! peptide fragments (protein digest) Mass spectrometry of biomolecules 2018 373 Mass spectrometry of biomolecules 2018 374 373 374

Peptide Mass Fingerprinting, PMF Example of Database Search: MS Fit (Peptide Mapping) 3. MS analysis and evaluation of results Input:

peptide fragments (protein digest)

MALDI MS separation - ESI MS ESI MS

m/z m/z

search in protein database ESI MS-MS (identification of known proteins) of selected peptides

structure (mutations, new proteins)

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Example of Database Search: MS Fit Drawbacks of PMF

Output: • Sample with a single protein needed, if possible, max. 2-3 proteins before cleavage

• m/z accuracy < 100 ppm or even better (<10 ppm) needed

• Better m/z accuracy  more confident answer from database search

• Absence of peptides in digest usually lower problem than presence of unexpected peptides

• Usually 4-5 peptides (in conjunction with high mass accuracy) sufficient for confident protein identification MS Fit – Protein Identification from m/z values of peptide fragments using MS Fit program on http://prospector.ucsf.edu

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63 PMF: Theory and Practice Sequence-Tag Method (MS-Tag)

Extra (unexpected) peaks • Protein identification based on knowledge of a portion of short portion of • non-selective cleavage (e.g. due to chymotryptic activity of trypsin) protein sequence (sequence-tag, MS-tag), usually sequence of a single • impurities (e.g. keratins) peptide • unsatisfactory protein isolation (additional proteins in band) • enzyme autolysis • Precise determination of amino acid sequence of a protein fragment from digestion – usually using ESI-MS/MS or MALDI-PSD-MS mass spectrum • post-translational modifications (PTM’s), artifacts or peak list

Missing peaks • Required length of sequence 3 or more amino acids • low-soluble peptides • adsorption of peptides • The longer sequence is known, the more unambiguous identification • mutual suppression of peptides • non-selective cleavage • The known portion of sequence is being searched for in protein databases • insufficient digestion (sterical restrictions) • PTM’s, artifacts • Other information of protein: organism, M.W., pI (both from 2D GE), etc.

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Basic Strategy of MALDI MS and LC - ESI MS 2D LC/MS/MS Schematics

peptide mass fingerprinting

MS/MS: MS-tag

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Experimental Setup for 2D LC/MS/MS (1) Experimental Setup for 2D LC/MS/MS (2)

1D: Chromatography on ionex 2D: RHPLC (2 HPLC columns ... higher productivity)

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64 Control of 2D LC/MS/MS Experiment Results of 2D LC/MS/MS Experiment

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LC-ESI MS and MS/MS: Accurate Mass Tag (AMT)

• Identification of protein on basis of knowledge of accurate mass of a Dynamic analysis LC-ESI MS/MS (data-dependent analysis): single peptide fragment (after enzymatic digestion of the protein) • One MS sken followed by few MS/MS scans of main precursors • In the case of more complex mixtures even MS3 scans possible (e.g. for • Accurate determination of protein based on mass of a single peptide detection of phosphorylation) using FT ICR MS atom H C N O S m [Da] 1.0078246 12.000000 14.00307 15.994915 31.972072

• Typical number of amino acid residues in peptide chain ~ 10. Accurate

mass of amino acids – e.g. mmono(G)=57.02146, mmono(A)=71.03711 etc.

• With precision of determination m/z < 1 ppm, there is high probability that the measured mass correspond only to a single amino acid composition as shown on the next slide

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AMT AMT početpeptidů

m/z m/z MS of mixture of all possible 10-mer peptide at various values of R (and m/z accuracy): 103 (1000 ppm) (A), 105 (100 ppm) (B), 106 (10 ppm) (C), and 107 (0.1 ppm) (D). Zdroj: T. P. Conrads, G. A. Anderson, T. D. Veenstra, L. Paša-Tolić and (T. P. Conrads, G. A. Anderson, T. D. Veenstra, L. Paša-Tolić and R. D. Smith R. D. Smith Anal Chem. 2000, 72, 3349-3354 Anal Chem. 2000, 72, 3349-3354.) Mass spectrometry of biomolecules 2018 389 Mass spectrometry of biomolecules 2018 390 389 390

65 Other Strategies of Proteome Analysis 10 • Not aimed at complex proteome analysis

• Based on column separation techniques (rather than on 2D GE)

• Simplification of the original mixture – selection of specific proteins/peptides, e.g. containing cystein (ICAT) or phosphorylated amino acid (IMAC) etc.

• Possible losses due to incomplete analysis are not dramatic and are compensated for by simpler and shorter analysis

• Additional tricks, such as use of isotope labels provide quantitative information about analyte Proteins and peptides. Isotope Labelling. ICAT, iTRAQ. Sequence Determination. Post-translational Modifications

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Isotope Labels MS of biomolecules Some Methods and Abbreviations

Unstable isotope labeling GIST Global Internal Standard Technology (2H, 13C, 15N) • in radiochemistry ICAT Isotope-Coded Affinity Tags (cystein-containing peptides capture on affinity columns) Stable isotope labeling PhIAT Phosphoprotein Istope-coded Afinity Tag (fosforylované peptidy) • common in MS IMAC Ion Metal Afinity Chromatography • basic approaches (phosphopeptide capture on affinity columns) - use of inner standards labeled with isotope AQUA Absolute QUAntification - incorporation of compound labeled with isotope into organism (synthesized isotopically labeled peptides as internal standards) (useful for differential proteomics of lower organisms) SILAC Stable Isotope Labeling with Amino acids in Cell culture - derivatization of analyte using reagent labeled with isotope (culture growth in normal and enriched media) MUDPIT Multidimensional Protein Identification Technology (SCX – RHPLC – MS/MS) etc. etc.

