mass spectrometry: methods & theory. 2 ms principles different elements can be uniquely...

Mass Spectrometry: Methods & Theory

2

MS Principles

• Different elements can be uniquely identified by their mass

3

MS Principles

• Different compounds can be uniquely identified by their mass

CH3CH2OH

NOH

HO

-CH2-

-CH2CH-NH2

COOH

HO

HO

Butorphanol L-dopa Ethanol

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

5

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)

6

Masses in MS

• Monoisotopic mass is the mass determined using the masses of the most abundant isotopes

• Average mass is the abundance weighted mass of all isotopic components

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Isotopic Distributions1H = 99.9% 12C = 98.9% 35Cl = 68.1%2H = 0.02% 13C = 1.1% 37Cl = 31.9%

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Isotopic Distributions1H = 99.9% 12C = 98.9% 35Cl = 68.1%2H = 0.02% 13C = 1.1% 37Cl = 31.9%

m/z

100

6.6

32.1

2.1 0.06 0.00

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Mass Calculation (Glycine)

NH2—CH2—COOH

R1—NH—CH2—CO—R3

Amino acid

Residue

Monoisotopic Mass1H = 1.00782512C = 12.0000014N = 14.0030716O = 15.99491

Glycine Amino Acid Mass5xH + 2xC + 2xO + 1xN= 75.032015 amuGlycine Residue Mass3xH + 2xC + 1xO + 1xN=57.021455 amu

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Amino Acid Residue Masses

Glycine 57.02147Alanine 71.03712Serine 87.03203Proline 97.05277Valine 99.06842Threonine 101.04768Cysteine 103.00919Isoleucine 113.08407Leucine 113.08407Asparagine 114.04293

Aspartic acid 115.02695Glutamine 128.05858Lysine 128.09497Glutamic acid 129.0426Methionine 131.04049Histidine 137.05891Phenylalanine 147.06842Arginine 156.10112Tyrosine 163.06333Tryptophan 186.07932

Monoisotopic Mass

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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 (iso.) • Aston Awarded Nobel Prize in 1922• 1920s - Electron impact ionization and

magnetic sector mass analyzer introduced

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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

Ionizer

Sample

+_

Mass Analyzer Detector

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Typical Mass Spectrometer

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Typical Mass Spectrum

aspirin

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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 ion’s ability to desorb or “fly” (some fly better than others)

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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:

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

MM

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Resolution in MS

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Resolution in MS

QTOF

783.455

784.465

785.475

783.6

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Inside a Mass Spectrometer

22

Mass Spectrometer Schematic

InletIon

SourceMassFilter Detector

DataSystem

High Vacuum SystemTurbo pumpsDiffusion pumpsRough pumpsRotary pumps

Sample PlateTargetHPLCGCSolids probe

MALDIESIIonSprayFABLSIMSEI/CI

TOFQuadrupoleIon TrapMag. SectorFTMS

Microch plateElectron Mult.Hybrid Detec.

PC’sUNIXMac

23

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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EI Fragmentation of CH3OH

CH3OH CH3OH+

CH3OH CH2O=H+ + H

CH3OH + CH3 + OH

CHO=H+ + HCH2O=H+

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Why You Can’t 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 sub-fragments and even peptides of 2,3,4… amino acids

• Result is 10,000’s of different signals from a single protein -- too complex to analyze

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Soft Ionization • Soft ionization techniques keep the molecule

of interest fully intact• Electro-spray ionization first conceived in

1960’s by Malcolm Dole but put into practice in 1980’s 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

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Soft Ionization Methods

337 nm UV laser

MALDI

cyano-hydroxycinnamic acid

Gold tip needle

Fluid (no salt)

ESI

+_

31

Electrospray (Detail)

32

Electrospray (Detail)

33

Electrospray Ionization• Sample dissolved in polar, volatile buffer (no

salts) and pumped through a stainless steel capillary (70 - 150 m) at a rate of 10-100 L/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)

34

Electrospray Ionization95%H2O/5%CH3CN 5%H2O/95%CH3CN

100 V

1000 V

3000 V

35

Electrospray Ionization• Can be modified to “nanospray” system

with flow < 1 L/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)

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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

• 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

37

Electrospray Ionization• Samples of MW up to 1200 Da usually

produce singly charged ions with observed MW equal to parent mass + H (1.008 Daltons)

• Larger samples (typically peptides) yield ions with multiple charges (from 2 to 20 +)

• Multiply charged species form a Gaussian distribution with those having the most charges showing up at lower m/z values

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Multiply Charged Ions

ESI spectrum ofHEW LysozymeMW = 14,305.14

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Peptide Masses From ESI

m/z = (MW + nH+)n

m/z = mass-to-charge ratio of each peak on spectrumMW = MW of parent moleculen = number of charges (integer)H+ = mass of hydrogen ion (1.008 Da)

Each peak is given by:

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Peptide Masses From ESI

1431.6 = (MW + nH+)n

Charge (n) is unknown, Key is to determine MWChoose any two peaks separated by 1 charge

1301.4 = (MW + [n+1]H+)[n+1]

