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Cytochrome c Digestion Standard
CONTENTS
I. INTRODUCTION
II. IDENTIFYING PARTS
III. USING THE WATERS
EXTRACTION MANIFOLD
IV. NEEDLE TIP INSTALLATION PROCEDURE
V. SPARE PARTS AND ACCESSORIES
I. INTRODUCTION
The Cytochrome c Digestion Standard was prepared by digesting
Bovine Heart Cytochrome c (Uniprot, P/N P62894) with sequencing
grade trypsin. Each batch of digestion standard is prepared under
strict quality control criteria confirmed on the included certificate
of analysis. Protein digestion standards may be used for a variety
of applications, including evaluation and benchmarking of HPLC
and UPLC® Columns or evaluation of mass spectrometry and
chromatography instrumentation performance.
Column
XBridge™ BEH300 C
XBridge BEH130 C
ACQUITY UPLC CSH™ and XSelect™
ACQUITY UPLC
ACQUITY UPLC BEH130 C
Atlantis® dC
Symmetry300™ C
BioSuite™ PA-B
BioSuite PA-A
®
BEH300 C
18
18
18
18
18
18
Table 1. Compatible chemistries that can be used with this standard.
II. STORAGE AND STABILITY
The lyophilized powder is stable at room temperature until the expiration
date printed on the label. Once reconstituted in 0.055% trifluoroacetic
acid in water, the solution is stable for one week when stored at 8 °C.
Stability can be extended by freezing samples at -20 °C.
III. RECONSTITUTION OF THE CYTOCHROME c
DIGESTION STANDARD FOR HPLC/UPLC
The protein digestion standards were prepared by digesting 1 nmol of
protein. For HPLC column evaluation, the recommended sample dilution
is 200 µl of 0.055% trifluoroacetic acid in water.
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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.
Peak Identification
Peptide Sequence Peptide Sequence
T1
N-AcGDVEK
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
Table 2. Peak identification key for each table
T8 TGPNLHGLFGR
Typical digest using Symmetry 300 C18
(formally BioSuite PA-B)
0.32
0.24
0.16
AU
0.08
0.00
T1
5101520253035404550 55 min
T14
T10
T9 T10
T4
T13 T14
Column: Symmetry300 C18, 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
T5
T8
T15
T19
T19C
T12 T13
T12
Typical digest using Atlantis dC18
(formally BioSuite PA-A)
0.35
0.30
0.25
0.20
AU
0.15
0.10
0.05
0.00
T1
510 15 20 25 30 35 40 45 50 min
T10
T10
T9 -
T14
T14
-
T4
T13
T19C
Column: Atlantis dC18, 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
T5
T8
T19
T15
T12
T13
-
-
T12
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Cytochrome c Digestion Standard
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6 10 14 18 22 26 30 34 38 min
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
AU
T1
T13-T14 and T14
T4
T9-T10
T10
T19C
T15
T8
T5
T19
T12-T13
T12
[ Care and Use ManUal ]
Typical digest using an ACQUITY UPLC BEH130 C18 Column
0.24
0.16
AU
0.08
0.00
4812 16 20 24 min
T4
T10
T14
T1
T13T14
T9T10
T19C
T5
T8
T15
T19
Column: ACQUITY UPLC BEH130 C18, 2.1 x 100 mm, 1.7 μm
Part number: 186003555
Mobile phase A: 0.045% TFA in water
Mobile phase B: 0.045% TFA in acetonitrile
Temp.: 35 °C
Wavelength: 214 nm
Injection vol.: 5 µL
