HP 241 User Manual

H

HP 241 Protein
Sequencer (N+C)

C-Terminal 5.0 Protein Sequencer Methods

Technical Note

The C-terminal 5.0 sequencer meth­ods implement the thiohydantoin
degradation chemistry (coupling
and cleavage) on the Hewlett­Packard column-based protein
sequencer. Reagents R1, R2A, R4,
and the PTH-Std are specific for N­terminal sequencing and are not
utilized for the C-terminal 5.0
sequencer methods.

These methods are suitable for
C-terminal sequence analysis of
most protein samples in the low
nanomole to 50 pmole range which
have been applied onto a Hewlett­Packard Zitex strip. Once dry the
strip is loaded into an empty
RP/SAX biphasic column. No col­umn preparation is required with
this method.

The methods are:

• C-Terminal 5.0

• C-Terminal 5.0 (Cycle 1)

• C-Terminal Flask 5.0

• TH Std 5.0

The column and flask methods
control all of the derivatization
and cleavage reactions for the
thiohydantoin chemistry.

The sequence program controls all
of the column and flask methods,
as well as any cycle exception
methods. The C-terminal 5.0
sequencer method uses a cycle 1
column exception method.

The cycle 1 method provides a
longer initial drying time and a
pause to provide time for proper
HPLC column equilibration after
switching from N-terminal sequenc­ing. There is no specialized cycle 1
method for the flask.

The TH Std 5.0 method delivers 50
pmol/100 µl TH-amino acid stan­dard solution to the on-line HPLC
allowing the quantitation and iden­tification of sequenced amino
acids. One may choose to run a
TH-standard by using TH-std 5.0
when scheduling a sample.

S2A Neat ethyl acetate

1R Diphenylphosphorylisothiocyanate (DPP-ITC) in toluene/heptane (23:27:50)
R1 Phenylisothiocyanate (PITC) in heptane (3:97)
2R Pyridine in ethylacetate (2:98)
R2 Diisopropylethylamine (DIEA) in 1-propanol/water (1:6:3)
S2/3 Acetonitrile/toluene (23:77)
R2A Ocylamine in heptane (3:97)
3R Potassium trimethylsilanolate (0.1M) in methanol/t-butanol (50:50)
S3 Acetonitrile/toluene (15:85)
R3 Neat trifluoroacetic acid (TFA)
L3 Acetic acid in methanol/water (1:74:25)
PTH Mixture of PTH amino acids (10 pmol/100 µl) in acetonitrile with DPTU

as a marker

TH Mixture of TH amino acids (50 pmol/100 µl) in acetonitrile
R4 Trifluoroacetic acid (TFA) in water (1:3)
S5 Phosphate buffer (pH 2.9), 0.2% ion pairing reagent

2

Method (Column):
C-Terminal 5.0

Total time: 75.2 min

All metering steps deliver the speci­fied reagent or solvent to waste.

Steps 3, 25, 47, 72: The volume of
R3 delivered during this step should
be just enough to wet the membrane.

Steps 7, 13, 19, 22, 29, 35, 41, 44,
51, 57, 63, 66, 69,76, 79, 82, 88,
91, 109, 112: The delivery of S2/3

and S3 should completely wet the
Zitex membrane. The solvent deliv­eries are held against a closed valve,
enabling 1/2 to 3/4 of the membrane­containing column compartment
(bottom) to remain filled with sol­vent for the duration of the step.

Steps 10, 32, 54: The delivery of
1R should completely wet the mem­brane, but not flood it. The initial
delivered volume (about 1/2 the vol­ume of the column) should blow
through the column wetting the Zitex
strip. The 1R reagent delivery is then
held against a closed valve, enabling
only a small volume of 1R to sit at
the bottom of the column (approxi­mately 1 slit distance as measured by
the slits on the column wings).

Method Details

The steps for the C-terminal 5.0 methods are described below. The steps indicate the ranges in volume that are
appropriate for the various reagents/solvent deliveries. All sequencer methods are accessed by choosing the top
menu item Edit/Method in the Protein Sequencer window. The sequencer column configuration used with the
C-terminal 5.0 methods consists of an empty reverse-phase (RP) sample column (top) mated with an empty
strong anion exchange (SAX) column (bottom).

