Mass Spectrometry

Principle, Instrumentation, Theory & Applications

Dr. Prafulla Kumar Sahu M. Pharm., Ph. D.

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Mass Spectrometry Needs

Ionization-how the analyte is injected in to the MS machine Separation-Mass and Charge is determined Activation-analytes/ protein are broken into smaller fragments (peptides/AAs) Mass Determination-m/z ratios are determined for the ionized analytes/ protein fragments/ peptides
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Mass Spectrometry (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 analyzer Detect ions

How does a mass spectrometer work?

Create ions

Separate ions Mass analyzer

MALDI-TOF
MW

Detections

Ionization method
MALDI Electrospray
(analytes must be charged and dry)

Triple Quadrapole
AA seq

MALDI-QqTOF
AA seq and MW

Mass spectrum Database analysis

QqTOF
AA seq and protein modif.

MS Principles
Mass spectrometry (MS) is based on generating ions in the gaseous state, separating them according to their mass-to-charge ratio (m/z) and detecting them.
MS is therefore useful for identification as it can elucidate chemical and structural information about molecules from their molecular weights and distinctive fragmentation patterns.
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MS Applications
In fact, MS provides more information about the composition and structure of a compnd. from less sample than any other analytical technique. MS is also very important for the quantitative measurement of atoms or molecules. The sensitivity of MS for identifying molecules is in the 10-12 10-15 molar range.
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MS Applications
But,
for proteins, mass measurement accuracy can be extremely low. Not all compounds are amenable to being ionized, especially from liquids and solids, so the challenge with mass spectrometry is often at the front end of the instrument, where:
Chemistry of the molecules Ionization chamber conditions play an imp. 8 Role.

MS Principles
Different elements can be uniquely identified by their mass

MS Principles
Different compounds can be uniquely identified by their mass
Butorphanol
N -CH2OH HO HO HO

L-dopa
COOH -CH2CH-NH2

Ethanol

CH3CH2OH

MW = 327.1

MW = 197.2

MW = 46.1
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Mass Spectrometry
Analytical method to measure the molecular or atomic weight of samples

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Weighing analytes
A mass spectrometer creates charged particles (ions) from molecules.
Common way is to add or take away an ions:

NaCl + e- NaClNaCl NaCl+ + eIt then analyzes those ions to provide information about the molecular weight of the compound and its chemical structure.

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Mass Spectrometry
For small organic molecules the MW can be determined to within 5 ppm or 0.0005% which is sufficiently accurate to confirm the molecular formula from mass alone
For large biomolecules the MW can be determined within an accuracy of 0.01% (i.e. within 5 Da for a 50 kD protein) Recall 1 dalton = 1 atomic mass unit (1 amu)
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MS 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 Designed to measure mass of elements Aston Awarded Nobel Prize in 1922
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MS History
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) 1968 - Tandem mass spectrometer appears Mass spectrometers are now one of the MOST POWERFUL ANALYTIC TOOLS IN CHEMISTRY

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MS Principles
Find a way to charge an atom or molecule (ionization) Place charged atom or molecule in a magnetic field or subject it to an electric field and measure its speed or radius of curvature relative to its mass-to-charge ratio (mass analyzer) Detect ions using microchannel plate or photomultiplier tube

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Mass Spec Principles

Sample

+ _

Ionizer

Mass Analyzer

Detector

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Instrumentation
A mass spectrometer consists:
A sample introduction system, An ion source, A mass-selective analyser, An ion detector and a computer.

Since mass spectrometers create and manipulate gaseous ions, they operate in a highvacuum system. The magnetic-sector, quadrupole, and time-offlight designs also require extraction and acceleration ion optics to transfer ions from the source region into the mass analyser.
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Mass Spectrometer Schematic

High Vacuum System
Turbo pumps Diffusion pumps Rough pumps Rotary pumps

Inlet

Ion Source
MALDI ESI IonSpray FAB LSIMS EI/CI

Mass Filter
TOF Quadrupole Ion Trap Mag. Sector FTMS

Detector

Data System
PCs UNIX Mac

Sample Plate Target HPLC GC Solids probe

Microchannel plate Electron Mult. Hybrid Detec.

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Sample and Inlet

The easiest sample: Gas or Volatile sample. However, there are now many ionisation and desorption techniques that can produce gas ions from a condensed liquid or solid phase. Thermo-labile molecules also can be ionised and separated according to their m/z values. Sample introduction: Two most common way by:
Direct inlet probe Infusion by syringe at a set (slow) flow rate. Another common means of delivery is straight from another instrument, (HPLC) where a stream of liquid is infused from the outlet of the HPLC into the MS via the 20 ion source chamber.

