Biomolecules and Mass Spectroscopy

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Biomolecules and Mass Spectroscopy
...wings for molecular elephants
Lecture 11 September 26th 2005
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Nobel Prize In Chemistry 2002
for his development of soft desorption
ionisation methods for mass spectrometric
analyses of biological macromolecules
electrospray wings for molecular elephants
John B. Fenn, Ph.D Yale 1940, Professor, Yale
University 1967-1987
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Mass Spectroscopy Some history and highlights.

• 1897 JJ Thompson first mass spec (1906 Nobel)
• Harold Urey discovers deuterium (1934 Nobel)
• H Kroto et al Fullerenes (1996 Nobel)

• Bieman first analysis of amino acids (1958)
• McFarlane spectrum of insulin 5750 Da (1982)
• Fenn First spectra of proteins above 20,000 Da
  (1988)
• Siuzdak Mass spec. of an intact virus (1996)

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Mass Spec The Basics
Produce gas phase ions

• 5 basic components Inlet, Source, Mass
  Analyzer, Detector, Signal Processor

Source converts a sample to a mixture of ions,
Analyzer resolves those ions based on their
m/z Detector detects those resolved ions.
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Best choice for Peptides and Proteins
MALDI Matrix-assisted Laser Desorption
ionization ESI ElectroSpray Ionization
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Matrix-assisted laser desorption ionization
(MALDI)

• Matrix is typically a small organic molecule
  with a desirable chromophore
• produce a crystalline lattice in which the
  analyte is integrated
• target is placed in the source
• laser is fired, matrix absorbs photons, gets
  excited!
• Excess energy is transferred to analyte in
  sample, which along with matrix
• is ejected, ionized into the gas phase
• produces and - ions. (peptides tend to pick up
  a single proton M H)

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Typical MALDI-TOF experiment

• First MALDI was on a protein so solvent was
  water
• Common to now use 70 water/30 acetonitrile
• 10 ?M peptide to 10 mM matrix solution
• 0.1 TFA to keep pH less than 4
• If using CHCA or SA as matrix 337 nm N2 Laser

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sinapinic acid (SA)(3,5-dimethoxy-4-hydroxycinnam
ic acid)   
alpha-cyano-4-hydroxycinnamic acid (CHCA)

• very efficient at ionizing proteins (intense
  signals)
• produces large fragments particularly in the
  500-5000 range where tryptic digests appear
  (Hence use for lt10,000 Da proteins
• Protein mass fingerprinting

• not strongly ionizing so fewer multiply charged
  ions detected
• high affinity for proteins
• very non-selective
• best for hydrophobic proteins
• best for crude biological extracts

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Concentration of sample contaminants tolerated
by MALDI of peptides and proteins
Mock et al., Rapid Commun. Mass Spectrom. 4
(1992) 233
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Pros and Cons of MALDI

• User friendly
• high accuracy
• very sensitive (down to attomole concs)
• excess of matrix molecules helps prevent cluster
  formation
• matrix means no need to adjust wavelength to
  match abs. freq. of each analyte

• Can be difficult to produce good matrix
  admixtures
• really only for detection of intact peptide ions
  (so not great for peptide-sequencing)

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Time-of-Flight (TOF)

• measures time it takes ions to fly from one end
  of the analyzer to the other.
• The greater the m/z the faster they fly (Newton)

• Original linear TOF detectors gave poor
  resolution

Average time in TOF 10-7 sec average speed 1-2
x 105 km/h
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Linear TOF vs Reflectron

• Linear poor resolution due to velocity
  variation of ions with the same m/z
• Reflectron Contact lens for a near sighted
  machine!

(a)
(b)
Insulin (a) with reflectron (b) linear TOF
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Electrospray

• an electrospray is produced by applying a strong
  electric field to
• a liquid passing through a capillary tube with
  a weak flux
• desolvation by gas flow (N2) or heated
  capillary-apply vacuum
• best for peptides/proteins at acidic pH
• produces multiply charged ions (unique
  characteristic)
• multiply charged species bring m/z into
  quadrupole limit range

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ESI Spectrum of Bovine apomyoglobin
(A) Multi-charge envelope
(B) After Charge-deconvolution algorithm
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Quadrupole detection (Q1)

• 4 metal rods arranged in parallel (hence the
  name!)

• DC/RF applied ? magnetic field
• causes corkscrew trajectory for ions
• depending on voltage applied, only specific m/z
  ions will pass through
• ions of greater or lesser m/z will fail to pass
  through
• by sweeping RF voltages, ions of increasing m/z
  values can be analyzed
• limited range (max 4 kDA) - Digestion??

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Why digestion?

• greater the mass-the greater the absolute error
• Not all proteins are amenable to intact mass
  measurement
• (e.g.v.large/v.hydrophobic)
• Modern computers can use algorithms to assist
  protein identification
• Programs can take into account specific cleavage
  sites of agents such as trypsin
• Digesting can increase ionization or even
  introduce it

Pros and Cons

• Resolution vs m/z intact mass window
• Sequencing vs mass fingerprinting
• Ease of use/sample preparation

Solution??
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Tandem MS (MS-MS)

• Triple Quadrupole Tandem mass analysis
• Q1-q2-Q3

Historically combined with ESI

• Q1-q2-TOF (Q-TOF)
• Best Resolution/large upper limit m/z/high mass
  accuracy
• Tandem HPLC (John R Hayes (Scripps))

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References

• D. C. Liebler, Introduction to Proteomics,
  Humana Press 2002.
• E. De Hoffmann, J. Charette and V. Stroobant,
  Mass Spectrometry
• 1st Ed., Wiley Press, 1996.
• R. M. Twyman, Advanced Molecular Biology,
  Springer Press, 1998.
• Proteome Research Mass Spectrometry James, P
  ed. Springer
• New York, 2001
• www.nobel.se
• http//massspec.scrips.edu/ G. Siuzdak Homepage
• http//fields.scripps.edu/ John R Yates
  Homepage