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Introduction to Mass Spectrometry

Table of Contents1. What is Mass spectrometry 2. Mass Spectrometry History3. Basic Components in a Mass Spectrometer 4. Sample Inlets 5. Ionization Technologies6. Mass Analyzers7. Mass Detectors8. Mass Resolution, and Accuracy9. Isotope Effect on Mass Spectra10. Tandem Mass Spectrometry11. Typical Mass Spectra

Ying Ge, Ph.D. Human Proteomics Program

School of Medicine and Public HealthUniversity of Wisconsin-Madison

* This presentation is solely used for public education purpose.

What is Mass Spectrometry?What is Mass Spectrometry?

“Mass spectrometry is the art of measuring atoms and molecules to determine their molecular weight. Such mass or weight information is sometimes sufficient, frequently necessary, and always useful in determining the identity of a species. To practice this art one puts charge on the molecules of interest, i.e., the analyte, then measures how the trajectories of the resulting ions respond in vacuum to various combinations of electric and magnetic fields.

Clearly, the sine qua non of such a method is the conversion of neutral analyte molecules into ions. For small and simple species the ionization is readily carried by gas-phase encounters between the neutral molecules and electrons, photons, or other ions. In recent years, the efforts of many investigators have led to new techniques for producing ions of species too large and complex to be vaporized without substantial, even catastrophic, decomposition.”

John B. Fenn, 2002 Nobel Laureate in Chemistry

Mass Spectrometry HistoryMass Spectrometry HistoryIn 1899, J.J. Thompson built the first mass spectrometer, a cathode ray tube

In 1918, Dempster developed electron impact (EI) ionization and magnetic focusing MS

In 1919, Aston weighed isotopes of inactive element using mass spectrometry

In 1946, Stephens described the first time-of-flight (TOF) mass spectrometer

In 1952, Johnson and Nier designed the first double focusing mass spectrometer

In 1953, Wolfgang Paul introduced quadrupole mass spectrometers and ion trap detectors

In 1956, McLafferty proposed a mechanism for γ-H transfer (McLafferty Rearrangement)

In 1966, Biemann, et. al. began peptide sequencing by mass spectrometry

In 1966, Munson and Field developed chemical ionization (CI)

In 1968, Dole introduced electrospray ionization (ESI) on macroions

In 1969, Beckey developed field desorption (FD) MS for organic molecules

In 1974, McFarlane and Torgerson developed plasma desorption (PD) MS

In 1974, Comisarow and Marshall developed FT-ICR MSIn 1978, Yost and Enke developed triple quadrupole MSIn 1981, Barber developed fast atom bombardment (FAB) In 1983, Tanaka, Karas, and Hillenkamp developed matrix-assistant laser desorption/ ionization (MALDI)

In 1984, Fenn et. al. developed electrospray ionization (ESI) on biomolecules

Nobel Prize History Nobel Prize History in Mass Spectrometryin Mass Spectrometry

• In 1899, J.J. Thompson built the first mass spectrometer awarded Physics Nobel Prize in 1906

• In 1919, Francis Anston discovered isotopes using mass spectrometry, awarded Chemistry Nobel Prize in 1922

• In 1953, Wolfgang Paul invented ion quadrupole and ion trap mass spectrometers, awarded Physics Nobel Prize in 1989

• In 1988-9, John Fenn and Koich Tanaka developed ESI for ionizing large molecules, awarded Chemistry Nobel Prize in 2002

http://almaz.com/nobel/2005-prizes.html

http://almaz.com/nobel/chemistry/chemistry.html

Basic Components in a Mass Spectrometer

SampleInlet

Ion Source Mass Analyzer

Detector

High Vacuum (10-6 – 10-9 Torr)

Ion transferRecorder

A mass spectrometer is composed of five essential parts: 1. Inlet: introducing samples from ambient room pressure into ion source2. Ion source: converting sample molecules to ions 3. Mass analyzer: separating ions according to their mass4. Detector: detecting ions and amplifying the signal5. Recorder: receiving signal from detector, further amplifying, recording,

creating mass spectrum

Sample Inlet Systems

An inlet system is needed to transfer the sample from the atmospheric pressure (760 Torr) into the source as mass spectrometers are operated in vacuum (~10-6 - 10-9 Torr).

