Waters Cytochrome c Digestion Standard User Manual

[ Care and Use ManUal ]

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.

[ Care and Use ManUal ]

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

2

Cytochrome c Digestion Standard