Ion source
The ion source is where the sample of interest is ionised with both positive or negative charge and converted into the gas phase. Number of ion sources available: Electron impact (EI) Chemical ionisation (CI) Electrospray ionisation (ESI) Atmospheric pressure chemical ionisation (APCI) Fast atom bombardment (FAB) Matrix assisted laser desorption ionisation (MALDI) 21 Surface enhanced laser desorption ionisation (SELDI)

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
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Electron Impact Ionization

Sample introduced into instrument by heating it until it evaporates
Gas phase sample is bombarded with electrons coming from rhenium or tungsten filament (energy = 70 eV)

Molecule is shattered into fragments (70 eV >> 5 eV bonds)

Fragments sent to mass analyzer
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Electron Impact Ionization

This means that the molecules are broken up into smaller fragments. The resultant mass spectrum can therefore be detailed but gives excellent information on the structure of a molecule. In fact, for a given set of conditions, the spectrum can be a fingerprint of the particular compound under investigation and hence can be compared to spectral 25 libraries for definitive identification.

Electron Impact Ionization

In El, a high vacuum (low pressure), typically 10-5 mbar, is maintained in the ion source so that any molecular ions (M*+) formed initially from the interaction of an electron beam and molecules (M) do not collide with any other molecules before being expelled from the ion source into the mass spectrometer analyzer. Decomposition (fragmentation) of a proportion of the molecular ions (M*+) to form fragment ions (A+, B+, etc.) occurs mostly in the ion source, and the assembly of ions (M*+, A+, B+, etc.) is injected into the mass analyzer. 26

Chemical Ionization (CI)

For CI, the initial ionization step is the same as in El, but the subsequent steps are different. In CI, the gas pressure is increased to 10-3 mbar (sometimes up to atm. pressure) by injecting a reagent gas (R). The analyte (M) is present as only a small fraction of the reagent gas pressure. Thus, the electrons in the electron beam mostly interact with the reagent gas to form reagent gas ions (R*+) and not M*+ ions. 27

Chemical Ionization (CI)

At high pressure, the initial reagent gas ions almost immediately suffer multiple collisions with neutral reagent gas molecules (R). During this process, new ions (RH+) are produced (step 2); these ions are reagent gas ions. Because many of these ions are produced, there is a high probability that they will collide with sample molecules (M) and that a proton (H+) will be exchanged to give protonated molecular ions [M + H]+, (step 3).
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CI vs EI
These quasi-molecular ions contain little excess of internal energy following CI and therefore tend not to fragment. Whereas El spectra contain peaks corresponding to both molecular and fragment ions. CI spectra are much simpler, mostly having only protonated molecular ion peaks. Negative reagent gases give abundant [M - H]- or [M + X]- ions.
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CI v/s EI

Comparison of basic El and Cl processes showing different types of molecular ions and the formation of fragment ions in El.

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Example: Chemical Ionization

The ion source, across which an electron beam passes, is filled with methane, the reagent gas. There is a high vacuum around the ion source, so, to maintain a high pressure in the source itself, as many holes as possible must be blocked off or made small. Interaction of methane (CH4) with electrons (e-) gives methane molecular ions (CH4*+). CH5+
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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?

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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 33

Soft Ionization Methods

337 nm UV laser Fluid (no salt)

+ _
cyano-hydroxy cinnamic acid

Gold tip needle

MALDI

ESI
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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 35

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Ionization methods
Electrospray mass spectrometry (ESI-MS) Liquid containing analyte is forced through a steel capillary at high voltage to electrostatically disperse analyte. Charge imparted from rapidly evaporating liquid.

Matrix-assisted laser desorption ionization (MALDI) Analyte (protein) is mixed with large excess of matrix (small organic molecule) Irradiated with short pulse of laser light. Wavelength of laser is the same as absorbance max of matrix.

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Electrospray Ionization
Sample dissolved in polar, volatile buffer (no salts) and pumped through a stainless steel capillary (70 - 150 mm) at a rate of 10100 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) 38

Electrospray (Detail)

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Electrospray Ionization
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)- (add40 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
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Matrix-Assisted Laser Desorption Ionization

337 nm UV laser

cyano-hydroxy cinnamic acid

MALDI
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MALDI
Sample is ionized by bombarding sample with laser light Sample is mixed with a UV absorbant matrix (sinapinic acid for proteins, 4hydroxycinnaminic acid for peptides) Light wavelength matches that of absorbance maximum of matrix so that the matrix transfers some of its energy to the analyte (leads to ion sputtering)

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HT Spotting on a MALDI Plate

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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)
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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
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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

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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

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MALDI = SELDI
337 nm UV laser

cyano-hydroxy cinnaminic acid

MALDI
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MALDI/SELDI Spectra
Normal

Tumor

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Mass spectrometers
ion source

