1. No category

N-terminal Edman Sequencing
Sample Preparation
ABRF 2002, Austin, Texas, March 9-12
John Neveu, Harvard University, Cambridge, MA
Bill Henzel, Genentech Inc., South San Francisco, CA
http://www.arbf.org
ABRF 2002, March 8 - 12, Austin, Texas
Objective: Get sample onto the instrument in
sufficient purity, quantity and cleanliness to
allow successful sequence analysis.
• Sample is the intact protein(s) or peptide(s) for analysis.
• Instrument defines the types of supports used for analysis.
• Purity describes the number of proteins or peptides in a sample;
assess by various methods including SDS-PAGE gel, HPLC.
• Quantity can be determined by AAA, Bradford/Lowry/BCA assay,
gel stain intensity, semi-quantitative HPLC.
• Cleanliness is a lack of common interfering compounds that could
impede the analysis.
These five factors will define what types of sample
preparation we must do for a successful analysis.
Supports for analysis
• Glass fiber filter, w/polybrene (quaternary
polyamine) (ABI)
• PVDF membrane by electroblotting, filtration,
adsorption, and covalent linkage.
• Bimodal reaction columns (RP/SAX) (HP)
Biobrene / Polybrene
Common Interferences
• TRIS and other amines cause large chemical artifacts.
• Buffering salts can interfere with the chemistry of the
analysis.
• Detergents can affect sample washout from reaction
cartridge and conversion flask dynamics. SDS can also
precipitate in the instrument.
• Free amino acids contribute to high background in early
cycles.
Quantitation basics
• Limited by the absolute instrument sensitivity,
about 1pmol loaded at ~50% IY for an average lab.
• Important to establish a reasonable level of
quantitation for the researcher and the lab.
• Rule: 1ug of 1kDa protein = 1 nmol
•
•
•
•
100ug of 100kDa protein = 1,000 pmol
10ug of 100kDa protein = 100 pmol
1.0ug of 100kDa protein = 10 pmol
0.5ug of 250kDa protein = 2 pmol
PVDF Stain Intensity by Amido Black
Common Sample Preparation
Techniques for Soluble Samples
•
•
•
•
•
•
•
•
•
•
•
Direct Adsorption to PVDF
Prosorb PVDF cartridge
Reversed-phase (other media) packed micro pipettor tip devices
Ultrafiltration membrane cartridge
Precipitation methods
1D / 2D gel electrophoresis to PVDF by electroblotting
HPLC separation and collection
Micro-spin type cartridges
Bimodal reaction cartridges
On-bead synthetic peptides
Covalent bonding to PVDF membrane
Direct absorption to PVDF
(and Zitex) membrane
• Use for samples with high purity.
• Suitable for samples of reasonably high concentration.
• Allows removal of salts, buffers, free amino acids and
some detergents.
• Sample remains bound to membrane for analysis
(addition of polybrene is recommended).
Prosorb PVDF membrane
• Use for samples with high purity.
• Can be used with dilute samples.
• Allows extensive washing to remove most
contaminants.
• Sample remains bound to membrane for analysis
(addition of polybrene recommended).
Reverse phase (or other media)
packed micropipettor-tip devices
• Suitable for samples of medium to high purity.
• Can be used with dilute samples, binding to media can
be a concern.
• Extensive desalting, detergent removal possible.
• Enrichment of specific species (i.e.. PO4), possible.
• Sample is eluted in high concentration in solution for
analysis or purification.
Ultrafiltration membrane cartridge
• Can be used to remove unwanted low MW
components, peptides and proteins from complex
mixtures by MW cutoff selection.
• Can be used with dilute samples.
• Allows extensive washing, buffer exchange, removal of
detergents.
• Sample is eluted in high concentration in solution for
analysis or other chemistries.
Precipitation methods
• Acetone, chloroform/methanol, TCA
• Low to high purity samples.
• Used to concentrate dilute samples from solution, as
little as 50ng total protein.
• Complete buffer exchange possible.
• Sample resolubilized in high concentration solution,
ideal for loading onto SDS-PAGE gel.
