Magnetic-Sector Mass Spectrometry

Document Sample
Magnetic-Sector Mass Spectrometry Powered By Docstoc
					Mass Spectroscopy

    Alireza Ghassempour (PhD)

Medicinal Plants and Drugs Research Institute
         Shahid Beheshti University
                 Evin, Tehran
            Operational sequence

introduce               separate by   detect ions
               ionize   mass/charge
Sample introduction

EI – direct interaction of electrons with sample
CI – electrons ionize reagent gases
DI – uses a pulse of energy to produce ions
SI – converts solvated molecules into ions
                  electron ionization

       There will be different degrees of fragmentation
       depending on the stability of the sample molecule

For a stable aromatic compound the primary peak is the parent ion
For a less stable cyclic compound fragmentation is predominant
             Chemical ionization

Chemical ionization is a more controlled method
of ionization than electron ionization
In CI a neutral analyte (M) reacts with a reagent
ion that is generated by EI to form a variety of
molecular ions

        Desorption ionization
• Energetic primary ions
      secondary ion mass spectrometry (SIMS)
• Energetic atoms
      fast atom bombardment (FAB)
• Nuclear fission fragments
      plasma desorption (PD)
4. Photons
      laser desorption (LD)
5. Very rapid heating
      desorption chemical ionization (DCI)
  FAB ionization matrices

Optimal matrix properties
    Strongly absorbs the energy provided
    Contributes few ions to the spectrum
    Interacts with the analyte to produce ions
    Effectively transfers energy to the analyte ions
Diagram of an FAB gun. 1, Ionization of argon; the resulting
ions are accelerated and focused by the lenses 2. In 3, the
argon ions exchange their charge with neutral atoms, thus
becoming rapid neutral atoms. As the beam path passes
between the electrodes 4, all ionic species are deflected.
Only rapid neutral atoms reach the sample dissolved in a
drop of glycerol, 5. The ions ejected from the drop are
accelerated by the pusher, 6, and focused by the electrodes,
7, towards the analyser, 8.
Depending on the nature of the matrix we can obtain different molecular ions

                                                    Why are there two peaks?
                                                          Ag and 109Ag

                      deprotonated        Ag salt
      species            species
            Spray ionization methods
Spray ionization achieves the direct conversion of non-volatile,
solvated molecules into gas phase ions
Electrospray (ESI)
                           Electrospray ionization

                                         ESI-MS of cytochrome c (mw = 12,360)
peak separation is 1/15
                          The isotope distribution also allows charge assignment, since each
                          isotopic peak is separated from the next by 1/n where n is the charge
Principles of MALDI
n   The sample is dispersed in a large excess of matrix material which will
    strongly absorb the incident light.
n   The matrix contains chromophore for the laser light and since the matrix
    is in a large molar excess it will absorb essentially all of the laser
n   The matrix isolates sample molecules in a chemical environment which
    enhances the probability of ionization without fragmentation
n   Short pulses of laser light (UV, 337 nm) focused on to the sample spot
    cause the sample and matrix to volatilize
n   The ions formed are accelerated by a high voltage supply and then
    allowed to drift down a flight tube where they separate according to
n   Arrival at the end of the flight tube is detected and recorded by a high
    speed recording device
Matrix Assisted Laser Desorption
          (Dithranol)                                polymers

    2,5-Dihydroxy­benzoic acid              proteins, peptides, polymers

a-Cyano-4-hydroxycinnamic acid                 peptides, (polymers)