Common Inlet Systems:1. Chromatography:

gas chromatography (GC)liquid chromatography (LC)capillary electrophoresis (CE)

2. Syringe for direct infusion in ESI

3. MALDI probe or MALDI plate

HPLC

NanoLC

CapLC

www.agilent.com

Ionization SourcesIonization Sources

Basic Type Name and Acronym Ionizing AgentElectron Ionization (EI) Energetic electrons

Chemical Ionization (CI) Reagent gaseous ions

Field ionization (FI) High-potential electrode

Field desorption (FD) High-potential electrode

Plasma desorption (PD) Fission fragment from 252Cf

Secondary ion mass spectrometry (SIMS)

Energetic beam of ions

Fast atom bombardment (FAB) Energetic atomic beam

Electrospray ionization (ESI) High electrical field

Atmospheric pressure chemical ionization (APCI)

High electric field Laser

Matrix-assisted desorption/ionization (MALDI)

Laser Beam

Condensed PhaseDesorption(for nonvolatile compounds)

Gas Phase(for Volatile compounds)

√

√

√

√

√

√

Electron Ionization (EI) - “Hard” Ionization

M + e- (60-70 ev) M+• +2e-

1. The sample is thermally vaporized -> gas phase sample.2. Electrons ejected from a heated filament are accelerated through an electric field

at 70 V to form a continuous electron beam.3. High energy electron beam passes through the gas phase sample molecules.4. The electrons collide with the neutral sample molecules -> knock off electron in

the sample molecule -> ionization, positively charged molecule ion M+·

5. Excess internal energy in the molecule leads to some degree of fragmentation.

Electron Ionization (EI) - “Hardest” Ionization

♠ EI, also known as electron impact ionization, is routinely used for analysis of small, hydrophobic, thermally stable molecules. ♠ The sample must be delivered as a gas from a probe via thermal desorption, or by introduction of a gas through a capillary- the output of a capillary column from gas chromatography instrumentation (GC/MS).♠ The utility of EI decreases significantly for compounds >400 Da as thermal desorption of the sample often leads to thermal decomposition before vaporization occurs.

Advantages:• Well-understood (“oldest” method)• Suitable for all volatile compounds • Reproducible mass spectra • Fragmentation provides structural information • Libraries of mass spectra available for EI mass spectral "fingerprint"

Disadvantages• Sample must be thermally

volatile and stable • Molecular ion may be weak or absent for many compounds• Low mass range (<1,000 Da)

Chemical Ionization (CI)

♣ CI - applied to samples similar to those analyzed by EI, primarily used to enhancethe abundance of the molecular ion.

♣ CI uses gas phase ion-molecule reactions within the vacuum of MS to produce ions from the sample molecule.

♣ CI is initiated with a reagent gas such as methane, isobutane, or ammonia, ionized by electron impact (EI).

♣ High gas pressure in the ionization source results in ion-molecule reactions between the reagent gas ions and reagent gas neutrals.

♣ Some of the products of the ion-molecule reactions can react with the analytemolecules to produce ions.

A possible mechanism for ionization in CI occurs as follows:Reagent (R) + e- → R+• + 2 e-

R+• + RH → RH+• + R

RH+• + Analyte (A) → AH+• + R

Fast Atom/Ion Bombardment (FAB) “Soft” ionization

Fast Atom/Ion Bombardment

1. The analyte is dissolved in a liquid matrix 2. Place a small amount (about 1 microliter) on a

target/probe. 3. The target/probe is bombarded with a fast atom

beam (e.g. 6 keV xenon atoms) that desorbmolecular-like ions and fragments from the analyte.

4. Cluster ions from the liquid matrix are also desorbed and produce a chemical background that varies with the matrix used.

