Peptide

1
Peptide Protein Analysis Part 1

• Micro 343
• David Wishart, Ath 3-41
• david.wishart_at_ualberta.ca

2
Objectives

• To learn about how AA composition is determined
  experimentally
• To learn about how polypeptides can be
  microsequenced
• To learn the basics of the chemistry and
  chromatographic separations associated with these
  techniques
• To compare contrast new MS methods with these
  older techniques

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Characterizing Proteins

• Amino Acid Analysis
• Used to identify a protein or peptide based on
  amino acid composition
• Can be used to ID unusual amino acids
• Edman Microsequencing
• Used to determine the N-terminal (5-10 residues)
  of a new protein
• Mass Spectrometry
• State of the art for protein ID

4
Protein Characterization

• All (or almost all) protein and peptide
  characterization methods require having small
  amounts of very pure samples
• Purification methods include column
  chromatography (HPLC, FPLC, SEC, IEC) or
  electrophoretic methods
• Electrophoresis is preferred since smaller
  quantities are required

5
Amino Acid Analysis
Model 420A PTC derivatizer with an on-line Perkin
Elmer Applied Biosystems Model 130A PTC Amino
Acid Analyzer.
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Amino Acid Analysis
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Amino Acid Analysis

• Amino acid analysis is a chemical separation
  process to determine the quantity of each amino
  acid in a protein
• There are four steps in amino acid analysis
• Hydrolysis
• Derivitization
• Separation (of derivatized amino acids)
• Data interpretation

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Hydrolysis

• A known amount of internal standard (norleucine)
  is added to the protein sample to be hydrolyzed
• Since norleucine does not naturally occur in
  proteins, is stable to acid hydrolysis and can be
  chromatographically separated from other protein
  amino acids, it makes an excellent internal
  standard

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Norleucine vs Leucine
CH3
CH2
CH2
CH2
Nle Leu
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Hydrolysis (Contd)

• The molar amount of internal standard should be
  approximately equal to that of most of the amino
  acids in the sample (5 nmoles of each amino acid
  (i.e. 10 µg of protein)
• The sample is transferred to a hydrolysis tube
  and dried under vacuum. The tube is placed in a
  vial containing 6 N HCl, a small amount of phenol
  and the protein is hydrolyzed by the HCl vapors
  under vacuum
• The hydrolysis is carried out for 65 minutes at
  150 oC

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Hydrolysis Tube
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Acid Hydrolysis Mechanism
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Hydrolysis Effects
N
Tryptophan
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Polar Amino Acids
OH
CH3
CONH2
N
T
CONH2
OH
Q
S
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Sulfo-Amino Acids
CH3
S
SH
M
C
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Derivatization

• Free amino acids cannot be detected by HPLC
  unless they have been derivatized
• Derivatization is performed automatically on the
  amino acid analyzer by reacting the free amino
  acids, under basic conditions, with
  phenylisothiocyanate (PITC) to produce
  phenylthiocarbamyl (PTC) amino acid derivatives
  which are detectable at 254 nm
• This process takes approximately 30 minutes per
  sample

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Amino Acid Derivitization with PITC
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HPLC Separation

• The PTC-amino acids are separated on a reverse
  phase C18 silica column and the PTC chromophore
  is detected at 254 nm
• Separation is done using an acetate and
  acetonitrile gradient under slightly acidic
  conditons
• All of the amino acids will elute in
  approximately 25 minutes

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AA Chromatogram
standard
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Data Analysis

• Chromatographic peak areas are identified and
  quantified using a data analysis system that is
  attached to the amino acid analyzer system
• A calibration file is used that is prepared from
  the average values of the retention times (in
  minutes) and areas (in (Au) of the amino acids in
  10 standard runs

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Data Analysis

• The amount of each amino acid (pmol) in the
  sample is calculated by dividing the peak area of
  each (corrected for the differing molar
  absorptivities of the various amino acids) by the
  internal standard (norleucine) in the
  chromatogram and multiplying this by the total
  amount of internal standard added to the original
  sample

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Calculation (Mole )

• Mole represents the amount of each amino acid
  present as a of the total amino acids recovered
  in the sample
• Mole percent can be useful for samples in which
  there is no known composition or molecular weight
• pmol of individual amino acid / total pmol of
  all amino acids in the sample X 100 mole
  percent of each amino acid

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Amino Acid Analysis

• Once a very common process in protein
  characterization but lacks sensitivity and is
  relatively slow and labor intensive
• Now largely replaced by MS methods which are
  faster, cheaper, more accurate, much more
  sensitive and far more reliable
• Occasionally used for unusual peptides

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Characterizing Proteins

• Amino Acid Analysis
• Used to identify a protein or peptide based on
  amino acid composition
• Can be used to ID unusual amino acids
• Edman Microsequencing
• Used to determine the N-terminal (5-10 residues)
  of a new protein
• Mass Spectrometry
• State of the art for protein ID

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Edman Microsequencing

• Edman degradation or Edman microsequencing refers
  to determining amino acid sequence by chemical
  degradation at the N-terminal of the protein
• Edman degradation, named after its developer Pehr
  Edman who worked the chemistry out in the 1950's
• First applied by Fred Sanger to insulin

