Mass Spectrometry

Introduction

A technique for "weighing" molecules

It measures mass better than any other technique

It can give information about chemical structures To identify, verify, and quantify:

metabolites, proteins, oligonucleotides, drug candidates, peptides, synthetic organic chemicals, polymers, pesticides, hormones, drugs, airport security, food safety

History

JJ Thomson built MS prototype to measure m/z of electron, awarded Nobel Prize in 1906 MS concept first put into practice by Francis Aston, a physicist working in Cambridge England in 1919 Aston Awarded Nobel Prize in 1922 1948-52 - Time of Flight (TOF) mass analyzers introduced 1955 - Quadrupole ion filters introduced by W. Paul, also invents the ion trap in 1983 (wins 1989 Nobel Prize) 1958: First GC/MS assembled by Roland Gohlke and Fred McLafferty and Time-of-Flight mass spectrometer by Bendix Corp.

History

1959: Multiple ion monitoring invented on TOF mass spectrometer by Donald Damoth 1960: First mass spectrometer with solids injection probe 1962: Total ion monitoring invented 1963: First commercial Quadrupole mass spectrometer, First Magnetic Sector mass spectrometer sold by HITACHI for GC/MS research. 1964: GC/MS designed and built by Ragnar Ryhage 1965: First pre-packaged Magnetic Sector mass spectrometer with Helium enrichment jet separator 1967:PERKIN ELMER introduces first magnetic double focusing GC/MS, Model 270. 1968 - Tandem mass spectrometer appears

Mass spectrometer schematic

High Vacuum System

Turbo pumps Diffusion pumps Rough pumps Rotary pumps

Inlet

Ion Source
MALDI ESI IonSpray FAB EI/CI

Mass Filter
TOF Quadrupole Ion Trap Mag. Sector FTMS

Detector

Data System
PCs UNIX Mac

Sample Plate Target HPLC GC Solids probe

Microch plate Electron Mult. Hybrid Detec.

Simplified MS

Introduce sample to the instrument Generate ions in the gas phase Separate ions on the basis of differences in m/z with a mass analyser Detect ions

High vacuum system

Ions are very reactive and short-lived Formation and manipulation must be conducted in a vacuum The pressure under which ions may be handled is roughly 1O-5 to 10-8 torr (less than a billionth of an atmosphere) High vacuum minimizes ion-molecule reactions, scattering, and neutralization of the ions Dual pumping system consisting of a mechanical or rough pump Oil diffusion pump Turbomolecular pump

Sample Introduction

Depends upon the sample and the sample matrix Inlet must transfer the analyte into the source as a gas phase molecule Gases and samples with high vapour pressure : Direct Liquids and solids : heating Thermally labile : direct ionization

Direct Vapour Inlet Gas Chromatography Liquid Chromatography Direct Insertion Probe Direct Ionization of Sample

Ionization Methods

Ionization excite the neutral analyte molecule which then ejects an electron to form a radical cation (M+) Ion molecule reactions that produce adduct ions (MH+) Ionization energy : controls the amount of fragmentation observed in the mass spectrum

Ionization Methods

Electron Ionization (EI)

EI is by far the most widely used ion-formation technique for analytes that can be put into the gas phase Electrons used for ionization are produced by passing a current through a tungsten filament Transfer of kinetic energy from the fast-moving electron to the electron cloud of the molecule

Ionization Methods

Chemical Ionization (CI)

Soft ionization technique that produces ions with little excess energy Increases the abundance of the molecular ion Reagent gas in the CI source is ionized with an electron beam to produce a cloud of ions Reagent gas ions donate a proton to the analyte molecule and produce MH+ ions Very useful for molecular mass determination Methane, isobutane and ammonia

Ionization Methods

Chemical Ionization Reactions

Ionization Methods

Fast Atom Bombardment and Secondary Ion MS

Use high energy atoms to sputter and ionize the sample in a single step Beam of rare gas neutrals (FAB) or ions (SIMS) is focused on the liquid or solid sample Compounds with molecular weights up to a few thousand Dalton and thermo labile FAB :

analyte is dissolved in a liquid matrix Glycerol or similar low vapor pressure liquids as matrix No matrix, ionizing beam is focused directly on the sample

SIMS

Ionization Methods

Fast Atom Bombardment Source

Ionization Methods

Electrospray Ionization

used for LC/MS of thermally labile and high molecular weight compounds Produces multiply charged ions, high molecular weight compounds are observed at lower m/z value Increases the mass range of the analyzer so that higher molecular weight compounds may be analyzed with a less expensive mass spectrometer liquid leaves the nozzle, the electric field induces a net charge on the small droplets. The droplet finally reaches a point where the coulombic repulsion from this electric charge is greater than the surface tension holding it together. This causes the droplet to explode and produce multiply charged analyte ions.