Advantages: 1. Suitable for polar thermally labile compound2. Rapid and simple sample preparation3. Relatively tolerant of sample variation4. Mass range from ~300 – 7000 Da5. Strong ion currents – good for high resolution

mass measuremnt

Disadvantages:1. High chemical background2. Difficult to distinguish low MW

compounds from chemical background 3. Analyte must be soluble in liquid matrix4. Not applicable for higher MW (>7 kDa)

molecules

©Gary Siuzdak,,Scripps Center for Mass Spectrometry

Matrix Assisted Laser Desorption Ionization (MALDI)

Matrix (M)Analyte (A) UV or IR laser

Desorption

Desolvation +

++

+ +++

++H+

Proton transfer

MH+ + A M + AH+

MH- + A M + AH-

1. Analyte is mixed with excess matrix acid 2. Laser pulses -> matrix molecules absorb photon energy -> excite stage,4. Localized heating caused microexplosion of the matrix material, producing matrix

neutral and matrix ions e.g. M+, MH+, (M-H)-

5. Collision with the neutral analytes facilitate charge transfer from matrix moleculesAnalyte molecules are ionized by gas phase proton transfer, AH+, (A+Na)+, (A-H)-

Common MALDI Matricies

As a resultThe matmost of

, both the matrix and any sample embedded in the matrix are vaporized. rix also serves to minimize sample damage from laser radiation by absorbing the incident energy.

MALDI plate for matrix/sample deposition

MALDI matrix --process by absorbing the laser radiation.

A nonvolatile solid material facilitates the desorption and ionization

©Gary Siuzdak,,Scripps Center for Mass Spectrometry


MALDI, good for higher MW compounds such as peptides, proteins, and oligonueotides. A MALDI mass spectrum mainly consists of singly charged ions, e.g. [M+H]+, [M+Na]+, [M+K]+ for each sample component.

Cytochrome C (12.3 kDa)

[M+H]+


Advantages:

• Practical mass range of up to 300 kDa• Soft ionization with little or no fragmentation• Good sensitivity (picomole-femtomole)• Tolerance of salts in millimolar concentration• Suitable for analysis of complex mixture

Disadvantages:• Relatively low resolution • Matrix interference, especially for low MW• Requires a mass analyzer compatible with pulsed ionization

techniques • MS/MS difficult • Not easily compatible with LC/MS

Surface Enhanced Laser Surface Enhanced Laser DesorptionDesorption/Ionization (SELDI) /Ionization (SELDI)

http://www.evms.edu/vpc/seldi/seldiprocess/index.html

SELDI, similar to MALDI, except the surface of SELDI protein chip contains arrays of chromatographic surfaces with different properties, e.g. hydrophobic, cation exchange, anion exchange & metal affinity.♣ Samples goes directly onto the ProteinChipTM Array (Ciphergen)♣ Protein captured and retained on the chip by affinity capture♣ Energy absorbing molecules added to the chip♣ Laser desorption/ionization of the molecules on the chipAdvantages: 1. on-chip analysis of proteins, 2 high throughputDisadvantages: 1. low coverage (mainly most abundant proteins)

2. poor reproducibility

Electrospray Ionization (ESI) – “Softest Ionization”

ESI Process

1. Droplet formation -> 2. Droplet shrinkage -> 3. Gaseous ion formation

A solution of the analyte passing through a capillary (needle) held at high potential. -> generate a mist of highly charged droplets -> traveling down a potential and pressure gradient towards MS analyzer -> the droplets reduced in size by evaporation of the solvent or by “Coulomb explosion” -> complete evaporation of the solvent results in fully desolvated gas phase ions.

3000

2500

2000

1500

1000

500

0120011001000900800700600 m/z

15+

14+

13+

ESI/MS ofCytochrome C (12.3 kDa)

Electrospray Ionization (Cont’d)

Advantages:• Good for charged, polar or basic compounds • Practical mass range of up to 100 kDa• Soft ionization, capable of observing MI and

non-covalent interactions • Good sensitivity (femtomole-attomole)• Excellent detection limits (low chemical

background)• Fragmentation controllable • Compatible with LC/MS & MS/MS

Disadvantages:• Multiply charged species require interpretation • No good for uncharged, non-basic, low-

polarity compounds (e.g.steroids) complementary to APCI.