2 equations with 2 unknowns - solve for n first

n = 1300.4/130.2 = 10

Substitute 10 into first equation - solve for MW

MW = 14316 - (10x1.008) = 14305.9 14,305.14

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ESI Transformation

• Software can be used to convert these multiplet spectra into single (zero charge) profiles which gives MW directly

• This makes MS interpretation much easier and it greatly increases signal to noise

• Two methods are available– Transformation (requires prior peak ID)– Maximum Entropy (no peak ID required)

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Maximum Entropy

43

ESI and Protein Structure

• ESI spectra are actually quite sensitive to the conformation of the protein

• Folded, ligated or complexed proteins tend to display non-gaussian peak distributions, with few observable peaks weighted toward higher m/z values

• Denatured or open form proteins/peptides which ionize easier tend to display many peaks with a classic gaussian distribution

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ESI and Protein Conformation

Native Azurin

Denatured Azurin

45

Matrix-Assisted Laser Desorption Ionization

337 nm UV laser

MALDI

cyano-hydroxycinnamic acid

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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, 4-hydroxycinnaminic 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)

47

MALDI Ionization

++

+

+

-

--

++

+

+

-

---+ +

Analyte

Matrix

Laser

+

++

• Absorption of UV radiation by chromophoric matrix and ionization of matrix

• Dissociation of matrix, phase change to super-compressed gas, charge transfer to analyte molecule

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

+

+

+

48

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 L of 1 pmol/L sample

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MALDI Sample Limits

• Phosphate buffer < 50 mM• Ammonium bicarbonate < 30 mM• Tris buffer < 100 mM• Guanidine (chloride, sulfate) < 1 M• Triton < 0.1%• SDS < 0.01%• Alkali metal salts < 1 M• Glycerol < 1%

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MALDI = SELDI

337 nm UV laser

MALDI

cyano-hydroxycinnaminic acid

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MALDI/SELDI SpectraNormal

Tumor

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MassFilter


InletIon

Source DetectorData

System






PC’sUNIXMac

53

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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Magnetic Sector Analyzer

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Mass Spec Equation (Magnet Sector)

mz

B2 r2

2V=

M = mass of ion B = magnetic fieldz = charge of ion r = radius of circleV = voltage

56

Quadrupole Mass Analyzer

57

Quadrupole Mass Analyzer• A quadrupole mass filter consists of four parallel

metal rods with different charges • Two opposite rods have an applied potential of

(U+Vcos(t)) and the other two rods have a potential of -(U+Vcos(t))

• 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 other ions are thrown out of their original path

58

Q-TOF Mass Analyzer

TOF

NANOSPRAY TIP

IONSOURCE

HEXAPOLECOLLISIONCELL

HEXAPOLE

HEXAPOLE

QUADRUPOLE

MCPDETECTOR

REFLECTRONSKIMMER

PUSHER

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Mass Spec Equation (TOF)

mz

2Vt2

=

m = mass of ion L = drift tube lengthz = charge of ion t = time of travelV = voltage

L2

60

Ion Trap Mass Analyzer• Ion traps are ion

trapping devices that make use of a three-dimensional quadrupole field to trap and mass-analyze ions

• invented by Wolfgang Paul (Nobel Prize1989)

• Offer good mass resolving power, and even MSn capability.

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Ion Trap Mass Analyzer

62

FT-Ion Cyclotron Analzyer

63

FT-ICR• Uses powerful magnet (5-10 Tesla) to

create 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• Will revolutionize proteomics studies

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MassFilter


InletIon

Source DetectorData

System






PC’sUNIXMac

65

MS Detectors• Early detectors used photographic film• Today’s 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

66

Mass Detectors

Electron Multiplier (Dynode)

67

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

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Different Types of MS

• GC-MS - Gas Chromatography MS– separates volatile compounds in gas column

and ID’s by mass

• LC-MS - Liquid Chromatography MS– separates delicate compounds in HPLC

column and ID’s by mass

• MS-MS - Tandem Mass Spectrometry– separates compound fragments by magnetic

field and ID’s by mass

69

Tandem Mass Spectrometer

TOF

NANOSPRAY TIP

IONSOURCE

HEXAPOLECOLLISIONCELL

HEXAPOLE

HEXAPOLE

QUADRUPOLE

MCPDETECTOR

REFLECTRONSKIMMER

PUSHER

70

Tandem Mass Spectrometry• Purpose is to fragment ions from parent ion to

provide structural information about a molecule

• Also allows separation and 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

71

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)

• Fragmentation experiments may also be performed on single analyzer instruments such as ion trap instruments and TOF instruments equipped with post-source decay

72

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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MS-MS & Proteomics

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Proteomics Applications• Protein sample identification/confirmation• Protein sample purity determination• Detection of post-translational modifications• Detection of amino acid substitutions• Determination of disulfide bonds (# & status)• De novo peptide sequencing• Mass fingerprint identification of proteins• Monitoring protein folding (H/D exchange)• Monitoring protein-ligand complexes/struct.

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Conclusions

• Mass spectrometers exist in many different configurations to allow different problems to be solved

• All mass spectrometers have a common architecture and relatively similar operating principles

• Understanding the applications and limitations of MS in proteomics will help in understanding and meeting the bioinformatics needs in proteomics

mass spectrometry: methods & theory. 2 ms principles different elements can be uniquely...

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