HP 241 User Manual
H
C-Terminal 5.0 Protein Sequencer Methods
Technical Note
HP 241 Protein
Sequencer (N+C)
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 N- terminal 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
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).
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).
|
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 |
|
|
|
|
|
|
|
|
|
|
|
|
|
2
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 |
53:Couple: Meter 1R
54:Couple: Deliver 1R UP
55:Couple: Dry column DOWN
56:Wash: Meter S2/3 and S2A
57:Wash: Deliver S2/3 DOWN (closed)
58:Wash: Dry column DOWN
59:Couple: Meter 2R
60:Couple: Deliver 2R UP
61:Couple: Dry column DOWN
62:Wash: Meter S2/3 and S2A
63:Wash: Deliver S2/3 DOWN (closed)
64:Wash: Dry column DOWN
65:Wash: Meter S2/3
66:Wash: Deliver S2/3 DOWN (closed)
67:Wash: Dry column DOWN
68:Wash: Meter S3
69:Wash: Deliver S3 DOWN (closed)
70:Wash: Dry column DOWN
71:Cleave: Meter R3
72:Cleave: Deliver R3 DOWN
73:Wash: Flush RV2 with S3
74:Cleave: Dry column DOWN
75:!Extract: Meter S3
76:!Extract: Deliver S3 DOWN to flask
77:!Extract: Dry column UP
78:!Cleave: Meter S2/3
79:!Cleave: Deliver S2/3 DOWN
80:!Cleave: Dry column
81:!Cleave: Meter S2/3
82:!Cleave: Deliver S2/3 DOWN
83:!Cleave: Dry column
84:!Cleave: Meter R2
85:!Cleave: Deliver R2 DOWN
86:!Cleave: Dry column
87:!Cleave: Meter S2/ 3
88:!Cleave: Deliver S2/3 DOWN
89:!Cleave: Dry column
90:!Cleave: Meter S2/3
91:!Cleave: Deliver S2/3 DOWN
92:!Cleave: Dry column
93:!Cleave: Meter L3
94:!Cleave: Deliver L3 to transfer flask
95:!Cleave: Meter 3R
96:!Cleave: Cleave and extract to flask
97:!Extract: Meter S3
98:!Extract: Extract to flask UP
99:!Cleave: Dry column UP
100:!Cleave: Meter 3R
101:!Cleave: Cleave and extract to flask
102:!Extract: Meter S3
103:!Extract: Extract to flask UP
104:!Cleave: Dry column UP
105:Wash: Meter R2
106:Wash: Deliver R2 DOWN
107:Wash: Dry column DOWN
108:Wash: Meter S2/3
109:Wash: Deliver S2/3 DOWN (closed)
110:Wash: Dry column DOWN
111:Wash: Meter S2/3
112:Wash: Deliver S2/3 DOWN (closed)
113:Wash: Dry column DOWN ! indicates system step
Primary Temperature
Time
5.080
30.080
10.080
9.080
15.080
5.080
5.080
10.080
5.080
9.080
15.080
5.080
9.080
15.080
5.080
9.080
15.080
30.080
7.080
400.080
5.080
160.080
9.080
15.080
10.060
10.050
30.050
10.050
10.050
30.050
30.050
10.555
120.055
60.050
10.050
30.050
30.050
10.050
30.050
30.050
7.040
25.040
9.050
30.050
10.050
10.050
30.050
9.050
30.050
10.050
10.050
20.055
10.555
120.055
60.060
9.060
15.060
15.060
9.060
15.060
20.060
3
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