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ICAT (Isotope-Coded Affinity Tags) ICAT

ICAT ... isotope-coded affinity tags (R. Aerbersold) Determination of differences in protein expression from relative signal of peptides

1. Protein fractionation 2. Enzymatic digestion of entire sample 3. Isotope labeling (ICAT) 4. Mixing and MS analysis

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66 ICAT ICAT Characteristics

Note Advantages • Proteins can be labeled before digestion (step # 2 and 3 exchanged) + Suitable for a range of protein samples (body fluids, cells, tissues, Drawbacks of 1st generation ICAT cultures) • Mass difference of the isotope labels was equal to 8, which might lead to + Significant simplification of mixture interferences in MS/MS spectra + Compatibility with other methods suitable for analysis of minor proteins • Slightly different retention of analytes labeled with light and heavy reagent. Result: the ratio b/w light and heavy form cannot be found from the ratio of ion intensities during HPLC MS/MS; integration across entire Disadvantages LC peak was necessary

- Relatively large label (~500 Da) 2nd generation ICAT - Not suitable for proteins without cystein (e.g. 8% in the case of yeast) • Use 9 13C atoms in the link (instead of 8 2H atoms) • The same elution profile of light and heavy forms and less interferences

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iTRAQ iTRAQ

- another technique of differential Example of two derivatization reagents proteomics with reporters 114 and 116: - increase of m/z of derivatized peptides the same (the same precursor, very similar separation) - different product ion m/z - 4 (or more) different reagents for peptide derivatization - the same mass of all labelled peptides, mass of “reporters“ 114, 115, 116 and 117

http://www.broadinstitute.org/scientific- community/science/platforms/proteomics/itraq http://www.proteome.soton.ac.uk/iTRAQ.htm

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Determinations of Protein/Peptide Sequence Determination of Peptide Sequence using MS

Classical analysis Combination of chemical cleavage and MS - Edman degradation 1. Generation of mixture of peptide fragments differing by one amino - Determination of terminals (N, C) acid:

a) phenyl isothiocyanate + A1A2A3.. → phenylisothihydantoinA2A3.. + A1

Disadvantages: analysis time, relatively high cost and sample consumption phenyl isothiocyanate + A2A3.. → phenylisothihydantoinA3.. + A2 etc. Current strategy: combination of more methods phenyl isothiocyanate in low amounts as terminating reagent forms - preparative separation small fraction of phenylcarbamate of each peptide - enzymatic digestion b) alternative strategy uses application of carboxypeptidase for different time periods or in different amounts  formation of different digests - isotope labeling - MS 2. MALDI MS of peptide fragment mixtures. - MS/MS, ISF, PSD amino acid is determined from distance of adjacent peaks of the same n - MS type, sequence from the peak order

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67 Determination of Peptide Sequence using MS/MS

CID • low-energy CID currently most popular fragmentation method of peptides • IT, TQ, QTOF

ISD • requires isolation of pure analyte • MALDI TOF

PSD • lower quality of spectra compared to CID, sometimes spectra stitching needed • MALDI TOF Note • high-energy CID, SID, photodissociation, dissociation after electron capture are not used routinely yet • Leu/Ile … isomers, Gln/Lys ... isobars Patterson, D. H., Tarr, G. E., Regnier, F. E. and Martin, S. A., Anal. Chem.1995, 67, 3971.

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Peptide Fragmentation Schematics of Peptide Fragmentation

Fragmentation of 1 or 2 bonds in peptide chain + - fragments containing N terminus (a, b, c) x y z x y z x y z H 3 3 3 2 2 2 1 1 1 - fragments containing C terminus (x, y, z) - these ions may be formed even after breakage of 2 bonds of the chain R O R O R O R 1 2 3 4 - loss of NH3, H2O, CO2 H N - CH - C - NH - CH - C - NH - CH - C - NH - CH - COOH 2 Fragmentation of side chain (amino acid residue) a b c a b c a b c - usually loss of a portion of side chain of amino acid 1 1 1 2 2 2 3 3 3 - fragments type d, w, v

Inner fragments immonium ions of amino acids: - do not contain either N or C terminus ... lower analytical significance

Immonium ions - useful information about amino acid presence; m(IM)= m(AKres) - 27 Roepstorff & Fohlman, Biomed. MS 1984, 11, 601.