• Very sensitive to contaminants• Low salt tolerance• Relatively low ion currents

ESI, routinely used for analysis of proteins, peptides, carbohydrates, lipids.oligonucleotides, and synthetic polymers, produces singly charged small molecules but well known for the formation of multiply-charged large molecules

16+

17+

18+

Nano Electrospray Ionization (NanoESI)

Flow rate: ESI (1-20 ul/min); NanoESI (1-50 nl/min)

http://www.newobjective.com/electrospray/index.html

In nanoESI, the spray needle has been made very small and is positioned close tothe entrance to the mass analyzer, resulting in the increased sensitivity and detection limit (up to attomole!).

Commercially available nanospray sources:1. The PicoView® nanospray source from New Objective2. Nanomate ® from Advion Biosciences3. Nanospray sources by Thermo and others.

Atmospheric Pressure Chemical Ionization (APCI)

Advantages:• Good for less-polar compounds

Better for compounds w/o N• Gentle vaporization of the analyte

intense MH+, minimal fragmentation• Enables coupling MS and LC with flow rate up to 1 ml/min, better for normal

phase LC• Compatible with MS/MS methods

Similar interface to that used for ESI. In APCI, a corona discharge is used to ionize the analyte in the atmospheric pressure region.

Disadvantages:• Low mass range (<2000 Da),not good for proteins• Sensitivity depends strongly upon analyte• Increased fragmentation compared to ESI• Complementary to ESI

Comparison of Sensitivity and Mass Ranges by Different Ionization Techniques

EI, APCI – for lower mass rangeESI, MALDI – for higher mass range the most common ionization sources for

biomolecular mass spectrometry/proteomics

NanoESI – highest sensitivity

©Gary Siuzdak,Scripps Center for Mass Spectrometry

General Comparison of Common Ionization SourcesGeneral Comparison of Common Ionization Sources

IonizationSource

Typical Mass Range

Matrix Interference

Degradation LC/MS Amenable

Sensitivity

EI 500 none Thermal degradation

Very limitedGC/MS

picomole

CI 500 None Thermal degradation

Very limited picomole

FAB 7,000 Yes, severe Matrix reaction & thermal

degradation

Very limited nanomole

MALDI 300,000 yes Photo degradation

possible Low to high

femtomole

APCI 1,200 none thermal degradation

excellent high femtomole

ESI(nanoESI)

~70, 000 none none excellent femtomole- attomole

Mass Analyzer Mass Analyzer -- Basic TypesBasic Types

(a). Magnetic Sector (b). Time of Flight (TOF)

(c). Quadupole (d). Ion Trap (e). Ion Cyclotron Resonance(ICR)

Magnetic field affect radius of curvature of ions -> m/z

Flight time - correlated directly with ion’s m/z

Scan radio frequency field-> m/z

Scan radio frequency field-> m/z

Image current– ion cyclotron frequency -> m/z

Mass Analyzer Mass Analyzer -- Hybrid InstrumentsHybrid Instruments1. Triple Quadrupole

API 4000™ from Sciex/Applied BiosystemsTSQ Quantum from ThermoMicromass® Quattro Premier™ from Waters Triple Quadrupole from Agilent

2. Quadrupole Time-of-Flight (Q-TOF)Q-Tof Premier™ from Waters QSTAR® from Sciex/Applied BiosystemsQ-q-TOF from BrukerQ-TOF from Agilent

3. Time-of-Flight/Time-of-Flight (TOF/TOF)4700 Proteomics Analyzer, 4800 TOF/TOF™ Analyzer from Sciex/ABITOF/TOF from Bruker

4. Quadrupole Fourier Transform Mass Spectrometer (Q-FTMS)Qq-FTMS from Bruker

5. Ion Trap Fourier Transform Mass Spectrometer (IT-FTMS)LTQ FT and LTQ-orbitrap from Thermo

?

Magnetic Sector - The Oldest Mass Analyzer

In magnetic sector, ions were separated with a magnetic field.