Linear Time Of Flight tube

Time of flight (TOF) (MALDI) Measures the time required for ions to fly down the length of a chamber. Often combined with MALDI (MALDI-TOF) Detections from Reflector Time Of Flight tube multiple laser bursts are averaged. Multiple laser
ion source

detector

time of flight

Tandem MS- MS/MS Separation and identification of compounds in complex mixtures Induce fragmentation and mass analyze the fragment ions. Uses two or more mass analyzers/filters separated by a collision cell filled with Argon or Xenon
detector

reflector

time of flight

Different MS-MS configurations

Quadrupole-quadrupole (low energy) Magnetic sector-quadrupole (high) Quadrupole-time-of-flight (low energy) Time-of-flight-time-of-flight (low energy)

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Typical Mass Spectrometer

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LC/LC-MS/MS-Tandem LC, Tandem MS

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Typical Mass Spectrum

Characterized by sharp, narrow peaks X-axis position indicates the m/z ratio of a given ion (for singly charged ions this corresponds to the mass of the ion) Height of peak indicates the relative abundance of a given ion (not reliable for quantitation) Peak intensity indicates the ions ability to desorb or fly (some fly better than others) 54

All analytes are sorted based on a

mass to charge ratio (m/z)

m/z ratio:
Molecular weight divided by the charge on this analyte

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Typical Mass Spectrum

Relative Abundance aspirin

120 m/z-for singly charged ion this is the mass 56

Resolution & Resolving Power

Width of peak indicates the resolution of the MS instrument The better the resolution or resolving power, the better the instrument and the better the mass accuracy Resolving power is defined as:

DM M

M is the mass number of the observed mass (DM) is the difference between two masses 57 that can be separated

Resolution in MS

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Resolution in MS
783.455

QTOF

784.465

785.475

783.6

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Mass Spectrometer Schematic

High Vacuum System
Turbo pumps Diffusion pumps Rough pumps Rotary pumps

Inlet

Ion Source
MALDI ESI IonSpray FAB LSIMS 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.

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Different Mass Analyzers

Magnetic Sector Analyzer (MSA)
High resolution, exact mass, original MA

Quadrupole Analyzer (Q)

Low (1 amu) resolution, fast, cheap

Time-of-Flight Analyzer (TOF)

No upper m/z limit, high throughput

Ion Trap Mass Analyzer (QSTAR)

Good resolution, all-in-one mass analyzer

Ion Cyclotron Resonance (FT-ICR)

Highest resolution, exact mass, costly
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Different Types of MS
ESI-QTOF
Electrospray ionization source + quadrupole mass filter + time-of-flight mass analyzer

MALDI-QTOF
Matrix-assisted laser desorption ionization + quadrupole + time-of-flight mass analyzer
Both separate by MW and AA seq
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Different Types of MS
GC-MS - Gas Chromatography MS
separates volatile compounds in gas column and IDs by mass

LC-MS - Liquid Chromatography MS

separates delicate compounds in HPLC column and IDs by mass

MS-MS - Tandem Mass Spectrometry

separates compound fragments by magnetic field and IDs by mass

LC/LC-MS/MS-Tandem LC and Tandem MS

Separates by HPLC, IDs by mass and AA sequence

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Magnetic Sector Analyzer

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Quadrupole Mass Analyzer

A quadrupole mass filter consists of four parallel metal rods with different charges Two opposite rods have an applied + potential and the other two rods have a potential The applied voltages affect the trajectory of ions traveling down the flight path For given dc and ac voltages, only ions of a certain mass-to-charge ratio pass through the quadrupole filter and all other65 ions are thrown out of their original path

Quadrupole Mass Analyzer

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Q-TOF Mass Analyzer

NANOSPRAY TIP MCP DETECTOR

PUSHER
HEXAPOLE HEXAPOLE COLLISION CELL

TOF
REFLECTRON

QUADRUPOLE
ION SOURCE SKIMMER

HEXAPOLE

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Mass Spec Equation (TOF)

2Vt2 m = z L2
m = mass of ion z = charge of ion V = voltage L = drift tube length t = time of travel

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Ion Trap Mass Analyzer

Ion traps are ion trapping devices that make use of a threedimensional quadrupole field to trap and massanalyze ions
invented by Wolfgang Paul (Nobel Prize1989) Offer good mass resolving power
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FT-ICR
Fourier-transform ion cyclotron resonance

Uses powerful magnet (5-10 Tesla) to create a miniature cyclotron Originally developed in Canada (UBC) by A.G. Marshal in 1974 FT approach allows many ion masses to be determined simultaneously (efficient) Has higher mass resolution than any other MS analyzer available
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FT-Ion Cyclotron Analzyer

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Common mass analyzers

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Atmospheric pressure chemical ionisation (APCI)

APCI is a technique that also creates gas phase ions from the liquid sample. It too takes place at atmospheric pressure and uses a similar interface to that in ESI. As in ESI, the sample solution is mixed with a nebulising gas and the sample arrives in the spray chamber as a fine mist of droplets or spray. In APCI, an extra component a corona discharge is used to further ionise the analyte droplets in a manner similar to CI. While a small amount of fragmentation may occur, the technique is still considered to be a soft ionisation one. The gas-phase ionisation in APCI is more effective than ESI for analysing less polar species. 73 ESI and APCI are complementary methods.