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Fragments of Peptides Schematics of Peptide Fragmentation

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68 Other Types of Peptide Fragments amino acid m(mono) m(immonium) m(accompanying ions) A 71.03712 44 m ... amino R 156.10112 129 59,70,73,87,100,112 acid residuum N 114.04293 87 70 D 115.02695 88 70 C 103.00919 76 E 129.04260 102 Q 128.05858 101 56,84,129 G 57.02147 30 H 137.05891 110 82,121,123,138,166 I 113.08407 86 44,72 L 113.08407 86 44,72 K 128.09497 101 70,84,112,129 M 131.04049 104 61 F 147.06842 120 91 P 97.05277 70 S 87.03203 60 T 101.04768 74 W 186.07932 159 77,117,130,132,170,171 Y 163.06333 136 91,107 V 99.06842 72 41,55,69

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Calculation of Molecular Weight CID Fragmentation of Peptide/Protein Types of ions in CID Sum of amino acid residua masses 1. b and y ion series + 2. accompanying peaks Mass of terminals: –17, NH3 loss from Gln, Lys, Arg –18, H O loss from Ser, Thr, Asp, Glu usually 1 Da (H) for N terminus (-NH2) 2 and 17 Da (OH) for C terminus (-COOH) a (tj. b – 28), satellite fragment series of b type (CO loss) 3. immonium ions of amino acids. + 4. internal fragments – especially from Pro in the direction of C terminus Mass of proton, 1 Da – ion is usually in [M+H]+ form (but e.g. 23 Da for adduct [M+Na]+ etc.) Precursor selection in CID MS/MS: z = 2: [M+2H]2+ Ion with two charges provides higher quality CID spectra compared to ions with z = 1 a 3

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ISD fragments

Fragmentation rules … according to relative bond strength. Typical products of peptide fragmentation:

c, y, z

H+ x3 y3 z3 x2 y2 z2 x1 y1 z1

R O R O R O R 1 2 3 4 ------H2N CH C NH CH C NH CH C NH - CH COOH

a b c a b c a b c 1 1 1 2 2 2 3 3 3

ISD. Analyte: 20 pmol KGF analog. Matrix: sinapinic acid. Laser: N2. (V. Katta, D. T. Chow, M. F. Rohde Anal. Chem., 70, 4410 -4416, 1998.) Mass spectrometry of biomolecules 2018 413 Mass spectrometry of biomolecules 2018 414 413 414

69 PSD Fragments

Typical products of peptide fragmentation: a, b, y, z, d.

Accompanying peaks –17 (loss of NH3) a –18 (loss of H2O). Immonium ions of amino acids. Internal ions – especially from Pro to the C terminus

(B. Spengler J. Mass Spectrom. 32, 1019-1036, 1997)

ISD Analyte: oxidized B chain of bovine insulin Matrix: thiourea Laser: Er:YAG (l = 2936 nm)

(http://medweb.uni- muenster.de/institute/impb/research/hillenkamp/publicat/abs-cm99.html) Mass spectrometry of biomolecules 2018 415 Mass spectrometry of biomolecules 2018 416 415 416

immonium ions of amino acids:

PSD, source: B. Spengler J. Mass Spectrom. 32, 1019-1036, 1997 Mass spectrometry of biomolecules 2018 417 Mass spectrometry of biomolecules 2018 418 417 418

Other dissociations

Electron capture dissociation, ECD Zubarev, R. A.; Kelleher, N. L.; McLafferty, F. W. Electron Capture Dissociation of Multiply Charged Protein Cations - a Nonergodic Process J. Am. Chem. Soc. 1998, 120, 3265-3266. McLafferty, F. W.; Turecek, F. Interpretation of Mass Spectra; Fourth ed.; University Science Books: Mill Valley, CA, 1993.

It provides different, often complementary fragments compared to CID and PSD

Electron transfer dissociation, ETD

PSD of bombesin, source: Bruker Daltonics, GmbH 2000 Mass spectrometry of biomolecules 2018 419 Mass spectrometry of biomolecules 2018 420 419 420

70 Evaluation of MS/MS spectra of peptides Determination of Protein Sequence

Manual and a semiautomatic deduction of sequence Procedure • localization of ion series of b and y type (accompanying series a) • identification of immonium ions 1. Enzymatic digestion • identification of inner fragments production of more types of digests to generate overlapping fragments • interpretation complicated by occurrence of other fragments - various length of digestion - various enzymes (trypsin, V8 ...) • not all peptides are product of specific digestion, e.g. not all tryptic fragments are terminated with with R or K on C-terminus - random cleavage, “shotgun sequencing” ... unambiguous determination of sequence of unknown peptide from CID is often impossible 2. Determination of at least partial sequence of peptides RPHPLC ESI MS/MS

Automated determination of sequence 3. Deduction of sequence using PC • MS/MS spectrum of unknown peptide is compared with a database of total sequence determined from combined portions of peptide sequences computer-generated spectra of all peptides, which have m/z of precursor • e.g. algorithm Sequest (J. R. Yates III et. al., 1994) ... much more successful than the deductive approach

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Helpful Techniques for Sequence Determination Determination of Protein Sequence

18 Protein hydrolysis in H2 O Example: time: identification of terminus: C-terminus will not be labeled Various extent of fragmentation Hydrolysis of protein in mixture of H 18O a H 16O (1:1) due to various length of 2 2 digestion and use of isotope 1. MS: dublets of tryptic peptides (except fragment of protein C-terminal) labels 2. MS/MS: both dublet peaks of a peptide selected as precursor for MS/MS; only fragments containing C terminus of a peptide will generate doublet Determination of terminus: 18 fragments ... easier to survey spectra hydrolysis in H2 O (C-terminus will not be labeled) Esterification (methylation) of carboxyl groups of a peptide

m = +14 / carboxyl group (-COOH → -COOCH3) comparison of original and resulting spectra similar technique based on derivatization of amino group of a peptide