1. Ions are accelerated into a magnetic field2. The radius (r) of an ion depends on the velocity of the ion (v), the magnetic field

strength (B), and the ion’s m/e. 3. MS Spectrum were obtained by scanning the magnetic field and monitoring ions

striking a fixed point detector.

Ek

Accelerating potential

Lorentz force law

Double Focusing Magnetic Sector Mass Analyzer

• They consist of a large magnetic sector, and an electrostatic sector.• The electric sector serves as a kinetic energy focusing point allowing only ions of a certain kinetic energy to pass through its field irrespective of m/z

©2005 Paul Gates, University of Bristol

Magneticsector

Electrostaticsector

Advantages:1. Classic mass spectrometer2. Very high reproducibility3. High resolution and sensitivity4. High dynamic range5. Reproducible high energy MS/MS

Disadvantages:1. Large size and high cost2. Not well-suited for pulsed ionization

method, i.e. MALDI3. Poor resolution in MS/MS spectra

Time of Flight (TOF) Mass SpectrometerTime of Flight (TOF) Mass Spectrometer

Advantages:• Simplest/fastest MS analyzer • Well suited for pulsed ionization methods

(e.g. MALDI) • High ion transmission • MS/MS information from post-source decay • Highest practical mass range of all analyzers

Disadvantages:• Requires pulsed ionization method or ion beam switching

• Limited dynamic range of fast digitizers

• Limited MS/MS experiments

Field-free regionFlight Tube

Ion beam

Time-of-flightm/z = 2Vt2/d2

m/z determined fromion’s time of arrival

Smaller ions - higher V reach the detector

earlier than larger ions

Laser pulseDetector

Sample

m/z = 2Vt2/d2

d

Time of Flight Time of Flight ReflectronReflectron Mass AnalyzerMass Analyzer


The reflectron combines time-of-flight technology with an electrostatic mirror. The reflectron serves to increase the amount of time (t) ions need to reach the detector while reducing their kinetic energy distribution, thereby reducing the temporal distribution ∆t. Since resolution is defined by the mass of a peak divided by the width of a peak or m/∆m(or t/∆t since m is related to t), increasing t and decreasing ∆t results in higher resolution.

Advantage: 1. Higher resolution (>5000); 2. MS/MS option - PSD (Post Source Decay) Disadvantage: Decreased sensitivity at higher masses (typically above 5000 m/z).

QuadrupoleQuadrupole Time of Flight (QTime of Flight (Q--TOF) Mass AnalyzerTOF) Mass Analyzer


Advantages:1. high resolving power (~10,000); 2. high accuracy (10 ppm) 3. upper m/z limit in excess of 10,000 4. high sensitivity; 5 MS/MS options

Quadrupole-TOF combines the quadrupole’s ability to select a particular ion and the ability of TOF-MS to achieve simultaneous and accurate measurements of ions across the full mass range. The quadrupole can act as any simple quadrupole analyzer to scan across a specified m/z range and also be used to selectively isolate a precursor ion and direct that ion into the collision cell. The resultant fragment ions are then analyzed by the TOF reflectron mass analyzer.

QuadrupoleQuadrupole Mass SpectrometerMass Spectrometer

Analyte ions of different M/Z

Source slitsQuadrupole rods

to detector

Resonant ions (detected)Nonresonant ions

Agilent MSD

©2000, Paul Gates

The quadrupole mass analyzer is a "mass filter". DC and RF potentials combination on the quadrupole rods set to only pass ions with selected mass-to-charge ratio -> focused on the detector. All other ions do not have a stable trajectory through the quadrupole mass analyzer, eventually collide with the quadrupole rods -> never reaching the detector. Varying strength and frequencies of the electric field (rf scan), different ions will be detected.

TripleTriple--QuadrupoleQuadrupole Mass SpectrometerMass Spectrometer

Advantages:• Classical mass spectra • Good reproducibility • Relatively small and low-cost systems • Low-energy CID MS/MS available

in triple-quadrupole

Disadvantages:• Limited resolution • Peak heights variable as a function of mass (mass discrimination).

• Not well suited for pulsed ionization methods • Low-energy MS/MS depend strongly on energy, collision gas, pressure, etc.