1D or 2D Gel Electrophoresis
Electroblotted to PVDF
• Used for low (1D) to very complex mixtures (2D) of
proteins or peptides.
• High sample concentration needed due to limiting gel
load volumes. Sample solubility issues may arise.
• Sample is strongly bound to PVDF membrane in a
highly concentration for analysis.
HPLC separation and collection
• Suitable for complex mixtures of proteins and
peptides.
• High sample concentration needed, injection volumes
can be limited by column and flow.
• Potential separation and concentration of individual
protein and peptide species.
• Sample is collected in high concentration solution.
Capillary RP HPLC Separation of Peptides
Micro-spin cartridges for protein
and peptide preparation
• Many new vendors, many chemistries and formats.
• Uses range from crude preparation of mixtures to final
clean up of single analyte for analysis.
• Match sample with media for optimal results.
• Multistage processes in convenient formats.
Hewlett Packard Bimodal
reaction columns
• Very convenient reverse phase sample loading,
concentration and washing.
• On-column chemistries possible, easy clean up.
On-bead synthetic peptides
•
•
•
•
Used to confirm synthesis efficiency and correctness.
Single beads can contain nanomoles of peptide.
In practice, cleavage from the resin is best.
Handling of single beads is a difficult task to do
reproducibly.
Covalent binding to
Sequelon membrane
• Chemically bind a single protein or peptide to
hydrophobic membrane for analysis.
• Allows efficient extraction of P32 labeled residues,
extended sequencing of immobilized proteins, and
other special chemistries to be applied.
• Detergents can inhibit efficient binding.
Sequelon DITC & Aryl Amine Chemistries
Solid Phase Sequencing of
DITC Coupled B e t a l a c t o g o l u b l i n
100
pmol value
10
1
0.1
0.01
0.001
0
20
40
60
Cycle
80
100
Peptide Standard: 10pmol (Tyr, Ala, Glu)
0226B PSS 10pmol
Standard 1
0226B PSS 10pmol
Residue 1
DPTU
Y
6.00
6.00
D
K
3.00
N
3.00
E
Q
T
H
G
S
0.00
D
A
R
W
MV
Y
P
F
L
I
T
G
0.00
DPTU
dpu
S
dpu
A
-3.00
-3.00
E
Q
N
R
P
6.0
8.0
10.0
12.0
14.0
0226B PSS 10pmol
6.0
4.0
16.0
Residue 2
8.0
10.0
12.0
14.0
6.00
3.00
3.00
KL
16.0
Residue 3
E
6.00
I
W
0226B PSS 10pmol
DPTU
A
F
M
H
4.0
V
DPTU
K
0.00
0.00
G
S
V
D
-3.00
T
N
Q
4.0
R
E
P
I
L
-3.00
N
8.0
10.0
12.0
S
H
T
14.0
16.0
4.0
P
R
V
dpu
Y
Q
M
H
6.0
F
A
D
K
dpu
Y
G
6.0
8.0
10.0
L
F
12.0
14.0
16.0
I
Dirty Peptide Run: No Sequence Data
0227A 10pmol PSS Dirty Boy
