Mass Spectrometry: Ionization methods

Mass Spec Principles

Sample

+ _

Ionizer

Mass Analyzer

Detector

How does a mass spectrometer work?

Create ions

Separate ions Mass analyzer

MALDI-TOF
MW

Detect ions

Ionization method
MALDI Electrospray

Triple Quadrapole
AA seq

MALDI-QqTOF
AA seq and MW

Mass spectrum Database analysis

QqTOF
AA seq and protein modif.

Ion sources for molecular mass spectrometry

Soft Ionization
Soft ionization techniques keep the molecule of interest fully intact Electro-spray ionization first conceived in 1960s by Malcolm Dole but put into practice in 1980s by John Fenn (Yale) MALDI first introduced in 1985 by Franz Hillenkamp and Michael Karas (Frankfurt) Made it possible to analyze large molecules via inexpensive mass analyzers such as quadrupole, ion trap and TOF

Soft Ionization Methods

337 nm UV laser Fluid (no salt)

+ _
cyano-hydroxy cinnamic acid

Gold tip needle

MALDI

ESI

Soft ion sources little excess energy in molecule reduced fragmentation

Hard ion sources leave excess energy in molecule-extensive fragmentation

Different Ionization Methods

Electron Impact (EI - Hard method)
small molecules, 1-1000 Daltons, structure

Fast Atom Bombardment (FAB Semi-hard)

peptides, sugars, up to 6000 Daltons

Electrospray Ionization (ESI - Soft)

peptides, proteins, up to 200,000 Daltons

Matrix Assisted Laser Desorption (MALDI-Soft)

peptides, proteins, DNA, up to 500 kD

Sample introduction/ionization method

Ionization method Typical Analytes Sample Introduction Mass Range Method Highlights Hard method versatile provides structure info

Electron Impact (EI)

Relatively small volatile

GC or liquid/solid probe

to 1,000 Daltons

Chemical Ionization (CI)

Relatively small volatile

GC or liquid/solid probe

to 1,000 Daltons

Soft method molecular ion peak [M+H]+

Electrospray (ESI)

Peptides Proteins nonvolatile

Liquid Chromatography or syringe

to 200,000 Daltons

Soft method ions often multiply charged

Fast Atom Bombardment (FAB)

Carbohydrates Organometallics Peptides nonvolatile

Sample mixed in viscous matrix

to 6,000 Daltons

Soft method but harder than ESI or MALDI

Matrix Assisted Laser Desorption (MALDI)

Peptides Proteins Nucleotides

Sample mixed in solid matrix

to 500,000 Daltons

Soft method very high mass

Electron Ionization
Also called as electron impact ionization Oldest and best-characterized of all the ionization methods. A beam of electrons passes through the gas-phase sample. An electron that collides with a neutral analyte molecule can knock off another electron, resulting in a positively charged ion.

Electron Ionization
The ionization process can either produce a molecular ion which will have the same molecular weight and elemental composition of the starting analyte, or it can produce a fragmention which corresponds to a smaller piece of the analyte molecule. Most mass spectrometers use electrons with an energy of 70 electron volts (eV) for EI. Decreasing the electron energy can reduce fragmentation, but it also reduces the number of ions formed.

EI Fragmentation of CH3OH
CH3OH
CH3OH

CH3OH+
CH2O=H+
+

+ H

CH3OH
CH2O=H+

CH3 + OH

CHO=H+ + H

Why wouldnt Electron Impact be suitable for analyzing proteins?

Why You Cant Use EI For Analyzing Proteins

EI shatters chemical bonds Any given protein contains 20 different amino acids EI would shatter the protein into not only into amino acids but also amino acid subfragments and even peptides of 2,3,4 amino acids Result is 10,000s of different signals from a single protein -- too complex to analyze

Electron Ionization
Sample introduction heated batch inlet heated direct insertion probe gas chromatography liquid chromatography (particle-beam interface)

Some typical reactions in EI source

Fragmentation patterns

Fragmentation patterns

Electron Ionization
Benefits well-understood can be applied to virtually all volatile compounds reproducible mass spectra fragmentation provides structural information libraries of mass spectra can be searched for EI mass spectral "fingerprint"

Electron Ionization
Limitations sample must be thermally volatile and stable the molecular ion may be weak or absent for many compounds. Mass range Low Typically less than 1,000 Da.