Note 1: exchange reaction may run even on C terminus Note: exchange reaction may Note 2: more complex isotope patterns with masses M (2x16O), M+2 run even on C terminus (1x16O,1x 2x18O) a M+4 (2x18O) HK

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Mutation Detection Post-Translational Modifications (PTM)

Detection of exchange/absence of an amino acid(s) in protein chain • modifications after translation RNA → protein on ribosome • cannot be explained based on genome Procedure: • related significantly with protein function 1. Enzymatic digestion • more than 200 PTM types described (database Delta Mass) 2. Analysis of mass spectra • often only small fraction of protein modified  sensitivity - missing peptides • requires selective isolation of peptides with modified amino acid - superfluous peptides • bond between peptide and modifying group often weak 3. MS/MS analysis of unknown (superfluous) peptides and their comparison with normal proteins/peptides, analysis of peak shifts

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71 Post-Translational Modifications (PTM) Phosphorylation

Common PTMs: • reversible PTM related to cellular regulatory mechanisms • phosphorylation • glycosylation • monoesteric bond of phosphoric acid group to side chain hydroxyl of • acylation (fat acid esters, acetyl) 1. serine (167 Da) O 2. threonine (181 Da) • attachment of glycosylphosphatidyl inositol 3. tyrosine (243 Da) ⎯ NH ⎯ CH ⎯ C ⎯ • proteolytic products • carboxylation (of glutamic acid) R • deamidation of asparagine and glutamine MS/MS identification of phosphorylation O 1. Loss of H3PO4 (neutral loss scan: 98 Da) Some modifications might be quite complex, e.g. oligosaccharide usually provides also ion [M+H-98]+ HO ⎯ P ⎯ OH modifications of glycoproteins (many types of sugars and many sites on - 2. Detection of ion PO3 (79 Da) in negative mode protein that can accommodate sugars – O, N). For structural elucidation, Parent ion scan for m/z (product) = 79 O n combined methods using MS and enzymatic cleavage might be used (N- Other negative ions: H PO - (97 Da), PO - (63 Da) glycosidase, O-glycanase etc.). 2 4 2 3. Fragment peak shifts in MS/MS spectra

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Phosphorylation Example: ESI MS/MS of Oligopeptide with Monophosphorylated Serine

y15 y14 y13 y12 y11 y10 y9 y8 y7 y6 y5 y4 y3 y2 y1 Identification procedure: 1. Preparation of mixture of proteolytic peptides (containing phosphopeptides) Phe Gln Pse Glu Glu Gln Gln Gln Thr Glu Asp Glu Leu Gln Asp Lys

2. Separation of phosphopetides using e.g. HPLC, CE, affinity b2 b3 b4 b5 b6 x2.5 chromatography, such as Immobilized Metal Affinity Chromatography 100 (IMAC), see Porath, J. Protein Expression Purif. 1992, 3, 263. 2+ MH2 H3PO4

3. MS/MS analysis of phosphopeptides b2 y y2 8 y y 3 4 MH 2+ Relatively stable monoester bond y6 2  peak shifts in MS/MS spectra (m = + 80 Da) % y5 y11

y7 y9 y12 amino acid M (residue) M (monoester H PO ) y10 3 4 y1 b3 b5 pSer serine 87 167 y 13 y b6 14 threonine 107 187 b4

tyrosine 163 243 y15 0 m/z 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 Mass spectrometry of biomolecules 2018 429 Mass spectrometry of biomolecules 2018 430 429 430

Example: ESI MS/MS of Oligopeptide with Monophosphorylated Serine

Information sources: 2+ 2+ 1. Molecular peaks MH2 a (M - H3PO4)H2 , mass difference m = 98 (m/z = 49)

2. b-ions: m/z (b3-b2)= 167

3. y-ions: m/z (y14-y13)= 167

(credit: K. R. Jennings) Disulfide Bridge Analysis. Proteins. MS databases. 11 DNA, Saccharides, Synthetic Polymers. Ion mobility spectrometry (IMS). Mass spectrometry imaging (MSI). Mass spectrometry of biomolecules 2018 431 Mass spectrometryIsotope of biomolecules ratio 2018MS, preparative MS 432 431 432

72 Disulfide Bridge Analysis Influence of S-S Bridges cysteine (-SH) → cystin (-S-S-), intra- and inter- molecular bridges in ESI-MS

Structures of lysozyme (b−sheet and a− Number of cysteines – using alkylation of cysteine helix) stabilized by 4 S-S bridges • e.g. alkylation of a single cysteine with vinylpyridine increase mass of protein/peptide by 105 Da  number of cysteines = m/105 Reduction with 1,4-dithiothreitol (DTT) removes S-S bridges Localization of cysteines in protein chain Enzymatic cleavage and splitting of the digest into 2 aliquots:  1. aliquot ... MS analysis „Unfolded“ protein with more exposed 2. aliquot ... reduction -S-S- (e.g. with dithioerythritol), then MS analysis basic amino acid residues Comparison of the 2nd spectrum with the 1st spectrum: 1. m = 2 Da  peptide with an intramolecular -S-S- bond  2. the first peak disappear, two other peaks with lower m/z show up  Higher number of charges intermolecular bridge between 2 peptides Knowledge of sequence of amino acid (MS/MS) enables exact localization of -S-S- bridge in protein chain.

