Waters MassPREP Protein Standard Mix User Manual
[ CARE AND USE MANUAL ]
MASSPREP PROTEIN STANDARD MIX
CONTENTS
I. INTRODUCTION
II. STORAGE AND STABILITY
III. RECONSTITUTION OF MASSPREP PROTEIN STANDARD MIX
FOR HPLC/UPLC USE
I. INTRODUCTION
The MassPREP™ Protein Standard Mix is a mixture of a variety of carefully
chosen proteins encompassing a wide range of properties. This mixture
contains proteins that vary in isoelectric points, molecular weights and
hydrophobicities. These characteristics provide the user with an attractive
intact protein mixture that can be used for a variety of applications.
Table 1 describes the composition of this mixture.
The standard was developed to provide a means to test Waters UPLC
and HPLC Protein Separation Columns such as the ACQUITY UPLC
BEH C41.7 μm and XBridge™ BEH300, C4 3.5 µm column offerings.
In our quest to offer useful analytical tools, we now offer the same
protein standard mix used at Waters to QC batch test of some of
our Protein Separation Technology Column offerings. The standard is
prepared under strict quality control criteria confirmed on the specific
certificate of analysis that is available upon request for each batch
of prepared MassPREP Protein standard. In addition to evaluation of
HPLC/UPLC columns, this mixture may also be used for a variety of
other applications, including evaluation of mass spectrometry and
chromatography instrumentation performance.
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Mass PREP P rotein Standard Mix 1
Table 1. Composition of MassPREP Protein Standard Mix
Protein Sample
Ribonuc lease A, bovin e pancreas 13.700 K P61823* R5500
Cytoc hrome c, horse heart, 96% 12. 384 K P00004* C7752
Albumin, bovine serum, 96-99% 66.430 K P02769* A8022
Myoglobin, horse heart >90%
Enolase, from baker’s yeast
(S. cerevisiae)
Phosphorylase b, rabbit muscle 97, 2 0 0 K P00489 P6635
*Best match from database for more information on sequence please
reference the raw material vendor information (Sigma)
Molecular
Weight, MW
16.950 K plus
the heme group
616 K
To tal :17.6 0 0 K
46.671 K P00924 E6126
SwissProt
Sequence
Information
P68082 M18 82
Part
Number
(Sig ma)
[ CARE AND USE MANUAL ]
II. STORAGE AND STABILITY
Upon receiving the sample, store immediately at -20 °C. The lyophilized
powder is stable at -20 °C until the expiration date printed on the label.
Once reconstituted, the solution should be used immediately for best
performance. Stability can be extended by freezing samples at -20 °C.
III. RECONSTITUTION OF MASSPREP PROTEIN STANDARD MIX
FOR HPLC/UPLC USE
Each vial of the MassPREP Protein Standard Mix contains approximately
123 µg of total protein. Table 2 outlines the individual concentra-
tions. For ACQUITY UPLC BEH C4 1.7 μm, 2.1 x 50 mm column evalu-
ation (P/N 186004495) the recommended reconstitution volume is
100 μL of 0.1% trifluoroacetic acid in water with a 5 µL load to make
a sample containing 1228 ng/ µL of total protein.
Table 2: Amount of Each Protein per Vial
Protein Amount (µg)
Ribonuclease A 4.15
Cytochrome c 5.65
Bovine Serum Albumin 20 .15
Myoglobin 12. 5 0
Enolase 21.45
Phosphorylase b 58.90
MSDS information: Waters Protein and Peptide 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 Protein and Peptide 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) and Appendix A and B, and are thus exempt from the
requirements of the Hazard Communication Standard, including the
requirement to supply MSDSs.
Figure 1: MassPREP Protein Standard Mix on an
ACQUITY UPLC BEH C4 1.7 μm 2.1 x 150 mm
Column: ACQUITY UPLC® BEH300 C4, 1.7 µm, 2.1 x 150 mm
Mobile Phase A: 0.1% TFA in water
Mobile Phase B: 0.1% TFA in 100% acetonitrile
Flow Rate: 0.2 mL/min
Gradient: 20-55% B in 60 min
Column Temp.: 40 ºC
Injection: 3.3 µL
Detection λ: 220 nm
Sample: MassPREP Protein Standard mixture prepared in 0.1% TFA in 5% acetonitrile
1. Ribonuclease A (0.04 mg/mL)
2. Cytochrome c (0.06 mg/mL)
3. Bovine Serum Albumin (0.20 mg/mL)
4. Myoglobin (0.13 mg/mL)
5. Enolase (0.22 mg/mL)
6. Phosphorylase b (0.59 mg/mL)
0.30
2
AU
0.15
1
0.00
12 24 36 48 min
3
5
4
Figure 2: MassPREP Protein Standard Mix on an
ACQUITY UPLC BEH C4, 1.7 μm, 2.1 x 50 mm
Column: ACQUITY UPLC BEH C4, 1.7 µm, 2.1 x 50 mm
Mobile Phase A: 0.1% TFA in Water
Mobile Phase B: 0.08% TFA in Acetonitrile
Flow Rate: 0.2 mL/min
Gradient: 22-90% B in 16 min
Column Temp: 40 °C
Injection: 5 µL of the MassPREP Protein Standard for a load of
0.615 µg of total protein
Detection λ: 220 nm
Sample: MassPREP Protein Standard mixture prepared in 0.1% TFA in 5% acetonitrile
1. Ribonuclease A (0.04 mg/mL)
2. Cytochrome c (0.06 mg/mL)
3. Bovine Serum Albumin (0.20 mg/mL)
4. Myoglobin (0.13 mg/mL)
5. Enolase (0.22 mg/mL)
6. Phosphorylase b (0.59 mg/mL)
0.24
0.20
0.16
AU
0.12
0.08
0.04
0.00
2 46810 12 14 16 18 20 22 24 Min
0
6
Mass PREP P rotein Standard Mix 2
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