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Edman Degradation

• Can be used to determine AA sequences for up to
  20 residues
• Can be used to determine the sequence of entire
  proteins as long as the protein is first broken
  up into small (lt20 AA) peptides (now replaced by
  DNA sequencing methods which are faster)
• Now primarily used to ID proteins from N-termini
  using database searches

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Edman Microsequencing

• Involves 3 basic steps
• Isolation or immobilization of pure protein (or
  peptide)
• Repeated PITC labelling and cleavage of N
  terminal (PTH-derivatized) amino acids
• Sequential separation (by HPLC) and
  identification of PTH amino acids
• Process takes 2 hours

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Microsequencing
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Edman Microsequencer
ABI 492 Procise cLC sequencer
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Sample Preparation

• 10-100 pmols is preferred, although a lower
  amount is acceptable
• Samples may be submitted in solution, as
  freeze-dried powder or on a PVDF (polyvinylidene
  fluoride) membrane
• If submitted as a solution, should be dissolved
  in 30-150 microliters of volatile solvents such
  as water, acetonitrile, acetic acid, or formic
  acid

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Sample Preparation for PVDF Immobilization
SDS Electrophoresis
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PVDF Immobilization
Electro-blotting on PVDF
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PVDF Membrane with Protein Bands
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Sample on PVDF Membrane

• On a PVDF membrane. The sample should be as
  concentrated as possible on the PVDF membrane
  (e.g. 1 µg/lane)
• The bands should be stained with Coomassie Blue,
  Ponceau S, or Amido Black
• After staining/destaining, a blotted membrane
  must be rinsed thoroughly with deionized water

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Edman Sequencing
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Edman Sequencing

• In this reaction phenylisothiocyanate (PITC)
  reacts with the amino acid residue at the amino
  terminus under basic conditions to form a
  phenylthiocarbamyl (PTC) derivative
• Trifluoroacetic acid then cleaves off the first
  amino acid as its anilinothialinone derivative
  (ATZ-amino acid) and leaves the new amino
  terminus for the next degradation cycle

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Edman Sequencing

• The ATZ amino acid is then removed by extraction
  with N-butyl chloride and converted to a more
  stable phenylthiohydantoin derivative (PTH-amino
  acid) with 25 TFA/water
• The PTH-amino acid is transfered to a
  reverse-phase C18 column for detection at 270nm

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Sequencing

• To determine the sequence of the protein, the
  HPLC chromatogram is compared with the
  chromatogram from the previous residue by laying
  one on top of the other
• From this, the amino acid for the particular
  residue can be determined
• This process is repeated sequentially to provide
  the N-terminal sequence of the protein/peptide

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Standard Run on 19 PTH AAs
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Residue 1 Leu
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Residue 2 Ile
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Microsequencing Summary

• Generates sequence info from N terminus
• Commonly done on low picomolar amounts of protein
  (5-50 ng)
• Newer techniques allow sequencing at the
  femtomolar level (100 pg)
• Up to 20 residues can be read
• Allows unambiguous protein ID for 8 AA
• Relatively slow, modestly expensive
• Now being replaced by MS/MS analyses

43
Characterizing Proteins

• Amino Acid Analysis
• Used to identify a protein or peptide based on
  amino acid composition
• Can be used to ID unusual amino acids
• Edman Microsequencing
• Used to determine the N-terminal (5-10 residues)
  of a new protein
• Mass Spectrometry
• State of the art for protein ID

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Protein ID by MS and 2D gel
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Protein ID by MS and 2D gel

• Requires gel spots to be cut out (tedious)
• Ideal for high throughput (up to 500 samples per
  day)
• Allows modifications to be detected
• MS allows protein identification by
• Intact protein molecular weight
• Peptide fingerprint molecular weights
• Sequencing through MS/MS

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Protein Identification

• 2D-GE MALDI-MS
• Peptide Mass Fingerprinting (PMF)
• 2D-GE MS-MS
• MS Peptide Sequencing/Fragment Ion Searching
• Multidimensional LC MS-MS
• ICAT Methods (isotope labelling)
• MudPIT (Multidimensional Protein Ident. Tech.)
• 1D-GE LC MS-MS
• De Novo Peptide Sequencing

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2D-GE MALDI (PMF)
Trypsin Gel punch
p53
Trx
G6PDH
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2D-GE MS-MS
Trypsin Gel punch
p53
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MudPIT
IEX-HPLC
RP-HPLC
Trypsin proteins
p53
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Typical Results

• 401 spots identified
• 279 gene products
• Confirmed by SAGE, Northern or Southern
• Confirmed by amino acid composition
• Confirmed by amino acid sequencing
• Confirmed by MW pI

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MS Analysis Software
Protein Prospector MS-Fit Mowse PeptideSearch PROW
L
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Conclusion

• Amino acid analysis and Edman degradation are
  powerful chemical techniques which have long
  provided molecular or residue-specific
  information about proteins, their composition,
  sequence and identity
• Both methods are now being replaced by newer,
  more sensitive MS methods
• Next time MS for protein ID