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Unformatted text preview: BIOC*2580
Lecture
6:
Analysis
of
proteins.
 
1 Synopsis:
In
addition
to
the
ion
exchange
chromatography
methods
already
described,
protein
 analysis
 involves
 methods
 for
 separation
 or
 analysis
 by
 gel
 filtration,
 metal
 affinity,
 centrifugation,
electrophoresis
and
mass
spectrometry.

 
 Reading:
Lehninger,
p.
85‐92
(4th
ed
p.
89‐95)
 
 Metal
affinity
chromatography
 
 This
 is
 a
 widely
 used,
 modern
 technique
 because
 it
 can
 yield
 almost
 pure
 proteins
 in
 a
 single
 efficient
 step.
 
 It
 relies
 on
 the
 fact
 that
 clusters
 of
 histidine
in
a
protein
have
high
affinity
for
binding
transition
metals
such
as
 Ni2+
or
Co2+
.

A
column
is
prepared
with
resin
containing
Ni
bound
to
a
 chelating
agent.
 
 Most
natural
proteins
do
not
contain
histidine
clusters,
so
will
not
bind
to
 the
 column.
 
 However,
 if
 the
 protein
 of
 interest
 is
 being
 artificially
 expressed
in
 yeast
or
bacteria,
its
gene
can
be
modified
to
add
a
cluster
of
 6‐8
histidines,
known
as
a
His‐tag,
either
at
the
N‐
or
the
C‐
terminus
of
the
 polypeptide,
 where
 they
 are
 less
 likely
 to
 interfere
 with
 the
 protein’s
 natural
function.
 
 Natural
protein:










































Met‐Pro‐Ser‐Leu‐Ser‐Tyr‐etc
 His‐tagged
protein:

‐His‐His‐His‐His‐His‐His‐Pro‐Ser‐Leu‐Ser‐Tyr‐etc
 
 Now
the
protein
of
interest
is
the
only
one
that
should
bind
to
the
column.

 The
 protein
 is
 then
 eluted
 by
 adding
 buffer
 containing
 imidazole,
 a
 molecule
that
 resembles
the
histidine
side
chain.
Imidazole
will
occupy
the
 Ni2+
sites
on
the
 resin,
allowing
the
His‐tagged
protein
to
pass
out
of
the
 column
and
be
collected.
 
 
 
 
 Page
1
of
5
 BIOC*2580
Lecture
6:
Analysis
of
proteins.
 
2 Gel
filtration
chromatography
 
 Another
widely
used
chromatography
technique
is
 called
 gel
 filtration
 or
 molecular
 exclusion
 chromatography.
 Separation
 is
 on
 the
 basis
 of
 molecular
 size,
 with
 the
 largest
 molecules
 emerging
 first
 (the
 term
 gel
 filtration
 is
 a
 bit
 misleading
 because
 it
 seems
 to
 imply
 smaller
 molecules
 might
 pass
 through
 more
 easily).
 The
 stationary
 phase
 of
 the
 column
 consists
 of
 a
 hydrated
 gel,
 formed
 from
 a
 polymer,
 which
 absorbs
 water
 to
 form
 an
 aqueous
 network
 of
 open
pores.
 The
 gel
 is
 in
 the
 form
 of
 beads
 or
 granules.
 Molecules
 in
 the
 sample
 that
 are
 small
 enough
 can
fit
into
pores
the
gel.

Since
the
gel
is
the
stationary
phase,
 these
molecules
progress
through
the
column
more
slowly.
 
 The
larger
molecules
are
excluded
from
the
pores
by
their
size,
 but
this
does
not
block
them
since
they
simply
stay
in
the
buffer
 that
 flows
 around
 the
 outside
 of
 the
 beads
 of
 gel.
 Therefore
 large
 molecules
 progress
 at
 a
 rate
 similar
 to
 the
 buffer,
 while
 smaller
molecules
are
slowed
down
according
to
 the
extent
that
 they
can
penetrate
the
gel.
 
 Gel
filtration
can
be
used
to
estimate
molecular
mass
of
proteins
 
 Elution
 volume
 of
 proteins
 is
 a
 negative
 slope
 linear
 function
 of
 log
 molecular
 mass.
 
 If
 the
 elution
 volume
 is
 measured
 for
 two
 or
more
proteins
of
known
molecular
mass,
 it
 is
 then
 possible
 to
 measure
 the
 elution
 volume
 of
 an
 unknown
 protein
 and
 estimate
 its
 molecular
 mass
 either
 by
 interpolating
 the
 graph
 or
 by
 deriving
 the
 equation
of
the
straight
line.
 