N2Ar2

Ion Beam

Q1 Q2 Q3

Collision Gas

(mass filter)Ion Selection Ion Fragmentation

(mass filter)Ion detection

(collision cell)

Detector

QuadrupoleQuadrupole Ion Trap (QIT) Mass AnalyzerIon Trap (QIT) Mass Analyzer

Three hyperbolic electrodes: • Ring electrode• entrance end-cap electrode• exit end-cap electrode

• The ions are trapped in a 3-D electrical field through entrance endcap electrode• The RF (applied on ring electrodes to create 3D quadrupolar potential field) is scanned to

resonantly excite and eject ions through small holes in the end-cap to a detectorAdvantages: 1. MSn capability 3. High sensitivity 2. Compatible with LC/MS/MSDisdvantages: 1. Low resolution 2. Limited dynamic range

QIT: Roughly the size ofa tennis ball!

Linear Ion Trap (LIT) Mass AnalyzerLinear Ion Trap (LIT) Mass Analyzer

The major difference between LIQ and 3D QIT:LIQ confines ions along the axis of a quadrupole mass analyzer using a two-dimensional radio frequency field with potentials applied to endelectrodes.

©Gary Siuzdak, Scripps Center for Mass Spectrometry

The primary advantage to the linear trap over the 3D trap: Larger analyzer volume => improves dynamic ranges =>good for quantitative analysis.

Fourier Transform Ion Cyclotron Resonance Fourier Transform Ion Cyclotron Resonance (FTICR) Mass Spectrometer(FTICR) Mass Spectrometer

CapillaryskimmerTurbo/Cryopumps

SuperconductingMagnet

ESI source

FTICR cell Ion guide

Advantages of FT-ICR MS:1) High resolution (> 1,000,000)2) High mass accuracy (< 1 ppm)3) High sensitivity (attomole)4) Simultaneous detection of

all ions5) Many MS/MS options

Disadvantages:•Limited dynamic range •Subject to space charge effects •Many parameters need to be tuned (excitation/trapping/detection)•Labor intensive – not yet automated!

A mass analyzer and a detector

How Does FTICR Work?How Does FTICR Work?

BmqB =ω

Excitation

Detection

TrappingMagnetic field

In FT MS, all the ions were trapped, excited and detected in the same ICR cell, therefore it is not only a mass analyzer but also a detector. The detected time-domain signal will be Fourier transform to frequency domain.• In the trapping mode, RF applied to excite all the ions to the coherent orbital.• In the detection mode, RF stopped, ions of the same m/z form an ion packet and induce image current which can be detected by the diode plates and amplified, digitized and Fourier transformed to frequency domain. The frequency is in inverse proportional to m/z. The amplitude depends on the total number of charges.

Magnetic Field Effect On FTMS Performance

FTMS attribute

Effect of Magnetic Field Strength B

Which means:

Resolution(m/∆m)

Directly proportional to B

Improves mass accuracy and the ability to get isotopic resolution on large macromolecules.

Kinetic energy Directly proportional to B2

Increases the fragmentation and also ability to fragment larger macromolecules.

Ion capacity Directly proportional to B2

Can store more ions before space-charge adversely affects performance

BrukerBruker 7T FTMS 7T FTMS

FTMS Console

7T SuperconductingElectromagnet

FT ICR Infinity Cell

NanoESISource

Thermo LTQ/FTThermo LTQ/FT

• Ion Trap based Fourier Transform ICR Mass Spectrometer• FTICR (FTMS) on LC timescale• Accurate Mass• High Sensitivity• Ultra-high Resolution• Above All -Easy to Operate

New Mass Analyzer New Mass Analyzer -- OrbitrapOrbitrapOrbitrap: a new type of mass analyzer employed trapping in an

electric field1. The field potential distribution is a

combination of quadrupole and logarithmic potentials.

