Waters Cytochrome c Digestion Standard - Manual (Page 2)

typical digest using Symmetry 300 c
18
(formally bioSuite Pa-b)
T1
T14
T13 T14
T4
T9 T10
T10
T19C T15
T8
T5
T19
T12 T13
T12
AU
0.00
0.08
0.16
0.24
0.32
5 10 15 20 25 30 35 40 45 50 55 min
IV. examPleS of uSIng the cytochrome c dIgeStIon Standard for Performance/
benchmarkIng teStIng
The below are reference chromatograms with conditions to help provide examples of the Cytochrome c standard used with each chemistry.
If the column size is different, the gradient/injection volume can be scaled accordingly.
Table 2. Peak identification key for each table
Peak Identification
Peptide sequence Peptide sequence
T1 N-AcGDVEK T8 TGPNLHGLFGR
T13-T14 KYIPGTK T15 MIFAGIK
T14 YIPGTK T19 EDLIAYLK
T4 IFVQK T5 CAQCHTVEK (heme attached)
T9-T10 KTGQAPGFSYTDANK T12-T13 GITWGEETLMEYLENPKK
T10 TGQAPGFSYTDANK T12 GITWGEETLMEYLENPK
T19C EDLIAY
Column: Symmetry300 C
18
, 3.9 x 150 mm, 5 μm
Part number: WAT106154
Mobile phase A: 0.05% TFA in water
Mobile phase B: 0.1% TFA in acetonitrile
Temp.:
35 °C
Wavelength: 220 nm
Injection vol.: 20 µL
Run time: 65 minutes
Gradient: Time Flow %A %B Curve
0 1.0 100 0 *
45 1.0 72 28 6
55 1.5 40 60 11
58 1.5 100 0 11
typical digest using atlantis dc
18
(formally bioSuite Pa-a)
AU
5 10 15 20 25 30 35 40 45 50 min
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.00
T1
T13
- T14
T14
T4
T9
- T10 T10
T19C
T8
T15 T19
T5
T12
- T13
T12
-
-
-
Column: Atlantis dC
18
, 3.9 x 150 mm, 3 μm
Part number: 186001317
Mobile phase A: 0.025% TFA in water
Mobile phase B: 0.05% TFA in acetonitrile
Temp.:
35 °C
Wavelength: 220 nm
Injection vol.: 20 µL
Run time: 65 minutes
Gradient: Time Flow %A %B %C %D Curve
0.7 100 0 0 0
45 0.7 64 0 0 36 6
55 1.5 40 0 0 0 11
58 1.5 100 0 0 0 11
65 1.5 100 0 0 0 11
68 0 100 0 0 0 11
2
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Cytochrome c Digestion Standard