Proteomics: Strategies for protein Identification

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Proteomics Strategies for protein Identification

• Yao-Te Huang
• Oct 12, 2009

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Two major methods to determine proteins ID

• Determine proteins ID by traditional chemical
  method (Edman degradation)
• Determine proteins ID by mass spectrometry

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• Part One
• Determine proteins ID by traditional chemical
  method (Edman degradation)

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1A complete hydrolysis

• The first step is to determine the amino acid
  composition of a protein.
• The protein is hydrolyzed into its constituent
  amino acids by heating it in 6 N HCl at 110C for
  24-72 hours. Amino acids in hydrolysates can then
  be labeled with ninhydrin or fluorescamine, and
  be separated by ion-exchange chromatography on
  columns of sulfonated polystyrene.
• The identity of the amino acid is revealed by its
  elution volume (which is the volume of buffer
  used to remove the amino acid from the column)
  and the height of the absorption peak is
  proportional to the number of times that
  particular amino acids occurs in the protein.

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If the unknown protein is A-G-D-F-R-G
Determination of Amino Acid Composition.
Different amino acids in a protein hydrolysate
can be separated by ion-exchange chromatography
on a sulfonated polystyrene resin (such as
Dowex-50).Buffers (in this case, sodium citrate)
of increasing pH are used to elute the amino
acids from the column. The amount of each amino
acid present is determined from the absorbance.
Aspartate, which has an acidic side chain, is
first to emerge, whereas arginine, which has a
basic side chain, is the last.
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1A complete hydrolysis (contd.)

• Amino acids treated with ninhydrin give an
  intense blue color, except for proline, which
  gives a yellow color because it contains a
  secondary amino group.
• The concentration of an amino acid in a solution,
  after heating with ninhydrin, is proportional to
  the optical absorbance of the solution. This
  technique can detect a microgram (10 nmol) of an
  amino acid. As little as a nanogram (10 pmol) of
  an amino acid can be detected by fluorescamine,
  which reacts with the ?-amino group to form a
  highly fluorescent product.

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1A complete hydrolysis (contd.)

• The complete hydrolysis gives the info of the
  amino acid composition of a protein, not the
  sequence info.
• However, there are algorithms such as
  AACompIdent, which attempt to predict protein
  sequences on the basis of amino acid compositions
  by searching protein sequence databases for
  entries that would give a similar composition
  profile.

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Polypeptides have characteristic amino acid
compositions
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1B protein sequencing by Edman degradation

• Edman degradation involves labeling the
  N-terminal amino acid of a protein or peptide
  with phenyl isothiocyanate .

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1B protein sequencing by Edman degradation
(contd.)

• Mild acid hydrolysis then results in the cleavage
  of the peptide bond immediately adjacent to this
  modified residue, but leaves the rest of the
  protein intact.
• The terminal amino acid can then be identified by
  chromatography, and the procedure is repeated on
  the next residue and the next, thus building up a
  longer sequence.

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1B protein sequencing by Edman degradation
(contd.)

• It is not suitable for sequencing proteins larger
  than 50 residues in a single run because each
  cycle of degradation is less than 100 efficient.
• This problem is addressed by cleaving large
  proteins into peptides, using either chemical
  reagents or specific endoproteases.

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1C Edman degradation in proteomics
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1C Edman degradation in proteomics (contd.)

• Disadvantages (a) laborious time-consuming
  (sequencing 10 residues per day) (b) in some
  proteins, the ?amino group of the N-terminal
  amino acid residue is modified, and then fails to
  react with phenyl isothiocyanate.
• Advantages (a) the most convenient method for
  determining the N-terminal sequence of a protein
  (b) also very sensitive method (that can sequence
  0.5-1 pmol of pure protein).

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• Part Two
• Determine proteins ID by mass spectrometry

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What is a mass spectrometer, and what does it do?

• A mass spectrometer is an analytical device that
  determines the molecular weight of chemical
  compounds by separating molecular ions according
  to their mass-to-charge ratio (m/z).
• The ions are generated by inducing either the
  loss or the gain of a charge (e.g., deprotonation
  or protonation).
• Once the ions are formed they can be separated
  according to the m/z and finally detected.
• The resulting mass spectrum may provide the info
  about MW of a chemical compound or even about its
  structural information.