Standard 1
0227A 10pmol PSS Dirty Boy
D
4.00
N
S
TG
E
Residue 1
H
A
R
DPTU
Y
dpu
4.00
D
K
2.00
2.00
E
N
M
Q
M
T
S
0.00
H
G
A
V
L
I
0.00
DPTU
P
Q
F
Y
R
K
V
W
L
P
dpu
F
I
-2.00
-2.00
-4.00
-4.00
W
6.0
4.0
8.0
10.0
12.0
14.0
0227A 10pmol PSS Dirty Boy
D
S
G
E
4.0
16.0
Residue 2
H
A
R
6.0
8.0
10.0
12.0
14.0
16.0
0227A 10pmol PSS Dirty Boy
DPTU
D
S
G
E
Residue 3
H
A
DPTU
T
4.00
4.00
R
Y
N
2.00
2.00
dpu
N
M
0.00
Q
K
K
dpu
0.00
V
P
T
M
P
L
-2.00
Q
Y
L
V
-2.00
F
I
F
W
-4.00
-4.00
4.0
6.0
8.0
10.0
12.0
14.0
16.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
I
Direct Adsorption to PVDF Membrane
0301C PSS via PVDF Direct
Standard 1
0301C PSS via PVDF Direct
G
4.00
Residue 1
H
DPTU
Y
4.00
S
D
K
2.00
2.00
dpu
D
E
N
K
W
Q
0.00
MV
T
S
H
G
A
F
Y
P
R
T
L
I
0.00
DPTU
A
N
dpu
L
F
V
-2.00
I
-2.00
E
R
P
-4.00
M
-4.00
6.0
4.0
8.0
10.0
12.0
14.0
16.0
0301C PSS via PVDF Direct
G
A
6.0
4.0
Residue 2
10.0
12.0
14.0
16.0
Residue 3
G
DPTU
4.00
8.0
PSS via PVDF Cycle 2
E
DPTU
4.00
2.00
2.00
H
K
K
N
0.00
D
N
0.00
S
D
dpu
V
Y
dpu
S
F
T
-2.00
I
L
P
H
-2.00
I
P
E
A
R
Q
V
R
M
Y
M
Q
-4.00
-4.00
4.0
6.0
8.0
10.0
12.0
14.0
16.0
4.0
L
F
T
6.0
8.0
10.0
12.0
14.0
16.0
Prosorb PVDF Filter Cartridge
0301B PSS Prosorb
Standard 1
0301B PSS Prosorb
Residue 1
Y
4.00
DPTU
4.00
D
K
D
2.00
2.00
E
N
W
Q
0.00
T
S
A
H
G
F
MV
Y
R
L
I
T
0.00
DPTU
P
G
dpu
dpu
K
S
-2.00
-2.00
A
Q
N
E
-4.00
6.0
8.0
10.0
12.0
14.0
16.0
0301B PSS Prosorb
6.0
4.0
Residue 2
A
R
P
8.0
10.0
12.0
14.0
16.0
0301B PSS Prosorb
Residue 3
DPTU
DPTU
E
4.00
4.00
2.00
2.00
L
I
H
-4.00
4.0
F
V
K
D
0.00
0.00
K
S
G
L
I
F
P
N
Q
R
E
4.0
6.0
-2.00
I
T
N
Q
10.0
12.0
14.0
16.0
4.0
R
H
-4.00
6.0
8.0
F
V
A
S
M
8.0
L
P
Y
H
-4.00
dpu
G
dpu
V
T
-2.00
D
Y
10.0
12.0
14.0
16.0
C18 Zip Tip Clean Up
0228C PSS by ZIP tip
Standard 1
0228C PSS by ZIP tip
Residue 1
Y
4.00
DPTU
4.00
D
K
2.00
N
Q
0.00
2.00
E
G
D
W
T
S
H
G
MV
A
R
P
L
I
F
Y
0.00
N
DPTU
S
dpu
dpu
-2.00
-2.00
T
V
A
E
L
R
P
Q
-4.00
6.0
8.0
10.0
12.0
14.0
4.0
16.0
PSS by ZIP tip Cycle 1
Residue 2
6.0
8.0
10.0
12.0
14.0
Residue 3
DPTU
E
4.00
4.00
2.00
2.00
K
16.0
PSS by ZIP tip Cycle 1
DPTU
A
I
M
H
-4.00
4.0
F
G
G
D
0.00
0.00
D
A
P
V
-2.00
-2.00
S
E
N
R
P
Y
QT
F
H
-4.00
4.0
K
dpu
6.0
10.0
12.0
14.0
16.0
K
S
N
I
Q
R
H
T
dpu
F
Y
M
-4.00
M
8.0
V
L
4.0
6.0
8.0
10.0
12.0
14.0
16.0
I
L
OK, so what if we get no data?
• Insufficient quantities.
• Highly heterogeneous sample leading to
uninterpretable results.
• A blocked n-terminus, leading into Bill
Henzel’s part of this presentation.