Janosch Hennig 2009-09-14 - Linköping University · Janosch Hennig 2009-09-14 Janosch Hennig Biom¨atteknik 2009-09-14. IntroductionPrinciplesIon SourceMass AnalyzerAppl.Refs 1 Introduction

Introduction Principles Ion Source Mass Analyzer Appl. Refs

Mass spectrometry

Janosch Hennig

2009-09-14

Janosch Hennig Biomatteknik 2009-09-14


1 Introduction2 Principles3 Ion Source

EICIFD/FIFABESIMALDI

4 Mass AnalyzerResolutionSector fieldQuadrupoleIon TrapFTICRTOF

5 Appl.ProteomicsSequencingNon-covalent



Tertiary, Quaternary Structure and Folding

6 RefsJanosch Hennig Biomatteknik 2009-09-14


What is mass spectrometry?

It is an analytical technique to measure the mass-to-charge (m/z) ofions!

=⇒

Primary information: mass of your analyteSecondary information: analyte indentification, structure ofanalyte, primary structure, tertiary structure information,interaction partners etc.

Informally mass-spec, or MS (used throughout this presentation)



A mass spectrum

A plot of ion abundance versus mass-to-charge ratio normalized tothe most abundant ion



Comparison to other techniques

Method Sample components determined Sensitvity (g)IR 1 10−3 − 10−6

NMR 1 10−3 (depends)GC Many 10−12

HPLC Many 10−9

MS Few 10−12

GC-MS A lot 10−12

CD, Fluorescence, NMR, UV, IR excites analyte viaelectromagnetic radiation (different energy levels) =⇒ analytecan be reusedanalytes in MS cannot be recycled due to a different kind ofexcitation



Work Scheme

Your analyte has to be ionized in order to get its mass


Introduction Principles Ion Source Mass Analyzer Appl. Refs EI CI FD/FI FAB ESI MALDI

Samples - Ion Sources

The sample types depend on the ion source used:

sample type ionization methodgases & vapors electron ionization (EI)

chemical ionization (CI)field desorption/ionization (FD/FI)

solids & liquids fast atom bombardement (FAB)electrospray ionization (ESI)matrix-assisted laser desorption/ionization (MALDI)

plus some more which are usually modifications of those aboveAdditionally distinguished whether hard or soft ionization



Why Vacuum?

All mass spectrometers work under high vacuum (low pressure),why?

to increase the mean free pathmean free path: the average distance that an ion will travel beforecolliding with another ion or molecule



Electron Ionization



Electron Ionization

Ionization of analyte depends on interaction between analyte andelectronthe efficiency of ionization depends on the energy of theelectrons

optimum for ionization 70 eV - de Broglie wavelength - matchestypical bond length (0.14 nm)=⇒ fragmentation occurs according to:

M + e− → M+• + 2e−

10 eV not enough energy is transferredhigher energy, electron wavelength smaller then bond length,efficiency decreasesenergy can usually be varied between 10-100 eV to optimizeionization



Electron Ionization Fragmentation

M + e− → M+• + 2e−

=⇒ a radical ion is produced, which leads to further fragmentationreactions:

σ-cleavageα-cleavageBenzyl-cleavageRetro-Diels-Alder RearrangementMcLafferty RearrangementOnium-Reaction

Show transparency now!



Electron Ionization - ResultsFinite number of possible fragmentations leads to reoccurring peakpatterns: e.g. 1-Propanol

Peak (m/z) Fragment15 CH327 C2H328 C2H429 C2H531 CH3O42 C3H6 or C2H2O59 C3H7O60 C3H8O

Huge library of EI-MS spectra to easily identify molecules in e.g. foodanalysis using GC/MS



Chemical Ionization

A further development of EI - softer ionization

Reagent gas usually methane



Chemical Ionization

Primary Ion Formation:

CH4 + e− → CH+•4 + 2e−

Secondary Reagent Ions:

CH+•4 → CH+

3 + H•

CH4 + CH+•4 → CH+

5 CH•3

CH4 + CH+3 → C2H+

5 H2

Product Ion Formation:

protonation: CH+5 + M → MH+ + CH4

abstraction: M + CH+5 → M+ + CH4 + H2

adduct formation: M + CH+5 → [M + CH+

5 ]charge exchange: M + CH+•

4 → CH4 + M+•



Chemical Ionization Spectra

Reagent gas protects analytes, but still some fragmentation



Field Desorption/Ionization

Very small filaments on sharp surface =⇒ Very high electric field =⇒Electron tunneling of the analyte leads to M+• and [M + H+] ions.