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Major components of a mass spectrometer (1)
(1) The ion source unit (including sample
introduction) in which, molecular ions are
generated, and then electrostatically
propelled into the mass resolution unit (2) the
mass resolution unit (the mass analyzer) in
which molecules ions can be resolved (separated
or filtered) according to their m/z ratios. (3)
the ion detector unit in which the signal is
detected, and transferred to a computer for
further processing.
e.g. MALDI or ESI
e.g., TOF (time-of-flight)
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Major components of a mass spectrometer (2)
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MALDI (Matrix-assisted laser desorption-ionization
)

• We may pump much energy into a solid matrix (in
  which macromolecules are embedded) to ionize and
  desorb (into a vacuum) the macromolecule without
  significant degradation.
• The best way to pump energy is through a laser
  pulse, and the matrix is chosen as a substance
  that absorbs strongly at the laser wavelength.
• The power density required to generate a
  significant ion current corresponds to an energy
  flux of 20mJ/cm2.
• Aromatic molecules such as 2,5-dihydroxybenzoic
  acid, which absorbs in the UV, are favorite
  matrices because of the common use of UV lasers
  in the MALDI method.
• The pulsed nature of the excitation (from a few
  tens of nanoseconds to a few hundred
  microseconds) simplifies the data analysis
  because all molecules begin their flight in
  nearly a synchronous fashion.

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MALDI (Matrix-assisted laser desorption-ionization
)

• In MALDI, the analyte is first co-crystalized
  with a large molar excess of a matrix compound,
  usually a UV-absorbing weak organic acid.
• Irradiation of this analyte-matrix mixture by a
  laser results in the vaporization of the matrix,
  which carries the analyte with it into the vapor
  phase. That is, both the matrix and any sample
  embedded in the matrix are vaporized.
• Ionization of the analyte results from exchanges
  of electrons (or protons) with the matrix
  compound.
• Once in the gas phase, the desorbed charged
  molecules are then directed electrostatically
  into the mass resolution unit (the mass
  analyzer).

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MALDI (Matrix-assisted laser desorption-ionization
)
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Commonly used MALDI matrices
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ESI (electrospray ionization)

• Charged microdroplets containing the
  macromolecules to be studied are sprayed into the
  mass spectrometer through a charged nozzle (which
  ionizes the drops that are exciting the tip).
• As the droplets accelerate away from the tip, the
  solvent evaporates until, at some point, the
  concentration of charges is so high that the
  coulombic forces overcome the surface tension of
  the drop, resulting in dispersion of the drop
  into a spray of smaller droplets.
• These droplets continue to evaporate and will
  themselves disperse into even finer sprays until
  all the solvent is gone, leaving the macroions
  they contained for analysis.

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ESI (electrospray ionization)
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ESI
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nanoESI

• The spray needle has been made very small, and is
  positioned close to the entrance to the mass
  analyzer.
• The flow rates for nanoESI sources are on the
  order of tens to hundreds of nanoliters per
  minute.
• The end result of this rather simple adjustment
  is increased efficiency, which includes a
  reduction in the amount of sample needed.
• NanoESI is more tolerant of salts and other
  impurities (because less evaporation means the
  impurities are not concentrated down as much as
  they are in ESI)

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nanoESI
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Mass Analyzers having many kinds, including TOF,
quadrupoles, etc

• Performance characteristics accuracy,
  resolution, mass range, tandem analysis
  capabilities, and scan speed

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Accuracy

• It is the ability with which the analyzer can
  accurately provide m/z information and is largely
  a function of an instruments stability and
  resolution.
• For example, an instrument with 0.01accuracy can
  provide info on a 1000.00 Da peptide to 0.1 Da
  or a 10000 Da protein to 1.0 Da.
• An alternative means of describing accuracy is
  using part per million (ppm) terminology, where
  1000.00 Da peptide to 0.1 Da could also be
  described as 1000.00 Da peptide to 100 ppm.

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Resolution

• It is the ability of a mass spectrometer to
  distinguish between ions of different
  mass-to-charge ratios. ResolutionM/(?M)
• Where ?M represents the peak width at half
  maximum,
• And M corresponds to the m/z

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Resolution (contd.)
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Mass Range

• It is the m/z range of the mass analyzer. For
  instance, time-of-flight (TOF) analyzers have
  virtually unlimited m/z range, and quadrupole
  analyzers typically scan up to m/z 3000.

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Tandem MS analysis

• It is the ability of the analyzer to separate an
  ion, generate fragment ions from the original
  ion, and then analyze the fragmentation ions.
• Typically tandem MS experiments are performed by
  generating the ion of interest and selecting it
  with an analyzer. The ion is then collided with
  inert gas molecules such as argon or helium, and
  the fragments generated by the collision are
  analyzed.