2. Ion stability is achieved only due to ions orbiting around an axial electrode in absence of magnetic or rf fields.

3. Orbiting ions also perform harmonic oscillations along the electrode with frequency in proportional to (m/z)-1/2

4. Oscillations are detected using image current detection similar to FT.

LTQ/Orbitrap

Alexander Makarov, Anal. Chem. 2000, 72, 1156-1162

Mass DetectorsMass DetectorsMass detector detects a current signal generated from the incident ions. Different mass detectors are used to detect ions depending on the type of mass spectrometer.

Most Commonly Used Detectors:

• Electron Multiplier• Faraday Cup• Photomultiplier Conversion Dynode• High-Energy Dynode Detector (HED)• Array Detector e.g. Microchannel Plate (MCP)• Charge (or Inductive) Detector e.g. FTMS (detect image current)

Mass DetectorsMass Detectors -- Electron Multiplier


Electron multiplier - the most commonly used detector, made up of a series (12 to 24) of aluminum oxide (Al2O3) dynodes maintained at ever increasing potentials.

Ions strike the first dynode surface causing an emission of electrons => These electrons are attracted to the next dynode held at a higher potential and generate more secondary electrons => A series of dynodes at increasing potential produce a cascade of electrons an overall current gain on the order of one million or higher

Mass DetectorsMass Detectors -- Faraday Cup


• Ions striking the dynode (BeO, GaP, or CsSb) surface causing secondary electrons to be ejected. • This temporary electron emission induces a positive charge on the detector = > a current of electrons flowing toward the detector.• Relatively high pressure tolerance• Offering limited amplification of signal, not particularly sensitive

•

Mass DetectorsMass Detectors -- Array Detector

An array detector - a group of individual detectors aligned in an array format, spatially detects ions according to m/z. Spatially differentiated ions can be detected simultaneously by an array detector.

Microchannel Plate(MCP)

MCP consist of an array of miniature electron multiplier channels (~10µm diameter, ~15 µm spacing between channels). The channels are parallel to each other and often enter the plate at a small angle to the surface (~8° from normal).

http://hea-www.harvard.edu/HRC/mcp/mcp.html

General Comparison of Mass DetectorsGeneral Comparison of Mass Detectors

Table 2.3. General comparison of detectors.Detector Advantages DisadvantagesFaraday Cup •Good for checking ion

transmission and low sensitivity measurements

•Low amplification (≈10)

Photomultiplier Conversion Dynode (Scintillation Counting)

•Robust •Long lifetime (>5 years) •Sensitive (≈gains of 106)

•Cannot be exposed to light while in operation

Electron Multiplier•Robust •Fast response •Sensitive (≈gains of 106)

•Shorter lifetime than scintillation counting (~3 years)

High Energy Dynodes with electron multiplier

•Increases high mass sensitivity •May shorten lifetime of electron multiplier

Array •Fast and sensitive •Reduces resolution •Expensive

Charge Detection • Detects ions independent of mass and velocity

•Limited compatibility with most existing instruments

Mass Resolution (Resolving Power) Mass Resolution (Resolving Power)

Mass Resolution: the ability to discriminate between adjacent ions in a spectrum. Greater resolution corresponds directly to the increased ability to differentiate/separate ions.

Resolution = M/∆MM: m/z∆M: the full width at half

maximum (FWHM)©Gary Siuzdak,Scripps Center for Mass Spectrometry

Isotope Effect Natural Isotopic Abundances of Common Elements

Isotope: are forms of an element containing same number of protonsbut different number of neutrons in the nucleus.

Element M, % M+1, % M+2, %

H 1, 100 2, 0.015

C 12, 100 13, 1.1N 14, 100 15, 0.37

O 16, 100 17, 0.04 18, 0.20

P 30, 100

S 32, 100 33, 0.79 34, 4.4

Cl 35, 100 37, 32

Br 79, 100 81, 97.3

I 127, 100

Theoretic Isotopic Distribution

Myoglobin (C769H1215N209O221S4)

Resolution: 100k

Resolution: 10k

Resolution: 1M

Mass AccuracyMass Accuracy

Mass accuracy - the ability with which the analyzer can accurately provide m/z information; a function of an instrument’s stability and resolution.

The accuracy varies dramatically from analyzer to analyzer depending on the analyzer type and resolution.