Fast Atom BombardementA soft ionization method:

Ion gun utilizes fast moving beam of atoms with high kinetic energy (Ar or Xe ions- 4-10 keV)After charge exchange, fast atoms will impact the liquid matrix, where analyte isdissolvedthereby, the analyte is sputtered as secondary ion from the surface (M + H+ orKat+)−→ largely intact molecular species, few structural fragmentationin low mass range many matrix peakssuitable for small biological molecules (nucleotides, oligosaccharides,oligopeptides)



Fast Atom Bombardement

Possible matrix substances for FAB-MS:GlycerinThioglycerin4-NitrobenzylalcoholDithioldiethanol

Matrix should dissolve the analyte, allowing diffusion to the surface,facilitate ionization, should be of low volatility, and inert to the analyte



Electrospray IonizationElectrospray describes the dispersion of a fluid into many smallcharged droplets using an electrostatic field

1 Production of small charged droplets from the electrolyte2 continous desolvation of droplets through evaporation −→

charge density increases at droplet surface3 repeated spontaneous decay of droplets into microdroplets

(Coulomb explosion)4 desolvation of analyte molecules during transfer into the mass

analyzerJanosch Hennig Biomatteknik 2009-09-14


Electrospray Ionization

analyte solution (liquid) is pushed through a conductive capillarypositive ions are pulled to the surface and negative ion inopposite directionaccumulated positively charged ions are pulled further to thecatodethe characteristic taylor cone is formedif electric field is high enough, then the cone emitts a continous,filamentous fluid flow of few micrometers in diameterfrom a certain distance to the anode it gets unstable and decaysinto tiny dropletspositive ions accumulate at surface without counter ions



Electrospray IonizationExperiments showed that the first droplets have a diameter of a fewmicrometer, but have 105 charges.Concerning their constitution, size and charge they are near theRayleigh limit.Rayleigh equation:

Q =√

64π2ε0γr3

ε0: dielectricity constant in vacuumr : radius of dropletγ: surface tension

−→ droplet size decreases through evaporation, while charge Q isconstant, until r is above rayleigh limit (repelling forces are strongerthan surface tension and droplet decays).

−→

Coulomb explosion of droplets into even smaller ones with only a fewnanometer of diameter (surface area increases over a certain volumeof a number of particles→ below rayleigh-limit again)




Two theories about how a sinlge analyte molecule gets finallyseparated from droplet into the mass analyzer:

charged-residue modelion evaporation model



ESI - Charged Residue Model

The above described progress repeats itself until droplet radii only1 nm big, containing one analyte molecule.Free gaseous analyte ions arise through desolvation by collision withnitrogen molecules at mass analyzer interface.



ESI - Ion Evaporation Model

Direct emission of ions from highly charged droplets of 8 nm radiusand 70 charges, which is above rayleigh-limit.




Some pros and cons for each. Proteins are unlikely to undergo direction emissions, but the ion evaporation model could explain the chargedistribution of the analyte molecule in ESI-MS spectra.



Electrospray IonizationHow to calculate the real analyte mass of the ESI-MS spectrum?The number of charges n of a multiple charged molecule ion and itmolecular weight M can be calculated from measuredmass-to-charge ratios m of two deliberately chosen peaks who followeach other:

m1 =M + nX

n(1)

m2 =M + (n − 1)X

(n − 1)(2)

where X is the mass of the charge added or subtracted (in case ofaddition of a proton X = 1).Rearranging (1) and (2) to get M:

M = n(m1 − X ) (3)

andM = m2(n − 1)− X (n − 1) (4)




From (3) and (4) you can write:

n(m1 − X ) = m2(n − 1)− X (n − 1)

divide by n:

m1 − X =m2(n − 1)

n− X (n − 1)

n⇐⇒

m1 − X = (m2 − X )(n − 1)

n⇐⇒

m1 − Xm2 − X

=n − 1

n⇐⇒

m1 − Xm2 − X

= 1− 1n




⇐⇒1n

= 1− m1 − Xm2 − X

⇐⇒1n

=m2 − Xm2 − X

− m1 − Xm2 − X

⇐⇒1n

=m2 −m1

m2 − X=⇒

n =m2 − Xm2 −m1

Now you have the number of charges of the peak corresponding tom1




And you can calculate easily the molecular weight M:

M = n(m1 − X )

Nowadays, the data analysis system (the computer basically) is doingthese calculation automatically, using all molecule ions present, whichleads to higher accuracy, and it presents the resulting spectra directly.