Electron Ionization
Advantages of EI: high ion currents - sensitive fragmentation aids identification Disadvantages of EI: weak or absent M+ peak inhibits determination of MW molecules must be vaporized (MW < 103 Da) molecules must be thermally stable during vaporization

Chemical Ionization (CI)

Chemical ionization uses ion-molecule reactions to produce ions from the analyte. The chemical ionization process begins when a reagent gas such as methane, isobutane, or ammonia is ionized by electron impact. A high reagent gas pressure (or long reaction time) results in ion-molecule reactions between the reagent gas ions and reagent gas neutrals. Some of the products of these ion-molecule reactions can react with the analyte molecules to produce analyte ions.

Chemical Ionization (CI)

Example (R = reagent, S = sample, e = electron, . = radical electron , H = hydrogen): R + e ---> R+. + 2e R+. + RH ---> RH+ + R. RH+ + S ---> SH+ + R (of course, other reactions can occur)

Chemical Ionization (CI)

Sample introduction heated batch inlet heated direct insertion probe gas chromatograph liquid chromatograph (particle-beam interface) Benefits often gives molecular weight information through molecular-like ions such as [M+H]+, even when EI would not produce a molecular ion. simple mass spectra, fragmentation reduced compared to EI

Chemical Ionization (CI)

Limitations sample must be thermally volatile and stable less fragmentation than EI, fragment pattern not informative or reproducible enough for library search results depend on reagent gas type, reagent gas pressure or reaction time, and nature of sample. Mass range Low Typically less than 1,000 Da.

Field Desorption (FD)

The sample is deposited onto the emitter and the emitter is biased to a high potential (several kilovolts) and a current is passed through the emitter to heat up the filament. Mass spectra are acquired as the emitter current is gradually increased and the sample is evaporated from the emitter into the gas phase. The analyte molecules are ionized by electron tunneling at the tip of the emitter 'whiskers'. Characteristic positive ions produced are radical molecular ions and cation attached species such as [M+Na]+ and [MNa]+. The latter are probably produced during desorption by the attachment of trace alkali metal ions present in the analyte.

Field Desorption (FD) Sample introduction

Direct insertion probe. The sample is deposited onto the tip of the emitter by . dipping the emitter into an analyte solution . depositing the dissolved or suspended sample onto the emitter with a microsyringe

Field Desorption (FD)

Benefits simple mass spectra, typically one molecular or molecular-like ionic species per compound. little or no chemical background works well for small organic molecules, many organometallics, low molecular weight polymers and some petrochemical fractions

Field Desorption (FD)

Limitations sensitive to alkali metal contamination and sample overloading emitter is relatively fragile relatively slow analysis as the emitter current is increased the sample must be thermally volatile to some extent to be desorbed Mass range Low-moderate, depends on the sample. Typically less than about 2,000 to 3,000 Da. some examples have been recorded from ions with masses beyond 10,000 Da.

Field Ionization (FI)

The sample is evaporated from a direct insertion probe, gas chromatograph, or gas inlet. As the gas molecules pass near the emitter, they are ionized by electron tunneling.

Sample introduction
heated direct insertion probe gas inlet gas chromatograph

Field Ionization (FI)

Benefits simple mass spectra, typically one molecular or molecular-like ionic species per compound. little or no chemical background works well for small organic molecules and some petrochemical fractions

Field Ionization (FI)

Limitations The sample must be thermally volatile. Samples are introduced in the same way as for electron ionization (EI). Mass range Low Typically less than 1000 Da.

Fast Atom Bombardment (FAB)

The analyte is dissolved in a liquid matrix such as glycerol, thioglycerol, m-nitrobenzyl alcohol, or diethanolamine and a small amount (about 1 microliter) is placed on a target. The target is bombarded with a fast atom beam (for example, 6 keV xenon atoms) that desorb molecular-like ions and fragments from the analyte. Cluster ions from the liquid matrix are also desorbed and produce a chemical background that varies with the matrix used.

Fast Atom Bombardment (FAB)

Sample introduction direct insertion probe LC/MS (frit FAB or continuous-flow FAB). Benefits rapid simple relatively tolerant of variations in sampling good for a large variety of compounds strong ion currents -- good for high-resolution measurements

Fast Atom Bombardment (FAB)

Limitations high chemical background defines detection limits may be difficult to distinguish low-molecular-weight compounds from chemical background analyte must be soluble in the liquid matrix no good for multiply charged compounds with more than 2 charges Mass range Moderate Typically ~300 Da to about 6000 Da.