 Gels
 of
 various
 kinds
 are
 commercially
 available,
and
have
different
pore
sizes
for
 different
sizes
of
protein:
 
 
 Page
2
of
5
 BIOC*2580
Lecture
6:
Analysis
of
proteins.
 
3 Methods
for
separating
protein
molecules
other
than
chromatography
 
 1.
Centrifugation
 In
 centrifugation,
a
sample
is
spun
in
an
 ultracentrifuge
at
speeds
from
10,000
to
75,000
rpm,
 producing
 a
 force
 from
 10,000
 to
 500,000
 x
 gravity.
 At
 these
 forces,
 individual
 molecules
 of
 proteins
 are
 large
 enough
 to
 sediment
 at
 a
 rate
 determined
 by
 their
 size.
 By
 measuring
 sedimentation
velocity,
it's
possible
to
derive
the
molecular
mass.
 It's
theoretically
possible
to
 measure
sedimentation
rates
of
proteins
down
to
10
kDa,
but
very
time
consuming
for
anything
 smaller
than
1000
kDa.
 
 2.
Electrophoresis
 Electrophoresis
 is
 separation
 based
 on
 movement
 of
 charged
 particles
 in
 an
 electric
 field.
 A
 mixture
 of
 proteins
 is
 placed
 between
 a
 pair
 of
 electrodes
 immersed
 in
 a
 conductive
 buffer
 solution,
and
a
voltage
of
100‐1000
volts
applied.
Positive
molecules
move
towards
the
negative
 electrode
 and
 negative
 molecules
 move
 to
 the
 positive
 electrode.
 Rate
 of
 movement
 is
 a
 function
of
 size,
shape,
and
charge.
See
Lehninger
Fig
3‐18,
p.89
(4th
ed
Fig
3‐19,
p.93)
for
an
 illustration
of
electrophoresis
apparatus.

 
 Since
 free
 solution
 is
 subject
 to
 disturbance
 by
 convection
 (local
 fluid
 motion
 caused
 by
 temperature
differences),
the
buffer
is
immobilized
in
a
gel.
On
the
molecular
scale,
the
gel
is
 sufficiently
porous
to
allow
protein
sized‐molecules
to
pass
through.

 
 Agarose
gels
are
best
for
very
high
mass,
especially
DNA
where
molecular
masses
may
be
>10
 MDa.
 
 Polyacrylamide
gels
are
easily
formed
from
simple
chemicals
in
the
lab,
and
are
often
used
for
 protein.
where
molecular
mass
is
in
the
range
10‐1000
kDa.

 
 A
typical
polyacrylamide
gel
is
5‐10%
polymer,
90‐95%
buffer.
 
 
 Page
3
of
5
 
 BIOC*2580
Lecture
6:
Analysis
of
proteins.
 
4 SDS‐PolyAcrylamide
 Gel
 Electrophoresis
 (SDS‐PAGE)
 is
 a
 modified
 form
 of
 electrophoresis
 in
 which
protein
is
treated
with
the
ionic
detergent
sodium
dodecyl
sulfate,
SDS.

 
 SDS
ions
coat
 the
protein
 molecules,
 which
 adopt
 rodlike
 shapes,
 so
 that
 with
 SDS
bound,
all
proteins
 have
the
 same
 rodlike
 shape.
 The
 strong
 negative
 charge
 of
 the
 bound
 SDS
 ions
overrides
the
 somewhat
variable
 charge
 of
 the
 polypeptide
 itself.
 The
 charge
 of
 the
 complex
 then
 depends
 on
 the
 number
 of
 SDS
 molecules
 bound,
which
in
turn
 depends
on
the
 size
 of
 the
 polypeptide.
 As
 a
 result,
 all
polypeptides
now
behave
as
if
they
 had
similar
charge
per
unit
length.
 
 The
 one
 remaining
 factor
 that
 distinguishes
 protein
 in
 the
 presence
 of
 SDS
 is
 the
 increased
 frictional
resistance
for
larger
proteins.
Hence
separation
is
based
on
size,
with
smaller
proteins
 being
most
mobile,
and
larger
proteins
being
retarded
(opposite
to
gel
filtration).