ESI - Solution Characteristics

Variables must be properly balanced:flow rateapplied voltageconductivityliquid surface tension

Ideal solvent depends on applicationimproves response and will not form clusters

Positive ion mode: 50 % MeOH or ACNNegative ion mode: Halogenated Solvents

=⇒ ESI-MS is very suitable in combination with HPLC (HPLC/MS)



Matrix-assisted Laser Desorption/Ionization

MALDI-TOF-MSmatrix-assisted laser desorption/ionization time-of-flight massspectrometry







Laser can be:Nitrogen Laser (λ = 337.1 nm)Nd/YAG Laser (neodynium-doped yttrium aluminium garnet,Nd:Y3Al5O12, λ = 256 nm)

mechanism of desorption and ionization not fully understoodlaser causes mainly electronic energy absorption to the matrixinstead of ionizationphase transfer theories:

thermal desorptionsurface layer-by-layer sublimation/evaporation

delayed extraction (0-2000 ns), that lets expanding plum ofanalytes, ions, and neutrals with different kinetic energy expandin a weak electric field⇒ higher chance for bigger molecules to be desorbed beforeaccelerating voltage transfer analytes into mass analyzeraccelerating voltage between 20-25 kV




Matrix is of crucial importance choice depends on the following:fairly low molecular weight to allow vaporization, large enough tonot evaporate during sample preparation or while in thespectrometerstrong optical absorption in the UV range to rapidly andefficiently absorb laser irradiation =⇒ desorps and protectsanalyte from fragmentationshould be acidic to act as a proton source to encourageionization of the analyte (acidic matrix solution is used, e.g. TFA,in addition during crystallization - it is not yet known whenionization occurrs)matrix must be able to crystallize in presence of the analytemolecules (to high concentration of analyte can prevent that)



Matrix Examples




Advantages:rapid, easy sample preparationlarge mass scan routine, up to 150000 Damixture analysis (low peak suppression, few multiple protonatedanalytes)quite tolerant to impurities (crucial thing in MS)

Disadvantages:difficult to use with on-line separation (e.g. HPLC/MS)difficult to analyze low molecular weight analytes (matrixinterference)



Biophysics - MALDI

A spectrum:


Introduction Principles Ion Source Mass Analyzer Appl. Refs Resolution Sector field Quadrupole Ion Trap FTICR TOF

Mass Analyzer

Mass Analyzer - Separation of ions, according to theirmass-to-charge ratio



Mass AnalyzerAll mass spectrometers are based on dynamics of charged analyteions in electric and magnetic fields in vacuum, where the followingtwo laws apply:

Lorentz force law:~F = q(~E + ~v × ~B)

Newton’s second law of motion:~F = m · ~a

~F : force applied to the ion (N = kg·m/s2)q: ionic charge (C = A · s)m: mass of the ion (kg)~a: acceleration experienced by the ion (m/s2)~E : electric field (N/C)~v : ion velocity (m/s)~B: magnetic field (T = kg/A·s2)



Mass Analyzer

Equating both expressions for the force applied to the ion yields:

mq· ~a = ~E + ~v × ~B

Classic equation of motion of charged particles. Basis of all massspectrometers.Data is represented as the m/z- ratio, where z = q/e with e being theelementary charge of 1.602176487 · 10−19C =⇒ z = charge number.



Mass Analyzer

Technically different mass analyzers are (most common):sector field (electric and/or magnetic)quadrupolequadrupole ion traptime-of-flight

Others are:Fourier transform ion cyclotron resonanceOrbitrap

All based on law above, whether they use a static or dynamic field,electric or magnetic field. All have strengths and weaknesses(especially different resolutions)



Resolution

Resolution in mass spectrometry



Resolution

R =m

∆m=

m1

m2 −m1



Sector Field

an electric and/or magnetic field to affect the path of the ionsbends the ion path dependent on their mass-to-charge ratios,deflecting the more charged and lighter ions moreemploying different geometries (how much the ions can be bend)very high resolution (≥ 100000), often used for a narrow range ofm/zpopular combination BEB (magnetic-electric-magnetic)modern analyzers use double-focusing, a combination offocusing the ion beams both in direction and velocity