Mass spectrometry of biomolecules 2018 433 Mass spectrometry of biomolecules 2018 HK 434 433 434

Examples: Nanospray MS

, , 273, 1199. 1996

pH increase: „Unfolded“ protein with Science

more exposed basic

F. W. W. F. Valaskovic, G. A.; Kelleher, Kelleher, A.; L.; N. McLafferty, G. Valaskovic, amino acid residues =carbonic CA anhydrase, Cy =cytochrome

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Noncovalent Interactions

• Interactions with low mass ligands (+metal ions), other proteins, oligomers etc.

• The relation between occurrence of the complex in (g) and (l) phase is not always clear

• Complexes dissociate during ionization and MS analysis

Example: 1. Complexes of bovine hemoglobine are stabilized in form of tetramer by cross-linking with glutaraldehyde (bridges between lysine residues). Then Tetramer MALDI MS (see next slide) known from solution

2. ESI for complexes of proteins with ligands (e.g. metaloproteins with drugs) ... soft ionization, which barely removes water molecules. Study of interactions between 2 proteins is more difficult – use of soft extraction conditions, favorable for formation of complexes in solution.

Mass spectrometry of biomolecules 2018 437 Mass spectrometry of biomolecules 2018 438HK 437 438

73 Scientific Databases on Internet Scientific Databases on Internet

The databases below are just a few examples of many: Internet sources for protein identification using MS

NCBI (National Center for Biotechnology Information) • Eidgenossische Technische Hochschule (MassSearch) Medline: www.ncbi.nlm.nih.gov www.cbrg.inf.ethz.ch Scirus: www.scirus.com • European Molecular Biology Laboratory (PeptideSearch) ScienceDirect: www.sciencedirect.com http://www.mann.embl- heidelberg.de/GroupPages/PageLink/peptidesearchpage.html • Swiss Institute of Bioinformatics (ExPASy) www.expasy.ch/tools Databases for organic chemistry • Matrix Science (Mascot) www.matrixscience.com NIST Chemistry WebBook (NIST) • Rockefeller University (PepFrag, ProFound) prowl.rockefeller.edu Spectra Online (ThermoGalactic) • Human Genome Research Center (MOWSE) www.seqnet.dl.ac.uk Spectral Data Base System, SDBS (NI AIST) • University of California (MS-Tag, MS-Fit, MS-Seq) prospector.ucsf.edu • Institute for Systems Biology (COMET) www.systemsbiology.org • University of Washington (SEQUEST) http://fields.scripps.edu/sequest/index.html

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Scientific Databases on Internet Useful Tools on Internet

Other links: Tools UMIST (pepMAPPER) http://wolf.bms.umist.ac.uk/mapper/ European Molecular Biology Laboratory, Heidelberg (PeptideSearch) MS-Comp – suggestion of possible combinations of amino acids, mass www.narrador.embl-heidelberg.de table of dipeptides Global Proteome Machine MS BLAST 2 – short sequences (6 amino acids) http://h112.thegpm.org/tandem/thegpm_tandem.html BLAST a FASTA – protein homology

Protein (peptide) databases GlycoSuite DB, GlycoSciences – saccharides analysis Genpept – NCBI GenBank NBRF - National Biomedical Research Foundation Mass calculators (GPMaw, SHERPA, PAWS, MW Calculator) Swissprot - Swiss Institute of Bioinformatics Owl - Leeds Molecular Biology Database Group etc. Delta Mass – protein posttranslational modification database

Mass spectrometry of biomolecules 2018 441 Mass spectrometry of biomolecules 2018 442 441 442

MS of Nucleic Acids and Oligonucleotides MALDI MS of Nucleic Acids and Oligonucleotides

• Ionization: usually more efficient for negative ions. Analysis typically in Typical matrices negative mode. 3-hydroxypicolinic acid 2’,4’,6’-trihydroxyacetophenon • Ionizaton technique: MALDI (usually IR MALDI) picolinic acid

• Less successful than in the case of proteomics Typical applications • characterization of synthetic and biologic oligonucleotides (determination of M.W.) • Higher extent of fragmentation and more salt adducts • analysis of PCR products, mutation analysis (absence or exchange of nucleotides) • Proper desalting essential … prevention of formation of a series of • DNA sequencing adducts with Na and K - Sanger sequencing (classical method) - Sequencing using exonuclease • MALDI of heavy DNA (>100 kDa): linear TOF MS, IR laser, pulse - Fragmentation (in gaseous phase) extraction

Mass spectrometry of biomolecules 2018 443 Mass spectrometry of biomolecules 2018 444 443 444

74 MALDI MS of Nucleic Acids and Oligonucleotides IR MALDI MS of RNA

• MALDI MS spectra of very heavy NA (>2 thousands nucleotides)

• m/z accuracy: < 1% ... the best of current methods

• excellent sensitivity: < 1 fmol

Berkenkamp, S; Kirpekar, F; Hillenkamp, F Science 1998, 281, 260-262.