Secondary Ion Mass Spectrometry (SIMS)

Dynamic SIMS is nearly identical to FAB except that the primary particle beam is an ion beam (usually cesium ions) rather than a neutral beam. The ions can be focused and accelerated to higher kinetic energies than are possible for neutral beams, and sensitivity is improved for higher masses. The use of SIMS for moderate-size (3000-13,000 Da) proteins and peptides has largely been supplanted by electrospray ionization.

Secondary Ion Mass Spectrometry (SIMS)

Sample introduction Same as for FAB Benefits Same as for FAB, except sensitivity is improved for higher masses (3000 to 13,000 Da).

Secondary Ion Mass Spectrometry (SIMS)

Limitations Same as for FAB except target can get hotter than in FAB due to more energetic primary beam high-voltage arcs more common than FAB ion source usually requires more maintenance than FAB Mass range Moderate Typically 300 to 13,000 Da.

Electrospray Ionization (ESI)

Sample dissolved in polar, volatile buffer (no salts) and pumped through a stainless steel capillary (70 - 150 mm) at a rate of 10-100 mL/min Strong voltage (3-4 kV) applied at tip along with flow of nebulizing gas causes the sample to nebulize or aerosolize Aerosol is directed through regions of higher vacuum until droplets evaporate to near atomic size (still carrying charges)

Electrospray Ionization (ESI)

Electrospray Ionization (ESI)

Can be modified to nanospray system with flow < 1 mL/min Very sensitive technique, requires less than a picomole of material Strongly affected by salts & detergents Positive ion mode measures (M + H)+ (add formic acid to solvent) Negative ion mode measures (M - H)- (add ammonia to solvent)

Positive or Negative Ion Mode?

If the sample has functional groups that readily accept H+ (such as amide and amino groups found in peptides and proteins) then positive ion detection is used-PROTEINS If a sample has functional groups that readily lose a proton (such as carboxylic acids and hydroxyls as found in nucleic acids and sugars) then negative ion detection is used-DNA

Electrospray Ionization (ESI)

Sample introduction flow injection LC/MS typical flow rates are less than 1 microliter per minute up to about a milliliter per minute

Electrospray Ionization (ESI)

Benefits good for charged, polar or basic compounds permits the detection of high-mass compounds at mass-to-charge ratios that are easily determined by most mass spectrometers (m/z typically less than 2000 to 3000). best method for analyzing multiply charged compounds very low chemical background leads to excellent detection limits can control presence or absence of fragmentation by controlling the interface lens potentials compatible with MS/MS methods

Electrospray Ionization (ESI)

Limitations multiply charged species require interpretation and mathematical transformation (can sometimes be difficult) complementary to APCI. No good for uncharged, non-basic, low-polarity compounds (e.g.steroids) very sensitive to contaminants such as alkali metals or basic compounds relatively low ion currents relatively complex hardware compared to other ion sources Mass range Low-high Typically less than 200,000 Da.

Atmospheric Pressure Chemical Ionization (APCI)

Similar interface to that used for ESI. In APCI, a corona discharge is used to ionize the analyte in the atmospheric pressure region. The gas-phase ionization in APCI is more effective than ESI for analyzing less-polar species. ESI and APCI are complementary methods.

Atmospheric Pressure Chemical Ionization (APCI)

Sample introduction same as for electrospray ionization Benefits good for less-polar compounds excellent LC/MS interface compatible with MS/MS methods

Atmospheric Pressure Chemical Ionization (APCI)

Limitations complementary to ESI Mass range Low-moderate Typically less than 2000 Da.

Matrix-Assisted Laser Desorption Ionization (MALDI)

The analyte is dissolved in a solution containing an excess of a matrix such as sinapinic acid or dihydroxybenzoic acid that has a chromophore that absorbs at the laser wavelength. A small amount of this solution is placed on the laser target. The matrix absorbs the energy from the laser pulse and produces a plasma that results in vaporization and ionization of the analyte.