Quadrupole

consists of four rod-like (or hyperbolic shaped) electrodesarranged around a circle with radius r on the z-axisRods have a DC-voltage U and a AC-voltage (V · cos 2πft) withfrequency fopposite rods have same polarity of DC-voltage and same phaseof AC-voltage=⇒ neighboring rods have thereby opposite polarity and 180◦

phase shift=⇒ near z-axis an electrical potential φ

φ(x , y , t) = (U + V cos 2πft) · x2 − y2

r2



Quadrupole

After acceleration in an electric field 10-20 V, the ions enter theelectric field of the quadrupole in z-axis direction. Their movementthrough the xy -plane can be described by following equation:

md2xdt2 = z · e δφ

δx=

z · er2 (U + V · cos 2πft) · x

md2ydt2 = z · e δφ

δy= −z · e

r2 (U + V · cos 2πft) · y

Can be arranged to Mathieus equations:

d2xd(πft)2 + (a + 2q cos 2πft)x = 0

d2yd(πft)2 − (a + 2q cos 2πft)y = 0

with a = 2zeUm(πfr)2 and q = zeU

m(πfr)2



QuadrupoleWith parameters a and q, the relation between a transferring ion withmass m and z elementary charges e and the properties of thequadrupole (r , U, V , and f ) can be determinedThere are two solutions to these equations:

finite amplitudes of oscillations, therefore stable movementthrough the quadrupole reaching the detectoramplitudes which grow in x- and y - directions exponentially,these ions do not reach the detector

For ions with a certain m/z ratio, there are certain values for a and q,that these ions acquire stable oscillations through the quadrupole.

=⇒

U and V will be increased simultaneously that the ratio U/V andtherefore the ratio a/q is constant (f is constant as well).

=⇒

Ions of increasing mass will experience a stable oscillation after eachother and can be detected



Quadrupole

Advantages:easy to couple to chromatography (low acceleration potential - afew Volts)can operate at higher pressuresMS/MS (triple resonance)compact design

Disadvantages:limited m/z range (up to 4000)Moderate sensitivity (few ions reach the entrance of thequadrupole)Moderate resolution (up to about 4000, if technically perfect itwould depend only on U/V, but in reality on entrance speed anddirection)



Ion Trap

Same principle as quadrupole:same equations valid but in three dimensionsions travel not linearly but are around the center of the ion trapions are trapped between 0.1-10 msmass-to-charge range increased to 6000resolution up to 10000



Fourier Transform Ion Cyclotron Resonance



Fourier Transform Ion Cyclotron ResonanceTechnically advanced method:

ions are trapped in a strong magnetic field and circulate(cyclotron) in a penning trap (assisted by electric trapping plates)

strong homogenous axial magnetic field to confine particles radiallyquadrupol electric field to confine particles axially

a radio frequency field perpendicular to the magnetic field isapplied in resonance to the angular frequency of the ion=⇒ ions are accelerated to a larger radius with same frequencyRF field is turned off, ions continue to cyclotron, but radiusdecreases again

ωc = qBm , in electric trapping: ωt =

√qαm




applied is a RF sweep to simultaneously accelerate all ions withdifferent resonant frequenciesget close to detection plates (but, as different to other MStechniques, never touch them)detection plates detect overlapping sine waves, called freeinduction decay (FID)FID is fourier transformed into the mass spectrum,







Advantages:high m/z (≥ 100 kDa)superior accuracy and resolving powerstable calibration with superconducting magnetsMSn (detection-isolation-fragmentation-detection....)non-destructive detectiongas phase ion chemistrygood sensitivity in wide mass range (zeptomole - 10−21M)blackbody infrared radiative dissociation (BIRD), to measuredissociation of proteins etc.

Disadvantages (there are some):resolving power is inversely proportional to m/z −→ not optimal forMALDIslowexpensive (6.000.000 SEK)



Time-of-Flight



Time-of-FlightAfter acceleration with accelerating Voltage U, the ions will have acertain kinetic energy:

Ekin =12

mv2 = zeU (5)

The velocity v results from the total flying time t the ion needs totraverse the flight tube of a certain length L:

v =Lt

(6)

Putting (6) into (5) yields:

12·m · (L

t)2 = zeU

After rearrangement to m/z we get:

mz

=2eUL2 t2



Time-of-Flight

Can be extended to reflectron-TOF and PSD-TOF



Time-of-Flight

Advantages:theoretically no upper mass limithigh ion transmissionfast mass spectrum acquisition (10-1000 ms)parallel ion detection (low peak suppression)

Disadvantages:rather low resolution (around 15000)


Introduction Principles Ion Source Mass Analyzer Appl. Refs Proteomics Sequencing Non-covalent Tertiary, Quaternary Structure and Folding

Applications

As mentioned in the beginning, MS can be used in many differentareas, such as proteomics, structural genomics, folding studies,chemical analytics, forensics and so on



Proteomics

The next great challenge after completion of HUGO!protein sequences are knowninteractions have to be determinedwhere are they in cells−→ analysis of complex mixturesseparation of proteinsMALDI-TOF, and ESI-MS to identify proteins

How?