HK

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Sequencing NA using Exonuclease and MALDI MS Oligonucleotides Fragmentation in Gaseous Phase

Schematics of fragmentation of oligonucleotides in gaseous phase and marking of fragments

w3 x3 y3 z3 w2 w2 x2 y2 w1 x1 y1 z1

B1 B2 B3 B4 O O O

HO O ⎯⎯ P ⎯⎯ O O ⎯⎯ P ⎯⎯ O O ⎯⎯ P ⎯⎯ O OH

OH OH OH

a1 b1 c1 d1 a2 b2 c2 d2 a3 b3 c3 d3

(Juhasz, P.; Roskey, M. T.; Smirnov, I. P.; Haff, L. A.; Vestal, M. L.; Martin, S. A.; Anal. Chem. ; 1996; 68(6); 941-946) Mass spectrometry of biomolecules 2018 447 Mass spectrometry of biomolecules 2018 448 447 448

Saccharides Analysis Nomenclature for MS/MS Oligosaccharides

• Soft ionization methods: MALDI, ESI • Nomenclature of MS fragments with charge on non-reducing side: A, B and C; fragments with charge on reducing side: X, Y and Z depending • MSn for determination of sequence and structure whether a circle or a glycosidic bond is cleaved.

• Enzymes used for partial cleavage of saccharides prior MS analysis • Lower index of B, C, Y and Z fragment ions determines number of cleaved glycosidic bonds, upper index of A and X fragment ions • Common monosacharides: glucose, manose, galactose, fucose, N- determines bonds that were cleaved. Lower indexes a, b etc. give acetylglucosamine, N-acetylgalactosamine and N-acetylneuramine acid cleaved branch of nonlinear saccharides. (sialic acid). • Fragments B, C, Y and Z together with mass differences can be used for • Determination of complete structure of oligosaccharides is more difficult determination of sequence and branching. than in the case of proteins and nucleic acids - result of isomeric nature of sugars and possible branching - knowledge of building blocks and sequence is not sufficient for • MS is suitable for determination of optical isomery of glycosidic bond. structural elucidation. Type, location and optical configuration of each Method is based on selective oxidation of b-anomer of derivatized glycosidic bond needed hexoses using CrO3; ketoester is formed.

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75 MSn of Oligosaccharides MSn of Oligosaccharides

Ion fragments produced upon (a) MS 2 , (b) MS 3 and (c) MS 4 of permethylated maltoheptaose.

Source: Weiskopf, A. S.; Vouros, P. and Harvey, D. J. Rapid. Commun. Mass Spectrom. 1997, 11, 1493–1504.

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MSn of Oligosaccharides

Mass spectrometry of biomolecules 2018 453 Mass spectrometry of biomolecules 2018 454 453 454

MSn of Oligosaccharides MSn of Oligosaccharides

→

isomer isomer

→

I

1333.7

–

→ →

1169.5 1169.5

937.7

m/z

–

→ →

1169.5 1169.5

m/z m/z

—

, MS 4(b)

→

characterizationtheHexNAc5of Hex5

n n

MS MS fromchicken ovalbumin. (a) MS 3 937.7

Mass spectrometry of biomolecules 2018 455 Mass spectrometry of biomolecules 2018 456 455 456

76 Lipids Analysis of Synthetic Polymers

Fat acids, acylglycerols, derivates of cholesterol, phospholipids and MALDI TOFMS glycolipids high Mmax, simple spectra, number of charges z = 1 alternative of GPC

FAB, DCI (desorption chemical ionization), MALDI Analysis Building unit (monomer) Negative mode ([M-H]- ions detected) Terminal groups Absolute molar weight Polydispersion (mean m, dispersion width) Fragmentation phospholipid negative ion Challenges + + fragments of sacharides Adduct formation with Na , K Mass discrimination … ionization and detection efficiencies = f(m) Correct transformation from time to mass domain

Example: Carman Jr, H.; Kilgore, D.; Eastman Chemical Company, USA, ASMS 1998

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Ion mobility spectrometry (IMS) 4046 – 1970 Cohen, Karasek Characteristics

, 4039, • A technique bridging mass spectrometry and electrophoresis 41 • Separation as a result of ion friction with surrounding gas

2000, • Ions are not separated based on their m/z, but according to their mobility • Isomers or number of charges can be resolved (different migration of M+ 2+ and M2 ) Polymer • Resolving power of IMS low in comparison with MS

et al. et • In practical applications, it is often use as one of the techniques of multidimensional analysis. In combination with common MS or MS/MS, it , E. ca substitute liquid phase separation; time demands conveniently:

separation: >s, mobility: >ms, MS:

Esser : :

Applications Source • safety (explosives), military (simple mobile instruments for war agents) • study of compound conformation (small molecules, proteins) in gas phase Mass spectrometry of biomolecules 2018 459 Mass spectrometry of biomolecules 2018 460 459 460