Step Description Primary Temperature

Time

1: Activation: Dry column DOWN 60.0 60
2: Activation: Meter R3 7.0 60
3: Activation: Deliver R3 DOWN 30.0 60
4: Wash: Flush RV2 with S3 5.0 80
5: Activation: Dry column DOWN 50.0 80
6: Wash: Meter S2/3 9.0 80
7: Wash: Deliver S2/3 DOWN (closed) 15.0 80
8: Wash: Dry column DOWN 5.0 80

9: Couple: Meter 1R 5.0 80
10: Couple: Deliver 1R UP 30.0 80
11: Couple: Dry column DOWN 10.0 80
12: Wash: Meter S2/3 and S2A 9.0 80
13: Wash: Deliver S2/3 DOWN (closed) 15.0 80
14: Wash: Dry column DOWN 5.0 80
15: Couple: Meter 2R 5.0 80
16: Couple: Deliver 2R UP 10.0 80
17: Couple: Dry column DOWN 5.0 80
18: Wash: Meter S2/3 and S2A 9.0 80
19: Wash: Deliver S2/3 DOWN (closed) 15.0 80
20: Wash: Dry column DOWN 5.0 80
21: Wash: Meter S2/3 9.0 80
22: Wash: Deliver S2/3 DOWN (closed) 15.0 80
23: Wash: Dry column DOWN 20.0 80
24: Activation: Meter R3 7.0 60
25: Activation: Deliver R3 DOWN 30.0 60
26: Wash: Flush RV2 with S3 5.0 80
27: Activation: Dry column DOWN 50.0 80
28: Wash: Meter S2/3 9.0 80
29: Wash: Deliver S2/3 DOWN (closed) 15.0 80
30: Wash: Dry column DOWN 5.0 80
31: Couple: Meter 1R 5.0 80
32: Couple: Deliver 1R UP 30.0 80
33: Couple: Dry column DOWN 10.0 80
34: Wash: Meter S2/3 and S2A 9.0 80
35: Wash: Deliver S2/3 DOWN (closed) 15.0 80
36: Wash: Dry column DOWN 5.0 80
37: Couple: Meter 2R 5.0 80
38: Couple: Deliver 2R UP 10.0 80
39: Couple: Dry column DOWN 5.0 80
40: Wash: Meter S2/3 and S2A 9.0 80
41: Wash: Deliver S2/3 DOWN (closed) 15.0 80
42: Wash: Dry column DOWN 5.0 80
43: Wash: Meter S2/3 9.0 80
44: Wash: Deliver S2/3 DOWN (closed) 15.0 80
45: Wash: Dry column DOWN 20.0 80
46: Activation: Meter R3 7.0 60
47: Activation: Deliver R3 DOWN 30.0 60
48: Wash: Flush RV2 with S3 5.0 80
49: Activation: Dry column DOWN 50.0 80
50: Wash: Meter S2/3 9.0 80
51: Wash: Deliver S2/3 DOWN (closed) 15.0 80
52: Wash: Dry column DOWN 5.0 80

3

Steps 16, 38, 60: The delivery of 2R
should completely wet the membrane.
The initial delivered volume (about
1/2 the volume of the column) should
blow through the column wetting the
Zitex strip. The 2R reagent delivery
is then held against a closed valve,
enabling only a small volume of 2R
to sit at the bottom of the column
(approximately 1 slit distance as mea­sured by the slits on the column wings).

Steps 12, 18, 34, 40, 56, 62: "Meter
S2/3 and S2A". The S2A flushes the
delivery lines and should not wet
the column. The S2/3 is involved in the
column wash and is sent to the column.

Steps 85, 106: The volume of R2
delivered during this step should not
wet the membrane.

Steps 86, 107: During the high
pressure dry step, the volume of R2
delivered should be just enough to
wet the Zitex membrane. Note: The
R2 reagent does not actually "wet"
the Zitex membrane like other
reagents and solvents. The mem­brane will not change its appearance
in the presence of R2.