An instrument with 0.01% accuracy can provide information on a 1000 Da peptide to ±0.1 Da or a 10,000 Da protein to ±1.0 Da.

An alternative means of describing accuracy is using part per million (ppm) terminology, where 1000 Da peptide to ±0.1 Da could also be described as 1000.00 Da peptide to ± 100 ppm.


The Effect of Resolution on Mass AccuracyThe Effect of Resolution on Mass Accuracy

Mass Accuracy is reduced by the uncertainty associated with identifying the center of the peak with the lower resolution.


Mass Range & Scan Speed

Mass Range: the m/z range of the mass analyzer.♣ Quadrupole analyzers typically scan up to m/z 3000♣ A magnetic sector analyzer typically scans up to m/z 10,000 ♣ Time-of-flight analyzers have virtually unlimited m/z range

Scan Speed: the rate at which the analyzer scans over a particular mass range.

♠ Scan speed varies with different analyzers. ♠ Most instruments require seconds to perform a full scan.♠ Time-of-flight analyzers, e.g. complete analyses in

milliseconds or less.

Tandem Mass Spectrometry (MS/MS or MSn)

Tandem mass spectrometry is the ability to isolate different molecular ions in the analyzer, generate fragment ions from the selected ion (parent ion or precursor ion), and then analyze the fragmented ions (product ion or daughter ion) spectrum. Either tandem in space or tandem in time.

The fragmented ions are used for structural determination of original molecular ions.

+

+

++

Fragmentation chamber

+

+

+

Ion Selection(parent ion/

precursor ion)MS1 MS2

Product ion/Daughter ion

Gas/Heat/Light/Electron

+

MS/MS Techniques in ESI/FTMS

SuperconductingMagnet

ESI source

Electron Capture Dissociation (ECD)

CapillaryskimmerBlackbody InfraredRadiative Dissociation(BIRD)

LaserIR multiphoton

Dissociation(IRMPD)

CollisionallyactivatedDissociation (CAD)

Turbo pumps

Post-Source Decay (PSD) in MALDI-TOF

MS/MS is possible with MALDI TOF reflectron mass analyzers. MALDI fragmentation occurs following ionization, or post-source decay (PSD).

Mass Spectrum - A Bar Graph Format

Molecular ion: An ion formed by the removal of one or more electrons to form a positive ion or the addition off one or more electrons to form a negative ion, also called parent ion or precursor ion.

Fragment ion: A product ion (or daughter ion) resulting from the dissociation of a precursor ion.

fragment ion(or daughter ion)

0

50

100 molecular ion (or parent ion)

m/z (mass to charge ratio)

13C isotope peak

13C2 isotope peak

Rel

ativ

e ab

unda

nce

ReferencesReferences1.Grayson, M.A. Measuring mass, from positive rays to proteins, Chemical Heritage Press,

Philadelphia, PA, 2002 2. Siuzdak, G. http://masspec.scripps.edu/MSHistory/whatisms.php#esi3. http://www-methods.ch.cam.ac.uk/meth/ms/theory/index.html4. Gates, P. http://www.chm.bris.ac.uk/ms/theory/sector-massspec.html5. Makarov, A. Electrostatic Harmonic orbital Trapping: A high-performance Technique of Mass

Analysis, Anal. Chem. 2000, 72, 1156-11626.Busch K.L., Glish G.L., McLuckey S.A. Mass Spectrometry/Mass Spectrometry: Techniques and

Applications of Tandem. John Wiley & Sons, 1989.7. Cotter R. Time-Of-Flight Mass Spectrometry: Instrumentation and Applications in Biological

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AcknowledgementsAcknowledgements

♠ The author would like to thank Dr. Gary Siuzdak for kindly granting the permission to use the materials from his website: http://masspec.scripps.edu/MSHistory/ ©2005 Scripps Center for Mass Spectrometry.

♠ The author thank Dr. Jeff Walker, Dr. Lingjun Li and Alice Puchalski for helpful discussions.

♠The financial support was provided by Wisconsin Partnership Fund for a Healthy Future.

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