    4-Hydroxypicolinic acid                       oligonucleotides

   Trans-Indol-3-acrylacid                           polymers
   Sample Preparation: Dried Droplet

solved Matrix                    solved sample

                 Mixing and Drying
Sample Preparation: Thin Layer

solved Matrix

                                  thin homogenuous
                         fast        layer of crytslas

solved sample

MALDI spectra of a monoclonal
  antibody (above) and of a
  polymer PMMA 7100
Secondary Ion Mass
Spectrometry (SIMS)
    Secondary ion
n   The sample is prepared
    in an ultra high vacuum.
n   A beam of primary ions
    or neutral particles
    impacts the surface with
    energies of 3-20 keV.
n   A primary ion or particle causes a collision cascade
    amongst surface atoms and between .1 and 10
    atoms are usually ejected. This process is termed
    sputtering. The sputter yield depends on the nature of
    the analyte.
Static SIMS
  n   Low ion flux is used. This means a small amount of
      primary ions is used to bombard the sample per area
      per unit time. Sputters away approximately only a
      tenth of an atomic monolayer.
  n   Ar+, Xe+, Ar, and Xe are the commonly used particles
      present in the primary particle beam, which has a
      diameter of 2-3 mm.
  n   The analysis typically requires more than 15 minutes.
  n   This technique generates mass spectra data well
      suited for the detection of organic molecules.
    Imaging SIMS
n   The mass spectrometer is                                        set
    to only detect one mass.
n   The particle beam traces                                         a
    raster pattern over the                                    sample
    with a low ion flux beam,                                  much like
    Static SIMS.
n   Typical beam particles consists of Ga+ or In+ and the beam
    diameter is approximately 100 nm.
n   The analysis takes usually less than 15 min.
n   The intensity of the signal detected for the particular mass is
    plotted against the location that generated this signal.
n   Absolute quantity is difficult to measure, but for a relatively
    homogeneous sample, the relative concentration differences are
    measurable and evident on an image.
n   Images or maps of both elements and organics can be
Images created using the Imaging SIMS mode.

        Scanning ion image of granite from the Isle of Skye.
        -University of Arizona SIMS 75 x 100 micrometers.
Mass Analyzers
Mass Analyzer divided into:
1. Scanning analysers transmit:
1.1 only the ions of a given mass-to-charge
ratio to go through at a given time (magnetic, qudrupole)
1.2. allow the simultaneous transmission of all ions (ion trap, TOF)

2. ion beam versus ion trapping types,
3. continuous versus pulsed analysis,
4. low versus high kinetic energies
The five main characteristics for mass analyser:
1. The mass range limit (Th)
2. The analysis speed (u s−1)
3. The transmission (the ratio of the number of ions reaching
  the detector and the number of ions entering the mass analyser, a
  quadrupole MS used in SIM mode has a duty cycle of 100 % but a
  quadrupole MS scanning over 1000 amu, the duty cycle is
  1/1000=0.1%. )

4. The mass accuracy (ppm)
5. The resolution.
Two peaks are considered to be resolved if the valley
between them is equal to 10% of the weaker peak           R=m/∆m
intensity when using magnetic or ion cyclotron
resonance (ICR) instruments and 50% when using
quadrupoles, ion trap, TOF, and so on.

  Low resolution or high resolution is usually used to describe analysers with a
  resolving power that is less or greater than about 10 000 (FWHM), respectively
The first example is human insulin, a protein having the
   molecular formula C257H383N65O77S6.
The nominal mass of insulin is 5801 u using the integer
   mass of the most abundant isotope of each element,
   such as 12 u for carbon, 1u for hydrogen, 14 u for
   nitrogen, 16 u for oxygen and 32 u for sulfur.
Its monoisotopic mass of 5803.6375 u is calculated using
   the exact masses of the predominant isotope of each
   element such as C=12.0000 u, H=1.0079 u, N=14.0031
   u, O=15.9949 u and S=31.9721 u.
Monoisotopic mass / Average Mass

                       Average mass

   Monoisotopic mass

The ion source accelerates ions to a kinetic energy given by:
                          KE = ½ mv2 = qV
Where m is the mass of the ion, v is its velocity, q is the charge on
the ion, and V is the applied voltage of the ion optics.