MALDI Ionization
+ + ++ + + + ++ + +
+
+ +

Matrix
Laser Analyte

Absorption of UV radiation by chromophoric matrix and ionization of matrix Dissociation of matrix, phase change to supercompressed gas, charge transfer to analyte molecule

Expansion of matrix at supersonic velocity, analyte trapped in expanding matrix plume (explosion/popping)

MALDI
Unlike ESI, MALDI generates spectra that have just a singly charged ion Positive mode generates ions of M + H Negative mode generates ions of M - H Generally more robust that ESI (tolerates salts and nonvolatile components) Easier to use and maintain, capable of higher throughput Requires 10 mL of 1 pmol/mL sample

Principal for MALDI-TOF MASS

peptide mixture embedded in light absorbing chemicals (matrix)

pulsed UV or IR laser (3-4 ns)

detector

+ + + + + + +

vacuum
+
+

strong electric field

Vacc

cloud of protonated peptide molecules

Time Of Flight tube

Principal for MALDI-TOF MASS

Linear Time Of Flight tube
ion source

detector

time of flight

Reflector Time Of Flight tube

ion source

detector reflector

time of flight

Matrix-Assisted Laser Desorption Ionization (MALDI)

Sample introduction direct insertion probe continuous-flow introduction Benefits rapid and convenient molecular weight determination

Matrix-Assisted Laser Desorption Ionization (MALDI)

Limitations MS/MS difficult requires a mass analyzer that is compatible with pulsed ionization techniques not easily compatible with LC/MS Mass range Very high Typically less than 500,000 Da.

How is the sample introduced into the

Mass Spectrometer?
For EI and CI there are four main options
Heated reservoir septum inlet for pure gases or volatile liquids. Basically this is a heated reservoir (~200oC) with a small restriction bleed into the ion source. Sample is injected into the reservoir through a septum. Direct insertion probe for volatile solids. Sample is loaded into a quartz tube at the end of a probe and inserted directly into the ion source. The end of the probe can then be heated, if required, up to temperatures in excess 400oC.

How is the sample introduced into the

Mass Spectrometer?
For EI and CI there are four main options
Gas chromatograph (GC) for volatile, non-polar mixtures. The mixture is injected into the top of the GC column the end of which is passed, via a heated interface, directly into the ion source. The components of the mixture separate out during their passage through the column and enter the ion source sequentially.

How is the sample introduced into the

Mass Spectrometer?
For EI and CI there are four main options
Particle Beam interface for semi-volatile compounds that are amenable to EI and CI. Samples are dissolved in a suitable solvent and the solution is introduced into the mass spectrometer using a suitable (e.g. HPLC) pump. The liquid is nebulised with helium gas to form an aerosol of solvent droplets. The stream of liquid droplets passes through a desolvation chamber and then a series of nozzles and skimmers that remove the solvent and helium, allowing a stream of solid particles (the sample) to enter the EI/CI ion source.

How is the sample introduced into the

Mass Spectrometer?
For FAB / LSIMS
Sample can be dissolved directly into the matrix on the target or applied dissolved in a miscible solvent. The probe is then inserted in to the mass spectrometer. A dynamic version of FAB exists, often referred to as continuous flow FAB. Here, the sample, in matrix, is introduced from outside the instrument and is then pumped to the target via a length of fused silica tubing. Interfacing chromatographic techniques such as HLPC and CZE has been successfully achieved with this option, although practical difficulties are often encountered. Dynamic FAB has largely been superseded by ESI, which is a more robust technique.

How is the sample introduced into the

Mass Spectrometer?
FOR MALDI: The sample is dissolved in matrix, spotted onto the target, allowed to crystallise and then inserted into the instrument

How is the sample introduced into the

Mass Spectrometer?
For ESI
For pure samples dissolved in the mobile phase, a direct injection loop can be used. The technique really comes into its own however, when interfaced to chromatographic procedures such as CZE, CEC and in particular HPLC. Historically, mass spectrometry and HPLC have been unhappy bedfellows. ESI has changed all that and the two are now routinely and reliably interfaced together. Also, with smaller columns and ever decreasing flow rates being used, the amount of sample required for successful analyses is also reduced, thus effectively increasing the sensitivity of the technique dramatically. With CEC being coupled to a nanospray (e.g. 50nl/min) ESI interface, attomole detection limits for certain compounds can be envisaged.