Proteomics



Proteomics



Sequencing

DNA:



Sequencing

Proteins:



Non-covalent

Non-covalent binding stochiometries can be determined



Tertiary, Quaternary Structure and Folding

The tertiary structure and quaternary structure, stability origins,folding, protein-ligand interactions, and protein-surface interactionscan be probed by MS



H/D exchange

The probably most powerful method to do that is H/D exchange:H −→ 1.007825... DaD −→ 2.014101... Da

≈ 1 Da per exchanged atomit can be followed how mass changes overtime



H/D exchange

to locate, which sites get exchanged, the protein is enzymaticallydigested and run in MS



Cross-linking



Limited Proteolysis and MS




Domain mapping only!




What we did:

Cx =

∑i Ii

2−∑

j Ij − 2 ¯IFL(7)

¯Ixm = Ix · (1 + ε(x)) (8)




Why?1. formula, to determine the “cleavage propensity” at one singlecleavage site2. formula, to be able to apply the 1. formula

=⇒

not only stable fragments, but now we have a gauge of exposure andburial of certain cleavage sites→ valuable tertiary structureinformation






References

MALDI:Karas, M., Bachmann, D., Hillenkamp, F. (1985), Influence of theWavelength in High-Irradiance Ultraviolet Laser Desorption MassSpectrometry of Organic Molecules. Anal. Chem., 57,2935-2939.Karas, M., Hillenkamp, F. (1988), Laser desorption ionization ofproteins with molecular masses exceeding 10,000 daltons. Anal.Chem., 60(20), 2299-301Tanaka, K., Waki, H., Ido, Y., Akita, S., Yoshida, Y., Yoshida, T.(1988), Protein and Polymer Analyses up to m/z 100 000 byLaser Ionization Time-of flight Mass Spectrometry. RapidCommun Mass Spectrom, 2, 151-153



References

ESI:Fenn, J. B., Mann, M., Meng, C. K., Wong, S. F., Whitehouse, C.M. (1989), Electrospray ionization for mass spectrometry of largebiomolecules. Science 246: 64-71

TOF:Stephens, W. E., (1946) A Pulsed Mass Spectrometer with TimeDispersion. Phys. Rev., 69, 691Wiley, W. C., MacLaren, I. H., (1955) Time-of-Flight Spectrometerwith Improved Resolution. Rev. Sci. Instr., 26, 1150



References

FTICR:Marshall, A. G., Hendrickson, C. L., Jackson, G. S., (1998)Fourier transform ion cyclotron resonance mass spectrometry: aprimer. Mass. Spectrom. Rev., 17, 1-35M.B. Comisarow, and A.G. Marshall, (1974). Chem. Phys. Lett.,25, 282

Quadrupole:Paul, W., Steinwedel, H., (1953) Ein neues Massenspektrometerohne Magnetfeld. Zeitschrift fur Naturforschung, A8, (7), 448-450



References

Proteomics and MS:Bantscheff, M., Schirle, M., Sweetman, G., Rick, J., and Kuster,B., (2007) Quantitative mass spectrometry in proteomics: acritical review. Anal. Bioanal. Chem., Epub ahead of print

H/D exchange:Carulla, N., Caddy, G.L., Hall, D.R., Zurdo, J., Gairi, M., Feliz, M.,Giralt, E., Robinson, C.V., Dobson, C.M. (2005) MolecularRecycling within amyloid fibrils. Nature, 436, 554-558Ruotolo, B.T., Giles, K., Campuzano, I., Sandercock, A.M.,Bateman, R.H., Robinson, C.V. (2005) Evidence formacromolecular protein rings in the absence of bulk water.Science, 310, 1658-1661



References

Cross-linking:Sinz, A., (2006) Chemical cross-linking and mass spectrometryto map three-dimensional protein structures and protein-proteininteractions. Mass. Spectrom. Rev., 25(4), 663-682

Tertiary structure probing using limited proteolysis and MS:Hennig, J., Ottosson, L., Andresen, C., Horvath, L., Kuchroo,V.K., Broo, K., Wahren-Herlenius, M., Sunnerhagen, M. (2005)Structural organization and Zn2+-dependent subdomaininteractions involving autoantigenic epitopes in theRing-B-box-coiled-coil (RBCC) region of Ro52. J. Biol. Chem.,280(39), 33250-33261


Documents

Janosch Hennig 2009-09-14 - Linköping University · Janosch Hennig 2009-09-14 Janosch Hennig Biom¨atteknik 2009-09-14. IntroductionPrinciplesIon SourceMass AnalyzerAppl.Refs 1 Introduction