Peptide & Protein

Analysis Part 1
Micro 343
David Wishart, Ath 3-41
david.wishart@ualberta.ca
 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
 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
 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
 Amino Acid Analysis

Model 420A PTC derivatizer with an on-line Perkin Elmer Applied Biosystems
Model 130A PTC Amino Acid Analyzer.
Amino Acid Analysis*
 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
 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
 Norleucine vs Leucine
CH3
CH2
CH3
CH2
CH3
CH2
C C
H2N COOH
H2N COOH
H
H
Nle Leu
 Hydrolysis (Cont’d)*
• 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
Hydrolysis Tube
Acid Hydrolysis Mechanism*
Hydrolysis Effects*

Tryptophan
C
H2N COOH
H
 Polar Amino Acids
CONH2 CH3 OH
N T
C C
H2N COOH H2N COOH
H H
CONH2
OH
Q S
C C
H2N COOH H2N COOH
H H
 Sulfo-Amino Acids
CH3
S
SH

C C
H2N COOH H2N COOH

H H

C M
 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
Amino Acid Derivitization
with PITC*
 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
AA Chromatogram
standard
 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
 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
 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
 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
 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
 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
 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 (<20 AA)
peptides (now replaced by DNA
sequencing methods which are faster)
• Now primarily used to ID proteins from N-
termini using database searches
 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
Microsequencing*
Edman Microsequencer

ABI 492 Procise cLC sequencer

 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
Sample Preparation for PVDF
Immobilization

SDS Electrophoresis
PVDF Immobilization

Electro-blotting on PVDF
PVDF Membrane with
Protein Bands
 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
Edman Sequencing*
 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
 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
 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
Standard Run on 19 PTH AAs
Residue 1 = Leu
Residue 2 = Ile
 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
 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
Protein ID by MS and 2D gel
 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
 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
 2D-GE + MALDI (PMF)

Trypsin
+ Gel punch

p53
Trx

G6PDH
 2D-GE + MS-MS

Trypsin
+ Gel punch

p53
 MudPIT
IEX-HPLC RP-HPLC

Trypsin
+ proteins

p53
 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
MS Analysis Software

Protein Prospector
MS-Fit
Mowse
PeptideSearch
PROWL
 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