1. 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
Butorphanol L-dopa Ethanol N OH HO
-CH2-
-CH2CH-NH2
COOH HO
CH3CH2OH
MW = MW = MW = 46.1

4. 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 % 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

7. Isotopic Distributions
1H = 99.9% C = 98.9% Cl = 68.1%
2H = 0.02% C = 1.1% Cl = 31.9%

8. Isotopic Distributions
1H = 99.9% C = 98.9% Cl = 68.1%
2H = 0.02% C = 1.1% Cl = 31.9%
100
32.1
6.6
2.1
0.06
0.00
m/z

9. Mass Calculation (Glycine)
NH2—CH2—COOH
Amino acid
R1—NH—CH2—CO—R3
Residue
Glycine Amino Acid Mass
5xH + 2xC + 2xO + 1xN
= amu
Glycine Residue Mass
3xH + 2xC + 1xO + 1xN
= amu
Monoisotopic Mass
1H =
12C =
14N =
16O =

10. Amino Acid Residue Masses
Monoisotopic Mass
Glycine
Alanine
Serine
Proline
Valine
Threonine
Cysteine
Isoleucine
Leucine
Asparagine
Aspartic acid
Glutamine
Lysine
Glutamic acid
Methionine
Histidine
Phenylalanine
Arginine
Tyrosine
Tryptophan

11. 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

12. MS History 1948-52 - Time of Flight (TOF) mass analyzers introduced
Quadrupole ion filters introduced by W. Paul, also invents the ion trap in 1983 (wins 1989 Nobel Prize)
Tandem mass spectrometer appears
Mass spectrometers are now one of the MOST POWERFUL ANALYTIC TOOLS IN CHEMISTRY

13. 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

14. Mass Spec Principles
Sample + _
Ionizer
Mass Analyzer
Detector

15. Typical Mass Spectrometer

16. Typical Mass Spectrum
aspirin

17. 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)

18. 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 (DM) is the difference between two masses that can be separated DM M

19. Resolution in MS

20. Resolution in MS
QTOF
783.6

21. Inside a Mass Spectrometer

22. Mass Spectrometer Schematic
Inlet
Ion
Source
Mass
Filter
Detector
Data
System
High Vacuum System
Turbo pumps
Diffusion pumps
Rough pumps
Rotary pumps
Sample Plate
Target
HPLC GC
Solids probe
MALDI
ESI
IonSpray
FAB
LSIMS
EI/CI
TOF
Quadrupole
Ion Trap
Mag. Sector
FTMS
Microch plate
Electron Mult.
Hybrid Detec.
PC’s
UNIX
Mac

23. Different Ionization Methods
Electron Impact (EI - Hard method)
small molecules, 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

25. 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

26. EI Fragmentation of CH3OH

27. 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

28. 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

30. Soft Ionization Methods
337 nm UV laser
Fluid (no salt) + _
cyano-hydroxy
cinnamic acid
Gold tip needle
MALDI
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 ( mm) at a rate of 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)

34. Electrospray Ionization
95%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 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)

36. 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

38. Multiply Charged Ions
ESI spectrum of
HEW Lysozyme
MW = 14,305.14

39. Peptide Masses From ESI
Each peak is given by:
m/z = (MW + nH+) n
m/z = mass-to-charge ratio of each peak on spectrum
MW = MW of parent molecule
n = number of charges (integer)
H+ = mass of hydrogen ion (1.008 Da)

40. Peptide Masses From ESI
Charge (n) is unknown, Key is to determine MW
Choose any two peaks separated by 1 charge
= (MW + nH+)
= (MW + [n+1]H+) n
[n+1]
2 equations with 2 unknowns - solve for n first
n = /130.2 = 10
Substitute 10 into first equation - solve for MW
MW = (10x1.008) =
14,305.14

41. 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)

42. 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

44. ESI and Protein Conformation
Native Azurin
Denatured Azurin

45. Matrix-Assisted Laser Desorption Ionization
337 nm UV laser
cyano-hydroxy
cinnamic acid
MALDI

46. 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
Matrix + +
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”) + - -
Laser - +
Analyte + + + - + + - - + - + + + + + +

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 mL of 1 pmol/mL sample

49. 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%

50. MALDI = SELDI
337 nm UV laser
cyano-hydroxy
cinnaminic acid
MALDI

51. MALDI/SELDI Spectra
Normal
Tumor

52. Mass Spectrometer Schematic
Turbo pumps
Diffusion pumps
Rough pumps
Rotary pumps
High Vacuum System
Inlet
Ion
Source
Mass
Filter
Detector
Data
System
Sample Plate
Target
HPLC GC
Solids probe
MALDI
ESI
IonSpray
FAB
LSIMS
EI/CI
TOF
Quadrupole
Ion Trap
Mag. Sector
FTMS
Microch plate
Electron Mult.
Hybrid Detec.
PC’s
UNIX
Mac

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

54. Magnetic Sector Analyzer

55. Mass Spec Equation (Magnet Sector)
B2 r2 = z 2V
M = mass of ion B = magnetic field
z = charge of ion r = radius of circle
V = 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(wt)) and the other two rods have a potential of -(U+Vcos(wt))
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 QUADRUPOLE NANOSPRAY TIP ION SOURCE MCP
HEXAPOLE
COLLISION
CELL
QUADRUPOLE
MCP
DETECTOR
REFLECTRON
SKIMMER
PUSHER

59. Mass Spec Equation (TOF)
2Vt2 = z L2
m = mass of ion L = drift tube length
z = charge of ion t = time of travel
V = voltage

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.

61. 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

64. Mass Spectrometer Schematic
Turbo pumps
Diffusion pumps
Rough pumps
Rotary pumps
High Vacuum System
Inlet
Ion
Source
Mass
Filter
Detector
Data
System
Sample Plate
Target
HPLC GC
Solids probe
MALDI
ESI
IonSpray
FAB
LSIMS
EI/CI
TOF
Quadrupole
Ion Trap
Mag. Sector
FTMS
Microch plate
Electron Mult.
Hybrid Detec.
PC’s
UNIX
Mac

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 MALDI-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

68. 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
ION
SOURCE
HEXAPOLE
COLLISION
CELL
QUADRUPOLE
MCP
DETECTOR
REFLECTRON
SKIMMER
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

73. MS-MS & Proteomics

74. 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.

75. 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