Step 94: After Step 94, the volume
of L3 delivered to the flask should be
50 µl (+/- 5 µl)

Steps 96, 101: The delivery of 3R
should completely wet the mem­brane, but not flood it. The 3R
reagent delivery is then held against
a closed valve, enabling only a small
volume of 3R to sit at the bottom of
the column (approximately 1 slit dis­tance as measured by the slits on the
column wings).

Step Description Primary Temperature

Time

53: Couple: Meter 1R 5.0 80
54: Couple: Deliver 1R UP 30.0 80
55: Couple: Dry column DOWN 10.0 80
56: Wash: Meter S2/3 and S2A 9.0 80
57: Wash: Deliver S2/3 DOWN (closed) 15.0 80
58: Wash: Dry column DOWN 5.0 80
59: Couple: Meter 2R 5.0 80
60: Couple: Deliver 2R UP 10.0 80
61: Couple: Dry column DOWN 5.0 80
62: Wash: Meter S2/3 and S2A 9.0 80
63: Wash: Deliver S2/3 DOWN (closed) 15.0 80
64: Wash: Dry column DOWN 5.0 80
65: Wash: Meter S2/3 9.0 80
66: Wash: Deliver S2/3 DOWN (closed) 15.0 80
67: Wash: Dry column DOWN 5.0 80
68: Wash: Meter S3 9.0 80
69: Wash: Deliver S3 DOWN (closed) 15.0 80
70: Wash: Dry column DOWN 30.0 80
71: Cleave: Meter R3 7.0 80
72: Cleave: Deliver R3 DOWN 400.0 80
73: Wash: Flush RV2 with S3 5.0 80
74: Cleave: Dry column DOWN 160.0 80
75: !Extract: Meter S3 9.0 80
76: !Extract: Deliver S3 DOWN to flask 15.0 80
77: !Extract: Dry column UP 10.0 60
78: !Cleave: Meter S2/3 10.0 50
79: !Cleave: Deliver S2/3 DOWN 30.0 50
80: !Cleave: Dry column 10.0 50
81: !Cleave: Meter S2/3 10.0 50
82: !Cleave: Deliver S2/3 DOWN 30.0 50
83: !Cleave: Dry column 30.0 50
84: !Cleave: Meter R2 10.5 55
85: !Cleave: Deliver R2 DOWN 120.0 55
86: !Cleave: Dry column 60.0 50
87: !Cleave: Meter S2/ 3 10.0 50
88: !Cleave: Deliver S2/3 DOWN 30.0 50
89: !Cleave: Dry column 30.0 50
90: !Cleave: Meter S2/3 10.0 50
91: !Cleave: Deliver S2/3 DOWN 30.0 50
92: !Cleave: Dry column 30.0 50
93: !Cleave: Meter L3 7.0 40
94: !Cleave: Deliver L3 to transfer flask 25.0 40
95: !Cleave: Meter 3R 9.0 50
96: !Cleave: Cleave and extract to flask 30.0 50
97: !Extract: Meter S3 10.0 50
98: !Extract: Extract to flask UP 10.0 50
99: !Cleave: Dry column UP 30.0 50

100: !Cleave: Meter 3R 9.0 50
101: !Cleave: Cleave and extract to flask 30.0 50
102: !Extract: Meter S3 10.0 50
103: !Extract: Extract to flask UP 10.0 50
104: !Cleave: Dry column UP 20.0 55
105: Wash: Meter R2 10.5 55
106: Wash: Deliver R2 DOWN 120.0 55
107: Wash: Dry column DOWN 60.0 60
108: Wash: Meter S2/3 9.0 60
109: Wash: Deliver S2/3 DOWN (closed) 15.0 60
110: Wash: Dry column DOWN 15.0 60
111: Wash: Meter S2/3 9.0 60
112: Wash: Deliver S2/3 DOWN (closed) 15.0 60
113: Wash: Dry column DOWN 20.0 60

! indicates system step