Run time: 61 minutes
Gradient: Time Flow %A1 %B1 Curve
0.2 100 0
6 0.2 85 15 6
26 0.2 64 36 6
40 0.2 40 60 11
50 0.2 100 0 11
70 0 100 0 11
T12
T12T13
Typical digest using XBridge BEH 130 C18 Column
0.50
0.40
0.30
AU
0.20
T1
0.10
0.00
6810 12 14 16 18 20 22 24 26 28 30 32 34 36 min
T13-T14
T14
T4
T10
T9-T10
T19C
Column: XBridge BEH130 C18, 4.6 x 150 mm, 3.5 μm
Part number: 186003569
Mobile phase A: 0.055% TFA in water
Mobile phase B: 0.055% TFA in acetonitrile
Temp.: 35 °C
Wavelength: 214 nm
Injection vol.: 36 µL
Run time: 65 minutes
Gradient: Time Flow %A %B Curve
0.96 100 0
9 0.96 85 15 6
39 0.96 64 36 6
43 0.96 40 60 11
58 0.96 100 0 11
67 0 100 0 11
T5
T8
T15
T19
T12
T12-T13
Typical digest using an ACQUITY UPLC BEH300 C18 Column
0.50
T15
T5
T8
T19
, 2.1 x 100 mm, 1.7 μm
18
0.40
0.30
AU
0.20
T1
0.10
0.00
4812 16 20 24 min
Column ACQUITY UPLC BEH300 C
Part number: 186003686
Mobile phase A: 0.055% TFA in water
T4
T13T14 T14
T9T10
T10
T19C
Mobile phase B: 0.055% TFA in acetonitrile
Temp.: 35 °C
Wavelength: 214 nm
Injection vol.: 5 µL
Run time: 61 minutes
Gradient: Time Flow %A1 %B1 Curve
0.2 100 0
6.6 0.2 85 15 6
28.6 0.2 64 36 6
30.0 0.2 40 60 11
40.0 0.2 100 0 11
62.0 0 100 0 11
Typical digest using XBridge BEH 300 C18 Column
T12
T12T13
Column: XBridge BEH300 C18, 4.6 x 150 mm, 3.5 μm
Part number: 186003613
Mobile phase A: 0.055% TFA in water
Mobile phase B: 0.055% TFA in acetonitrile
Temp.: 35 °C
Wavelength: 214 nm
Injection vol.: 36 µL
Run time: 65 minutes
Gradient: Time Flow %A %B Curve
0.96 100 0
9.9 0.96 85 15 6
42.9 0.96 64 36 6
45.0 0.96 40 60 11
50.0 0.96 100 0 11
83.0 0 100 0 11
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Cytochrome c Digestion Standard
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Typical digest using ACQUITY CSH130 C
18
1.2
1.1
1.0
0.9
0.8
0.7
0.6
AU
0.5
T8
0.4
0.3
0.2
0.1
0.0
2 3 4 5 6 7 8 9 10 11 12 min
T4
T4
T14
T14
T1
T1
T13 - T14
T13 - T14
T10
T10
T9 - T10
T9 - T10
T8
T15
T15
T19
T19
T19C
T19C
ACQUIT Y CSH with Cytochrome c Various Conditions
1.5
Column: ACQUITY CSH C
Mixer: 380 µL
1.0
AU
0.5
0.0
2 3 4 5 6 7 8 9 10 11 12 min
1.5
Column: ACQUITY CSH C
Mixer: 50 µL
1.0
AU
0.5
0.0
2 3 4 5 6 7 8 9 10 11 12 min
1.5
Column: XSelect CSH C
Mixer: 50 µL
1.0
AU
0.5
0.0
2 3 4 5 6 7 8 9 10 11 12 min
1.7 µm
18,
T8
T10
T4
T14
T9-T10
T1
T13-T14
1.7 µm
18,
T14
T1
T13-T14
T14
T1
T13-T14
T10
T4
T9-T10
5 µm
18,
T10
T4
T9-T10
T15
T19C
T19
T8
T15
T19C
T19
T8
T15
T19
T19C
See above figure for conditions.
T12
T12-T13
T5+Heme
T12
T12-T13
T5+Heme
T12
T12-T13
T5+Heme
Colum: ACQUITY CSH130 C18, 1.7 µm, 2.1 x 50 mm
T12
T12
Part number: 186006936
Mobile phase A: 0.1% Formic Acid in 100% Water
Mobile phase B: 0.085% Formic Acid in 75% Acetonitrile
Temp.: 40 °C
Wavelength: 214 nm
Reconstitution: 200 µL of Mobile Phase A
Injection Vol: 5 µL
T12 - T13
T12 - T13
Run Time: 15 minutes
Gradient: Time Flow Rate %A %B Curve
T5+Heme
T5+Heme
0.00 0.200 99.0 1.0 *
5.00 0.200 79.0 21.0 6
7.50 0.200 68.6 31.4 6
11.00 0.200 5.0 95.0 6
12.50 0.200 5.0 95.0 1
15.00 0.200 99.0 1.0 1
ACQUIT Y CSH with Cytochrome c MS Conditions.