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Tandem MS analysis

• Information obtained via tandem analysis can be
  used to sequence peptides, or structurally
  characterize carbohydrates, small
  oliogonucleotides, and lipids.

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Scan speed

• It refers to the rate at which the analyzer scans
  over a particular mass range. Most instruments
  require seconds to perform a full scan, however
  this can vary widely depending on the analyzer.
  Time-of-flight analyzers, for example, complete
  analyses in milliseconds or less.

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The principles of a time-of-flight (TOF) mass
spectrometer
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The principles of a time-of-flight (TOF) mass
spectrometer
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reflector time-of-flight (TOF)
In reflector time-of-flight(TOF) instruments,
the ions are accelerated to high kinetic energy
and are separated along a flight tube as a
result of their different velocities. The ions
are turned around in a reflector, which
compensates for slight differences in kinetic
energy, and then impinge on a detector that
amplifies and counts arriving ions.
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Quadrupoles
Quadrupole mass spectrometers select by
time-varying RF fields between four rods, which
permit a stable trajectory only for ions of a
particular desired m/z.
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Triple Quadrupoles
Again, ions of a particular m/z are selected in
a first section (Q1), fragmented in a collision
cell (Q2), and the fragments separated in Q3.
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Triple quadrupoles (contd.)

• The first quadrupole (Q1) is used to scan across
  a preset m/z range or to select an ion of
  interest.
• The second quadrupole (Q2), also known as the
  collision cell, transmits the ions while
  introducing a collision gas (argon) into the
  flight path of the selected ion. After colliding
  with Ar, the selected ion is fragmented ( a
  process called CID (collision-induced
  dissociation).
• The third quadrupole (Q3) serves to analyze the
  fragment ions generated in the collision cell
  (Q2).

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Peptide Mass Fingerprinting (PMF)

• More recently, MS has been combined with protease
  digestion to enable peptide mass fingerprinting.
• (1) Sequence specific proteases or certain
  chemical cleaving agents are used to obtain a set
  of peptides from the target protein that are then
  mass analyzed
• (2) The observed masses of the proteolytic
  fragments are compared with theoretical in
  silico digests of all the proteins listed in a
  sequence database.
• (3) The matches or hits are then statistically
  evaluated and ranked according to the highest
  probability

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Protein identification by PMF
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• Various databases are available on the web, and
  can be used in conjunction with such computer
  programs such as Profound, ProteinProspector, and
  Mascot.

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PMF possible causes of incorrect protein
identification

• An error in the sequence database
• An inaccurate experimental mass determination
• Existence of two or more polymorphic variants
  (e.g., SNPs)
• Post-translational modifications
• Occasional nonspecific cleavage of the protein by
  trypsin

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Protein identification using Tandem Mass
Spectrometry
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Protein identification using Tandem Mass
Spectrometry (contd.)

• Tandem mass spectrometry has the ability to
  induce fragmentation and perform successive mass
  spectrometry experiments on these ions. It is
  generally used to obtain this structural info.
  (Abbreviated MSn, where n refers to one the
  number of generations of fragment ions being
  analyzed).

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CID (collision-induced dissociation)

• CID is accomplished by selecting an ion of
  interest with the mass analyzer and then
  subjecting that ion of interest to collisions
  with neutral atoms or molecules. The selected ion
  will collide with the collision gas (e.g., Ar,
  He, or Xe), resulting in fragment ions which are
  then mass analyzed. CID can be accomplished with
  a variety of instruments, including triple
  quadrupoles or TOF/TOF mass analyer.

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CID (contd.)

• The fragment ions produced in this process can be
  separated into two classes
• (1) One class retains the charge on the
  N-terminal and occurs at three different
  positions, designated as types an, bn, and cn.
• (2) The second class of fragment ion ions retains
  the charge on the C-terminal and fragmentation
  occurs at three different positions, types xn,
  yn, and zn.

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CID (contd.)

• Most fragment ions are obtained from cleavage
  between a carbonyl and a nitrogen (the amide
  bond).
• Thus, if the charge is retained on the N-terminal
  end of the molecule, the cleavage is a b-type. If
  the charge is retained on the C-terminal end of
  the peptide, the cleavage is y-type.

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Ladder sequencing by mass spectrometry
The differences in mass between consecutive ions
in either series should correspond to the masses
of individual amino acids
E Glu T Thr
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Ladder sequencing by mass spectrometry
Two pairs of residues that are hard to
be distinguished by MS (1) Gln (128.13) Lys
(128.17) (2) Leu (113) Ile (113)