•The ions enter the flight tube and are deflected by the magnetic
field, B.
•Only ions of mass-to-charge ratio that have equal centripetal and
centrifugal forces pass through the flight tube:
           mv2 /r = BqV, where r is the radius of curvature

                           mv2 /r = BqV
•By rearranging the equation and eliminating the velocity term using
the previous equations, r = mv/qB = 1/B(2Vm/q)1/2
•Therefore, m/q = B2r2/(2V)
•This equation shows that the m/q ratio of the ions that reach the
detector can be varied by changing either the magnetic field (B) or
the applied voltage of the ion optics (V).
        Basis of Quadrupole Mass Filter
n   consists of 4 parallel metal
    rods, or electrodes
n   opposite electrodes have
    potentials of the same sign
n   one set of opposite electrodes
    has applied potential of
n   other set has potential of
     - [U+Vcosωt]
n   U= DC voltage, V=AC voltage,
    ω= angular velocity of
    alternating voltage
The trajectory of an ion will be stable if the values of x and y never reach r0, thus if it never hits
   the rods. To obtain the values of either x or y during the time, these equations need to be
   integrated. The following equation was established in 1866 by the physicist Mathieu :
                Mass analyzers

An ion trap is a device that uses an oscillating electric
field to store ions.
The ion trap works by using an RF quadrupolar field that
traps ions in two or three dimensions (2D and 3D).
    Ion Trap MS
n   Ions are trapped by applying
    rf frequencies on the ring
    electrode and endcaps
n   Then ions are scanned out
    of the trap by m/z as the
    base mass voltage is
    increased over time
n   This is accomplished by
    maintaining in the trap a
    pressure of helium gas
    which removes excess
    energy from the ions by
    Time of Flight Schematic

 • The Back Plate and Grid are used to accelerate the ions
         • The Ion source is used to ionize the Sample
   • The Sample Inlet introduces the sample to the source
       • The vacuum is used to maintain a low pressure
• The Drift Region separates the ions according to their mass
     • The Detector outputs current as each ion strikes it
        • The Oscilloscope displays the detector output
Time-of-Flight Converted to Mass
n   An accelerating potential (V) will give an ion of charge z an energy of zV. This can be
    equated to the kinetic energy of motion and the mass (m) and the velocity (v) of the ion

n   zV = 1/2mv2

n   Since velocity is length (L) divided by time (t) then

n   m/Z = [2Vt2]/L2

n   V and L cannot be measured with sufficient accuracy but the equation can be rewritten

n   m/Z = B(t-A)2

    where A and B are calibration constants that can be determined by calibrating to a known
Reflectron TOF-MS Improved mass resolution in MALDI TOF-MS has been obtained by the utilization of a
single-stage or a dual-stage reflectron (RETOF-MS). The reflectron, located at the end of the flight tube, is
used to compensate for the difference in flight times of the same m/z ions of slightly different kinetic
energies by means of an ion reflector. This results in focusing the ion packets in space and time at the
A typical MALDI mass spectrum of substance P in CHCA (see Table 1) employing both linear and
reflectron TOF-MS in the continuous ion extraction mode with a 500 MS/s transient digitizer is
shown. The maximum mass resolution observed in the linear mass spectrum of substance P
employing continuous ion extraction is about 600 which is typical for a peptide of this size. Only the
average chemical mass can be determined from this mass spectrum. In the reflectron mass
spectrum, the isotopic multiplet is well resolved producing a full width half maximum (FWHM) mass
resolution of about 3400
FT-ICR-MS instrument general scheme
    FTICR: New Dimensions of High
    Performance Mass Spectrometry

Ions are trapped and oscillate with low,
incoherent, thermal amplitude
Excitation sweeps resonant ions into a large,
coherent cyclotron orbit
Preamplifier and digitizer pick up the induced
potentials on the cell.
     FTICR: New Dimensions of High
     Performance Mass Spectrometry

The frequency of the cyclotron gyration of an ion
is inversely proportional to its mass-to-charge
ratio (m/q) and directly proportional to the
strength of the applied magnetic field B.
Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

              Detection electrodes
               Intensity [%]