9
6 x 10
Column: ACQUITY CSH C
Mixer: 380 µL
9
4 x 10
9
Intensity
2 x 10
0
2 3 4 5 6 7 8 9 10 11 12 min
9
6 x 10
Column: ACQUITY CSH C
Mixer: 50 µL
9
4 x 10
9
Intensity
2 x 10
0
2 3 4 5 6 7 8 9 10 11 12 min
9
6 x 10
Column: XSelect CSH C
Mixer: 50 µL
9
4 x 10
9
Intensity
2 x 10
0
2 3 4 5 6 7 8 9 10 11 12 min
1.7 µm
18,
T4
T4
T14
T14
T9-T10
T9-T10
T1
T1
T13-T14
T13-T14
1.7 µm
18,
T4
T9- T10
T14
T1
T13- T14
T14
T1
T13- T14
T10
5 µm
18,
T4
T9- T10
T10
T8
T8
T15
T15
T19
T19
T19C
T19C
T12-T13
T12-T13
T12
T12
T5+Heme
T10
T10
T8
T15
T19
T19C
T8
T15
T19C
T12- T13
T12
T19
T12- T13
T12
T5+Heme
T5+Heme
T5+Heme
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Cytochrome c Digestion Standard
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V. ORDERING INFORMATION
Part Number Product Description Qty
186006371 Cytochrome c Digestion Standard 1/pk
176003064 ACQUITY UPLC PST (CSH) C
186 003613 XBridge BEH300 C18, 4.6 x 150 mm, 3.5 μm Column 1/pk
186003569 XBridge BEH130 C
186003686 ACQUITY UPLC BEH300 C
186003555 ACQUITY UPLC BEH130 C18, 2.1 x 100 mm, 1.7 µm Column 1/pk
1860 01317 Atlantis dC
WAT106154 Symmetry300 C18, 3.9 x 150 mm, 5 µm Column 1/pk
186002325 MassPREP™ Digestion Standard, Enolase 1/pk
186002326 MassPREP Digestion Standard, Phosphorylase b 1/pk
186002327 MassPREP Digestion Standard, Bovine Hemoglobin 1/pk
186002328 MassPREP Digestion Standard, Alcohol Dehydrogenase 1/pk
186002329 MassPREP Digestion Standard, Bovine Serum Albumin 1/pk
186002330 MassPREP Digestion Standard Kit (one of each digestion std.) 5/pk
18
, 4.6 x 150 mm, 3.5 μm Column 1/pk
18
, 3.9 x 150 mm, 3 µm Column 1/pk
Column Kit, 130Å, 1.7 µm, 2.1 mm X 50 mm 1/pk
18
, 2.1 x 100 mm, 1.7 µm Column 1/pk
18
MSDS Information:
Waters Digestion Standards contain various non hazardous peptides in powder form. They are
for laboratory use only; solely for purposes of scientific experimentation, analysis, research or
development. Waters Digestion Standards should be used only as directed and in accordance
with good laboratory practice. All Waters Digestion Standards are non hazardous as defined by
OSHA’s Hazard Communication Standard, 29 CFR 1910.1200(c) Appendix A and B, and are thus
exempt from the requirements of the Hazard Communication Standard, including the requirement
to supply MSDSs.
Waters, ACQUIT Y UPLC, UPLC, Symmetr y and Atlantis are registered
trademarks of Waters Corp oration. BioSuite, XBrid ge, XSelect, CSH,
Mass Prep, Symmetry300 and T he Scienc e of What's Possible are
trademarks of Waters Corp oration. All other trademar ks are the pro perty
of their respective owners.
©2013 Waters Corporation. Produced in the U.S.A.
May 2013 720004341EN Rev. C KP-PDF
Waters Corporation
34 Maple Street
Milford, MA 01757 U.S.A.
T: 1 508 478 2000
F: 1 508 872 1990
www.waters.com
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