Ionization Methods

Electrospray Ionization Source

Ionization Methods

MALDI

Analysis of compounds with molecular weights up to 200,000 Dalton is possible and this high mass limit is continually increasing Desorption and ionization : single laser pulse Analyte and a matrix compound chosen to absorb the laser wavelength Mass spectrum is acquired for each laser pulse Typical MALDI spectra include the molecular ion, some multiply charged ions, and very few fragments

Ionization Methods

MALDI ionization

Mass Analyzers

Separates ions according to their m/z value depends upon the resolution, mass range, scan rate and detection limits required for an application Continuous

Transmit a single selected m/z to the detector Quadrupole filters and magnetic sectors

Pulsed

Collect an entire mass spectrum from a single pulse of ions Time-of-flight, ion cyclotron resonance, and quadrupole ion trap

Mass Analyzers

Quadrupole

compact size, fast scan rate, high transmission efficiency, and modest vacuum requirements have a mass range of m/z 1000-4000 Radio Frequency (RF) and Direct Current (DC) voltages applied to the electrodes Amplitude of this three dimensional wave depends upon the m/z value of the ion Acts like a high pass filter, transmitting high m/z ions and rejecting low m/z ions Often called mass filters

Mass Analyzers

Quadrupole filter

Mass Analyzers

Magnetic Sector

Mass Analyzers

Magnetic sector

Mass Analyzers

Time-of-Flight

separates ions in time as they travel down a flight tube poor mass resolution, usually less than 500 high transmission efficiency, no upper m/z limit, very low detection limits, and fast scan rates packet of ions is formed by a very fast ionization pulse and are accelerated into the flight tube by an electric field An electric field accelerates all ions into a field-free drift region with a kinetic energy of qV Kinetic energy=0.5mv2, lighter ions have a higher velocity than heavier ions and reach the detector at the end of the drift region sooner

Mass Analyzers

Reflectron is a series of rings or grids that act as an ion mirror. This mirror compensates for the spread in kinetic energies of the ions as they enter the drift region and improves the resolution of the instrument. The output of an ion detector is displayed on an oscilloscope as a function of time to produce the mass spectrum.

Mass Analyzers

Fourier-transform mass spectrometry

Takes advantage of ion-cyclotron resonance to select and detect ions Ions are trapped in a cell (Three distinct sets of plates - trapping , transmitter, and receiving plates) The ions in the trap move in circular orbits (cyclotron motion) in a plane perpendicular to the magnetic field Excited ions pass near the receiver plates, the frequency of their passage is detected as an induced current in the plates called 'image' current frequency of the motion of an ion is inversely proportional to its mass

Mass Analyzers

maximum m/z value that can be trapped is a function of the magnetic field strength lasers to fragment ions in these traps to perform MS/MS To produce desired magnetic field strength, it is necessary to use a super-conducting magnet

Detectors

Channeltron

A channeltron is a horn-shaped continuous dynode structure that is coated on the inside with a electron emissive material. An ion striking the channeltron creates secondary electrons that have an avalanche effect to create more secondary electrons and finally a current pulse.

Daly detector

A Daly detector consists of a metal knob that emits secondary electrons when struck by an ion. The secondary electrons are accelerated onto a scintillator that produces light that is then detected by a photomultiplier tube.

Detectors

Electron multiplier tube (EMT)

similar in design to photomultiplier tubes Consist of a series of dynodes that eject secondary electrons when they are struck by an ion. Multiply the ion current and can be used in analog or digital mode.

Faraday cup

Metal cup that is placed in the path of the ion beam. It is attached to an electrometer, which measures the ion-beam current. Since a Faraday cup can only be used in an analog mode it is less sensitive than other detectors that are capable of operating in pulsecounting mode.

Detectors

Microchannel plate:

Consists of an array of glass capillaries (10-25 um inner diameter) that are coated on the inside with a electronemissive material. The capillaries are biased at a high voltage and like the channeltron, an ion that strikes the inside wall one of the capillaries creates an avalanche of secondary electrons. This cascading effect creates a gain of 103 to 104 and produces a current pulse at the output.

Data Interpretation

First fragments identified were produced by the cleavage of a bond in the main chain CC, CN or NC Yields six types of fragments that are respectively labelled an, bn, cn when the positive charge is kept by the N-terminal side and xn, yn, zn when the positive charge is kept by the C-terminal side

The mass difference between consecutive ions within a series allows the identity of the consecutive amino acids to be determined

Data Interpretation

Two other types of fragments

internal fragment : lost the initial N- and C-terminal sides

multiple cleavage of the peptide chain appears among the low masses in the spectrum, immonium ions of the amino acids, labelled by a letter corresponding to the parent amino acid code

Data Interpretation

MS/MS fragment spectrum of a peptide with sequence GlyIleProThrLeuLeuLeuPheLys

complete series of bn ions, thus allowing one to deduce the peptide sequence from the N-terminal acid to the C-terminal acid, whereas the series of yn ions allows identification of the sequence in the reverse direction

Data Interpretation

mass difference of 97 Da between peak b2 and b3 indicates amino acid in position 3 is proline 147 Da difference between peaks y1 and y2 indicates amino acid in the next-to-last position is a phenylalanine The m/z values of ions w3, w4, w5 and w8 imply that the amino acid in positions 3, 4 and 5 starting from the C-terminal side are leucines, whereas the amino acid in position 8 is an isoleucine The presence of a proline induces the formation of internal fragments labelled PT, PTL and PTLL that allow one to verify the deduced sequence. The peaks labelled P, F and X represent the immonium ions and indicate the presence of proline, phenylalanine and leucine and/or isoleucine.

Data Interpretation

Several algorithms have been developed to interpret tandem mass spectra of peptides de novo spectral interpretation that involves automatically interpreting the spectra using the table of amino acid masses Searches the database to find the best sequence that matches the spectrum Bottom-up protein sequencing : fragmentation in the gas phase of peptides derived from protein digestion Top-down protein sequencing : direct analysis of intact protein ions by MS/MS