 
 We
 can
 use
 SDS
 gel
 electrophoresis
 to
 get
 information
 about
 the
 size
 or
 molecular
 mass
 of
 a
 polypeptide.
 In
 addition
 to
 the
 unknown
 protein
 samples,
 a
 set
 of
 proteins
 of
 known
 size
 are
 included
 in
 a
 separate
 lane
 of
 the
 gel.
 These
 proteins
 are
 used
 to
 create
 a
 calibration
 plot
 to
 match
distance
migrated
to
molecular
mass.
This
is
one
of
the
standard
laboratory
methods
for
 determining
protein
size
or
molecular
mass.
See
Lehninger
Fig
3‐19,
p.
90
(4th
ed
Fig.
3‐20,
p.
93).
 
 Another
variant
on
electrophoresis
is
called
isoelectric
 focusing.
 This
is
electrophoresis
in
a
pH
 gradient,
and
separates
proteins
on
the
basis
of
charge.
 
 Every
 protein
 has
 a
 specific
 pH
 called
 the
 isoelectric
 point,
 at
 which
 the
 sum
 of
negative
charges
is
exactly
equal
to
the
sum
of
positive
charges.
 Then
 its
 net
 charge
 is
 zero.
 If
 a
 protein
 starts
 at
 the
 high
 pH
 end
 of
 the
 gradient,
it
will
have
negative
charge.
If
the
positive
electrode
is
placed
at
 the
low
pH
end
of
the
gradient,
the
protein
migrates
towards
the
positive
 electrode,
and
passes
 through
 buffer
 of
 gradually
 decreasing
 pH.
 As
 the
 pH
 decreases,
 different
 side
 chains
 in
 the
 protein
 become
 protonated,
 causing
 the
 net
 negative
 charge
 to
 decrease.
 At
 some
 point
 the
 protein
 reaches
the
pH
equal
to
its
isoelectric
point,
where
it
has
no
charge
and
 stops
migrating
since
there
is
no
attraction
to
either
electrode.
Separation
 occurs
because
each
protein
in
a
mixture
has
a
different
isoelectric
point.
 
 
 Page
4
of
5
 BIOC*2580
Lecture
6:
Analysis
of
proteins.
 
5 Two
 dimensional
 electrophoresis
 involves
 separation
 a
 protein
first
by
isoelectric
focusing
in
a
thin
capillary
tube.
 The
 spaghetti‐like
 gel
 contains
 the
 partly
 separated
 proteins,
 and
 is
 then
 laid
 on
 the
 top
 edge
 of
 a
 conventional
 SDS‐PAGE
 gel.
 A
 second
 separation
 by
 electrophoresis
 is
 then
 carried
 out
 at
 90o
 to
 the
 original
 isoelectric
 focusing.
 See
 Lehninger
 Fig
 3‐20,
 p.90
 (4th
 ed
 Fig
3‐21,
p.94‐95).
 
 
 Mass
 spectrometry
 is
 a
 technique
 often
 used
 in
 conjunction
 with
 electrophoresis
 to
 identify
 proteins
 (Lehninger
Box
3‐2,
p.
98‐100
(4th
ed
p.102‐103)).
 
 A
pure
protein
sample
is
obtained
as
a
band
cut
out
from
 gel
electrophoresis.
The
sample
is
either
 introduced
into
 a
 high
 vacuum
 chamber
 as
 a
 superfine
 spray,
 or
 vaporized
by
laser
bombardment
on
a
positively
charged
 electrode.
 
 This
 yields
 charged
 protein
 ionic
 particles
 which
 are
 accelerated
 by
 attraction
 towards
 a
 negative
 electrode.
 A
 small
 hole
 in
 the
 negative
 electrode
 allows
 some
 of
 the
 ions
 to
 pass
 through,
 forming
 a
 beam
 of
 positively
charged
protein
ions.

The
 velocity
of
the
ions
depends
inverselyon
m/z,
the
 ratio
of
 mass
to
charge.
Since
the
unit
charge
on
an
electron
or
proton
is
known,
the
exact
mass
of
the
 protein
can
be
 calculated
by
measuring
 the
time
of
flight,
 the
time
it
takes
the
beam
to
 travel
 down
 a
tube
of
known
length
from
negative
electrode
to
detector.

The
protein
mass
can
then
 be
compared
with
a
catalog
of
proteins
of
known
mass.


 
 The
 laser
 method
 is
 known
 as
 MALDI‐TOF
 mass
 spectrometry,
 for
 Matrix‐Adsorption,
 Laser
 Desorption
Ionization‐Time
of
Flight
mass
spectrometry.
 
 
 
 
 Page
5
of
5
 ...
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This note was uploaded on 09/21/2011 for the course BIOOC 2580 taught by Professor Douger during the Fall '10 term at University of Guelph.

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