Example 1: IMS – Q IM spectra of lysozyme depending on its Drift tube: equilibrium b/w electric and friction forces conformation rings with decreasing voltage similarly as in reflector elevated pressure

p ~ 10 Torr (He, Ar, N2), E ~ 10-50 V/cm, l ~ 10 - 100 cm

Clemmer D. E., Jarrold J. F. Mass Spectrom 1997, 32, 577- Clemmer D. E., Jarrold M. F. J. Mass Spectrom 1997, 32, 577-592. 592. Mass spectrometry of biomolecules 2018 461 Mass spectrometry of biomolecules 2018 462 461 462

77 Example 2: ESI-IT-IM-TOF MS of horse albumin digest Example 3: IMS – TOF

Another design of the drift tube: travelling waves of potential on the rings, similarly to waves on sea Smaller ions (with higher mobility) „surf“ more readily on the potential „waves“ and reach end of the drift zone in shorter time

Credit: waters.com Myung S. et al. Anal. Chem. 2003, 75, 5137-5145. Mass spectrometry of biomolecules 2018 463 Mass spectrometry of biomolecules 2018 464 463 464

IMS - TOF Example 4: DMS, FAIMS

Differential (ion) mobility spectrometry Field assymetric waveform ion mobility spectrometry - incorporation of a simple cell in front of the MS inlet - various designs of the cell

Triwave: ciprofloxacin, m/z = 332,141 Credit: waters.com

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IMS applications Mass spectrometry imaging (MSI)

MSI ... mass spectrometry imaging • Isomer separation according to conformation - direct visualization of analytes without any derivatization • Sample matrix removal - optional MS/MS • Available in combination with hi-res MS and MS/MS (TOF) - mass analyzers: TOF (speed), FT-ICR or O (resolving power) • Multidimensional analysis - resolution: lateral, depth profile and mass (resolving power) • More specific analysis - 3D MSI • Separation of intramolemolecular protonated species • Study of protonation location Ionization techniques (and limit of lateral resolution): • Various types of fragmentation SIMS: nm • Calculation of ion cross-section MALDI: mm DESI: 100 mm

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78 Laterak resolution in MSI MSI

1873 Ernst Abbe: diffraction limit

n sinθ numeric aperture (NA), ~ 1.4 - 1.6 in modern microscopes Abbe limit d ~ λ/2 ~ hundreds nm

Louis de Broglie (1929 Nobel prize) duality particle-wave

Vis photon ... 400 - 800 nm ... 2 - 3 eV ... optical microscopy, LDI, MALDI e-, Xe+ ... 5 keV ... 1 nm ... electron microscopy, SIMS

Stoeckli, M.; Staab, D.; Staufenbiel, M.; Wiederhold, K.H. Anal. Biochem., 2002, 311, 33-39 Note: DESI MSI is not limited by particle focusing

Mass spectrometry of biomolecules 2018 469 Mass spectrometry of biomolecules 2018 470 469 470

Isotope-ratio mass spectrometry (IRMS) Determination of isotope ratio 13C:12C

• accurate determination of “stable” isotope ratios • ideally using multicollector, i.e. magnetic sector with simultaneous detection of at more m/z or even more sophisticated instruments • ionization: EI, TI, SIMS, ICP • biosamples: usually gaseous analytes, sample combustion products etc.

(H2, N2, CO2, SO2) • standard for C: Pee Dee Belemnite (PDB), fossil belemnite, 13C:12C = 0.0112372

Examples 1. radiocarbon dating method, e.g. 14C (b counter, AMS – accelerator MS) 2. determination of biological sample origin based on isotope ratio C (d13C)

plant metabolism: C3 (85% plants: rice, wheat, potatoes, beet), C4 (corn, cane), CAM (desert plants)

Mass spectrometry of biomolecules 2018 471 Mass spectrometry of biomolecules 2018 472 471 472

Př.: Preparative mass spectrometry Preparative mass spectrometry

A. L. Yergey, A. K Yergey: Preparative Scale Mass Spectrometry: A Brief 1941 Project Manhattan – production of atomic bomb, Oak Ridge, TN History of the Calutron J. Amer. Soc. Mass Spectrom. 8, 1997, 943–953. 1942 E. Lawrence: possibility of use of a modified cyclotron for separation of isotopes A kittle bit of history: Calutron – California University at Berkeley + tron (instrument) 1931 Ernest Lawrence: cyclotron – proton accelerator, 1 MeV Homogeneous magnetic sector 180º 1938 Otto Hahn a Fritz Strassman: atom nuclei fission 1939 annexation of Austria and seizure of Czechoslovakia, brain drain to Up to 208 g >85% 235U/day in two stages (natural abundance 0.72%) USA (Leo Szilard, Enrico Fermi, Edward Teller, Eugene Wigner) Totally 43 kg/bomb during 18 months from > 5 tons of natural uranium 2. 8. 1939 Alberta Einstein letter to Franklin D. Roosevelt After 1945 used for preparation of isotopes for scientific purposes 1. 9. 1939 beginning of World War II and emigration 11. 10. 1939 the letter delivered to the president 12. 10. 1939 E. Fermi - $3000 grant on slow n0 interactions with U Alfred O. Nier prepared 235U a 238U using MS John Dunning proved that 235U is responsible for fission ... theoretical base for atomic bomb laid down