Atmospheric pressure chemical ionisation (APCI)

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Fast atom bombardment (FAB)

FAB is an ionisation technique that involves bombarding a solid spot of the analyte/matrix mixture on the end of a sample probe by a fast particle beam. The matrix (a small organic species like glycerol or 3-nitro benzylalcohol) is used to keep a homogenous sample surface. The particle beam is incident onto the surface of the analyte/matrix spot, where it transfers its energy bringing about localised collisions and disruptions. Both analyte ions and matrix ions are ejected (sputtered) from the surface as secondary ions by this process. These ions are then extracted and focused before passing to the mass analyser.
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Fast atom bombardment (FAB)

In FAB, the particle beam is a neutral inert gas (argon or zenon) at 410 keV. The process is a comparatively soft one. The resulting spectra consist largely of protonated and sodiated molecular species (e.g. [M+H]+ and [M+Na]+) with some minor structural fragmentation. The low mass region of the spectra are, however, dominated by matrix and matrix/salt cluster ions. 76

Fast atom bombardment

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Tandem Mass Spectrometry

Purpose is to fragment ions from parent ion to provide structural information about a molecule Also allows mass separation and AA identification of compounds in complex mixtures

Uses two or more mass analyzers/filters separated by a collision cell filled with Argon or Xenon Collision cell is where selected ions are sent for further fragmentation

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Tandem Mass Spectrometry

Different MS-MS configurations Quadrupole-quadrupole (low energy) Magnetic sector-quadrupole (high) Quadrupole-time-of-flight (low energy) Time-of-flight-time-of-flight (low energy)

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MS-MS & Proteomics

Advantages
Provides precise sequence-specific data
More informative than PMF methods (>90%) Can be used for denovo sequencing (not entirely dependent on databases)

Disadvantages
Requires more handling, refinement and sample manipulation Requires more expensive and complicated equipment Requires high level expertise
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Can be used to ID post- Slower, not generally trans. modifications high throughput

Mass Spectrometer Schematic

High Vacuum System
Turbo pumps Diffusion pumps Rough pumps Rotary pumps

Inlet

Ion Source
MALDI ESI IonSpray FAB LSIMS 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.

81

MS Detectors
Early detectors used photographic film Todays detectors (ion channel and electron multipliers) produce electronic signals via 2o electronic emission when struck by an ion Timing mechanisms integrate these signals with scanning voltages to allow the instrument to report which m/z has struck the detector Need constant and regular calibration
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Mass Detectors

Electron Multiplier (Dynode)

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Different MS-MS Modes

Product or Daughter Ion Scanning
first analyzer selects ion for further fragmentation most often used for peptide sequencing

Precursor or Parent Ion Scanning

no first filtering, used for glycosylation studies

Neutral Loss Scanning

selects for ions of one chemical type (COOH, OH)

Selected/Multiple Reaction Monitoring

selects for known, well characterized ions only
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Applications
Mass spectrometry is an excellent technique for both qualitative and quantitative analysis. On the basis of molecular weight alone, many compounds can be identified (with the exception of isomers). Using a combination of molecular weight and fragmentation patterns, unambiguous identification is possible as well as structural elucidation of unknown compounds. MS can also be used for quantitative work using either internal or external standards for reference. Limits of detection are very low so sensitivity is 85 excellent.

 Mass spectrometry is a suitable technique for both large and small molecules. It has important applications in proteomics for both the analysis of peptides and proteins. Proteins can be analysed directly or their structure elucidated by peptide sequencing. There are extensive search libraries now available for characterising the protein or peptide of interest in the sample. MS is very useful for following the synthesis/quality control of oligonucleotides. Rapid, unambiguous and comprehensive identification of metabolic changes in drug discovery can be carried out using MS experiments. Fast detection of heroin on banknotes in drug trafficking cases.
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 Other applications include: the determination of rosuvastatin in brain tissue, drug distribution in whole body sections using MALDI MS and tandem mass spectrometric (MS2), determination of dydrogesterone, an orally active synthetic progestogen, in human plasma. One very important application of tandem mass spectrometry is its ability to distinguish between molecules of identical molecular weight on the basis of their fragmentation patterns; and the daughter ions can then be used for subsequent quantitative measurement. The anthracyclines, doxorubicin and epirubicin are epimers of each other and both have a molecular weight of 543. MS2 can be employed to quantitate them individually.
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Thank you

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