    Excitation                     Fourier Transform
Fourier Transform

High Resolution of FTICR MS

                              Ubiquitin (14+)
                        Mass resolution: ~170,000
                            (up to 5,000,000)
Finnigan LTQ FT Ultra

Finnigan LTQ FT Ultra

Biological samples
Ceribrospinal fluid (CSF), 1µL Injection volume
200 nL/min, Nanospray,

     TIC Þ Separation of peptides

                                   Retention Time, min
  MS Þ Peptide mass                            MS/MS Þ Peptide sequence

                     m/z                                        m/z
Finnigan LTQ FT Ultra – MS/MS

Biological samples
Ceribrospinal fluid (CSF), 1µL Injection volume
200 nL/min, Nanospray,

     TIC Þ Separation of peptides

                                   Retention Time, min
  MS Þ Peptide mass                            MS/MS Þ Peptide sequence

                     m/z                                        m/z

- a new type of FTMS
Principle of Trapping in the Orbitrap
      Click to edit Master title                               style
§ The Orbitrap is an ion trap – but there are no RF or
  magnet fields!
• Moving ions are trapped around an electrode
    - Electrostatic attraction is compensated by centrifugal
      force arising from the initial tangential velocity.
      Analogon: a satellite is “trapped” around the Earth;
      gravitational attraction is compensated by centrifugal
      force arising from initial tangential velocity.
• Potential barriers created by end-electrodes confine
  the ions axially
• The frequencies of oscillations (especially the axial
  ones) could be controlled by shaping the electrodes
• Thus we arrive at …
                                                               Orbital traps
                                                               Kingdon (1923)

   LTQ Orbitrap™ Hybrid Mass Spectrometer
   Launched in Summer 2005

                Finnigan LTQ™ Linear Ion Trap

     API Ion source                                     Linear Ion Trap                 C-Trap

                                 Differential pumping

                                                                                Differential pumping

Inventor: Dr. Alexander Makarov, Thermo Fisher Scientific (Bremen, Germany)
LTQ Orbitrap Operation Principle
1. Ions are stored in the Linear Trap
2. …. are axially ejected
3. …. and trapped in the C-trap
4. …. they are squeezed into a small cloud and injected into the Orbitrap
5. …. where they are electrostatically trapped, while rotating around the central electrode
       and performing axial oscillation

The oscillating ions induce an image current into the two
outer halves of the Orbitrap, which can be detected using
a differential amplifier

Ions of only one mass generate a sine
wave signal
Frequencies and Masses

  The axial oscillation frequency follows the formula
  Where       w = oscillation frequency
              k = instrumental constant
              m/z = …. well, we have seen this before

Many ions in the Orbitrap generate a complex
signal whose frequencies are determined using a
Fourier Transformation
What‘s the size of the Orbitrap?
LTQ Orbitrap™ Hybrid Mass
        Tandem mass spectrometers

Uses of tandem mass spectrometry:
   (1) Characterize individual compounds in complex mixtures
   (2) Completely identify the molecular structure of a compound

   To accomplish either of these goals mass analysis
   must be carried out twice in a tandem instrument
   This can be achieved either by separating the
   mass analysis operations in space or in time
Tandem mass spectrometers
Information obtained from MS experiments
          GC-MS                                         LC-MS
Chemical Ionization                           MS
                               Molecular weight

  Electron impact Ionization
             (EI)                             MS/MS

                               Structural information
Separation in space can be achieved by coupling two mass analyzers

     For example, a sector magnet (MS 1) can be coupled to a
     quadrupole mass filter (MS 2)
     A parent ion is selected by the sector magnet and
     separated from the other ions in the sample
     Each selected ion is activated by a collision process
     The resulting set of product ions are analyzed by the
     quadrupole mass filter
     Tandem mass spectrometers
The most common tandem mass spectrometer
  is a triple quadrupole mass spectrometer
Mass Spec Ion Detectors

n   Faraday Cup
n   Electron Multiplier and Channel Electron
n   Microchannel Plate
n   Daly Detector (Scintillation Counter or
Electron Multipliers
Continuous Electron Multiplier
Photomultiplier (Daly detector)