Mass spectrometry of biomolecules 2018 473 Mass spectrometry of biomolecules 2018 474 473 474

79 Scheme of 2nd stage Mass spectrometry of biomolecules 2018 475 Mass spectrometry of biomolecules 2018 476 475 476

Mass spectrometry of biomolecules 2018 477 Mass spectrometry of biomolecules 2018 478 477 478

Ultra-high “resolution” MS Resolving power vs. m/z

Convention R Mass analyzer R

Standard resolution 1 000 – 10 000 Q, IT, LIT High resolution 10 000 – 100 000 EB, TOF Ultra-high resolution 100 000 - … FT MS (ICR, O)

Note: TOF 1 “Resolution” = Resolving power O: ~ 푚/푧 Resolving power is function of m/z 1 FT-ICR: ~ Resolving power vs. scan speed (especially for FT-MS) 푚/푧 S/N increases with scan accumulation (all MS) 0 1000 2000 3000 m/z

Trend of the functions is important, not the absolute R value

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80 Example of a commercial FT-MS specifications Example of ultra-high mass resolution

Example: Q-O-LIT (Q-Exactive) Source: ThermoFisher Scientific

Mass spectrometry of biomolecules 2018 481 Mass spectrometry of biomolecules 2018 Source: ThermoFisher Scientific 482 481 482

Ultra-high resolution … a new dimension in MS Other topics

Fragmentation – gas phase chemistry Preparative MS - soft landing • Formula ID Izotopic dilution, quantification in MS • Isotope label techniques

Mass spectrometry of biomolecules 2018 Source: ThermoFisher Scientific 483 Mass spectrometry of biomolecules 2018 484 483 484

V. Questions and Consultation Questions and Consultation 12 Jan Preisler Dept. of Chemistry 43, tel.: 541 129 271, [email protected]

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Mass spectrometry of biomolecules 2018 485 Mass spectrometry of biomolecules 2018 486 485 486

81 Exemplary Questions Exemplary Questions

1. Compare MALDI and ESI (advantages vs. disadvantages). 7. Why instruments that can monitor both isotopes at the same time are 2. What can be said about 2 different spectra of the same (single) peptide? preferred for isotope ratio determination? (m/z-axis origin is not in zero, only a portion of the spectra is displayed.) 8. Can a quadrupole filter be used for analysis of a protein with M.W. 30 kDa? S S 9. What ionization method and what mass spectrometer would you suggest for: a) explosive detection on airport m/z m/z b) sequencing of short peptides 3. For 2 adjacent peaks of the same compound in ESI-MS, following values c) semiquantitative determination of ~70 samples in geologic sample were determined: (m/z)1~ 1001 and (m/z)2 ~ 1501. Determine m. + d) identification of elemenal impurities in thin surface layer of sample 4. Time of flight of ion C2H8O2N in TOFMS is 14.8 ms. Calculate the mass of a singly-charged ion, which hits the detector at 8.94 ms? What organic 10. What is the origin of Lorentzian peak profile in FT-ICR-MS? ion can it be? 11. What is mutual orientation of equipotential level of U and electric field 5. What variables do have impact on resolution in TOFMS? Positive or intensity vector E? negative influence? 12. What will be the difference between velocities of two ions with m = 100 6. Compare numbers of ions that can be detected during acquisition of a a.m.u., z = 2, initial velocity v = 100 m/s and v = 200 m/s after single spectrum using FT-ICR-MS and quadrupole filter? 01 02 acceleration by 1 kV and 10 kV? Mass spectrometry of biomolecules 2018 487 Mass spectrometry of biomolecules 2018 488 487 488

Exemplary Questions Exemplary Questions

13. What determines the practical upper m/z limit in TOF MS? 17. Explain the plateau on the graph of number of peptides vs. m/z 14. Compare challenges in determination of peptides and DNA oligomers accuracy (AMT method) between 200 and 700 ppm accuracy. using MS. 18. Compare advantages and disadvantages of 2DGE and column 15. You are about about to analyze peptides/proteins in a soup. Adding of separation techniques in proetomics. what compound will you try to avoid prior to MS analysis? How would 21. What parameters of mass spectrum may improve if signal is recorded you modify the soup and what ionization technique will you use? using a) FT ICR MS, b) TOF MS, c) IT for longer period? 16. What detector fits ion trap better: MCP, channeltron, electron multiplier, 22. Why does mass spectrometer need to be evacuated prior to any photographic plate or Faraday cup? measurement? 17. What is the influence of time dispersion (of ion formation) on resolution 23. What is the most significant reason of limited resolution in MALDI TOF of ion trap? MS? Explain principles of techniques leading to resolution 18. What is m of amino acid A, its residuum (in peptide chain) and its improvement in MALDI TOF MS. immonium ion? 24. What is the difference between units u, Da a Th? 19. MSI vs. IMS? 25. What is the difference between energy and velocity dispersions of ions? 20. Describe step-by-step identification of isolated protein if you have a) 26. In what region of TOF mass spectrometer are analytically important MALDI TOF MS, b) ESI Q, c) ESI QqQ fragments generated during a) MALDI ISD, b) MALDI PSD?

Mass spectrometry of biomolecules 2018 489 Mass spectrometry of biomolecules 2018 490 489 490

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