 n   Also called daly detector or scintillation counter
 n   Metal dynode emits secondary electrons
 n   Secondary electrons hit phosphorus screen and
     trigger photon emission; photon abundance
     measured by photomultiplier
 n   Advantage: keep detector in vacuum -- no
     contamination = low noise and long lifetime
 n   Disadvantage: cannot be exposed to light
Data Collection

n   Typically, the computer controls:
    q   Scanning of mass spec
    q   Data acquisition
    q   Data processing
    q   Interpretation of data (generation of spectra;
        possible comparison to spectra in database)
Sequencing Using MALDI
When the molecular mass of a peptide is known, for example Gly2Tyr = 296, we may use a
digested, or fragmented effect of MALDI to learn the sequence of the peptide.
Backbone (peptide bond) cleavages in the mass spectrometer generate two types of ions.
Acylium ions are produced when the charge is retained on the N-terminal side of the
peptide, while protonated peptides are produced when the charge is retained on the C-
terminal side of the peptide.
These ion fragments are known as bn and yn fragments respectively. In straightforward
cases, all possible fragments are produced using prior peptide digestion. This means that
for a given peptide sequence, two unique sets of fragment ions are produced (the bn and yn
In the following table, the “predicted” fragments for a given peptide are listed. Comparison
of these predicted fragments with experimentally observed peptide fragments allows for a
given peptide mass to be assigned to a unique peptide sequence.
  Formulas for prediction of mass spectrometer fragments during
  the analysis of peptides.

  Two series (bn and yn) are produced, depending on the peptide
  end on which cleavage occurs

b1             Mresidue + H          y1           Mresidue + 19

b2             Mresidue + b1         y2           Mresidue + y1

bn-1        MH+ - 18 - Mresidue     Yn-1         MH+ - Mresidue
  Consider the peptide GlyTyr. Two possible sequences are possible:

                              H-Gly-Tyr-OH        H-Tyr-Gly-OH

  The predicted fragments for these two sequences would be:

                   H-Tyr-Gly-OH              H-Gly-Tyr-OH
                   b1 = 164                  b1 = 58
                   b2 = 221                  b2 = 221
                   y1 = 76                   y1 = 182
                   y2 = 239                  y2 = 239

Suppose experimentally, we detected fragments at mass 164, 221 but none at 239.
This missing mass is sufficient to identify the sequence as H-Tyr-Gly-OH.
Peptide Fingerprinting

n   MALDI may be used to determine an exact
    “peptide fingerprint” of a section of a protein
    that carries a “disease” or differs from the
    normal protein in some way.
Image Analysis for Differential Protein Expression

                                                             Digitized PDQuestâ image
                                                             following matchset comparison,
    C-1                Digitized Image               Tox-1   which allows comparison of all
                                  Standard                   control versus all toxicant
                                Control 2D Gel               images. Each black spot
                                                             represents a protein; spots
                                                             circled in yellow indicate prot.
    C-2                                              Tox-2   eins with at least a 2-fold change
                                                             based on spot intensity or
                                                             statistical test

     C -3                                            Tox-3

Results: 196 features detected; 40 ( ) at 2-fold change.
Protein Identification by MALDI-MS
                             • Differentially expressed proteins
                             identified by image analysis are
                             excised from 2D gels and trypsin
                             digested. The resulting peptide
                             fragments are analyzed on a MALDI
                             mass spectrometer (MS).
                             • The MALDI spectra displays a
                             “peptide fingerprint” of the protein
                             using corresponding peptide masses.
  Database Searching using MALDI-MS Ions for
  Protein Identification
                                            • Proteins are identified by entering the
                                            masses (ions from MALDI spectrum) of
                                            the peptides into a peptide mapping
                                            database such as ProFound.
                                            • Search parameters are refined by
                                            including experimental mass and
                                            isoelectric point (pI) determined by 2D
                                            PAGE, as well as by taxonomic category
                                            and any modifications.

Shared By: