[ CARE AND USE MANUAL ]
Reverse Peptide Kit
CONTENTS
I. INTRODUCTION
a. Parts Required
b. Additional Materials Required But Not Supplied
II. RECOMMENDED SAMPLE
PREPARATION PROCEDURES
a. Sample Preparation Guidelines
b. Health and Safety Precautions
c. Storage and Handling
III. REVERSE PEPTIDE STANDARD
PREPARATION PROTOCOL
a. Diluent
b. Reverse Peptide Stock Solutions
c. Reverse Peptide Standard Solution Working Concentration
IV. MOBILITY MODE COMMISSIONING TEST
a. Resolution Mode (Positive Ion)
b. Saving System Settings
I. INTRODUCTION
The Reverse Peptide Kit (P/N 700005089) contains two wellcharacterized samples (Ser-Asp-Gly-Arg-Gly and Gly-Arg-Gly-Asp-Ser)
used to calibrate ion mobility spectrometry/mass spectrometry
(IMS/MS) systems in high-resolution positive-ion mode. Analysts
can also use the reverse peptide standard solution prepared
from these samples as a reference standard for verifying IMS
performance.
The instructions in this manual describe the recommended
procedure for preparing the reverse peptide standard solution and
performing the mobility mode commissioning test.
a. Parts Required
Table 1: Kit components
Serial Sample
1
2
Ser-Asp-
Gly-Arg-Gly
Gly-Arg-
Gly-Asp-Ser
Supply
format
Powder
Powder
Concentration/
weight
1 mg
(–0 +0.2 mg)
1 mg
(–0 +0.2 mg)
Number of
vials
1
1
V. ORDERING INFORMATION
b. Additional Materials Required But Not Supplied
Table 2: Additional materials
Solvents Additives Labware
Acetonitrile
(Fisher Scientific
high-purity or
equivalentquality solvents)
Water
(ELGA or
equivalentquality
(18 MΩ purity)
lab water)
>98% purity formic
acid (Aristargrade additives;
avoid GPR-grade
additives)
20-mL Class A
volumetric flasks (2)
30-mL Nalgene
container (w/cap)
100-mL graduated
measuring cylinder
250-mL Duran laboratory
glass bottle (w/cap)
200-μL pipette (and tip)
(Gilson or equivalentquality pipettes)
1000-μL pipette (and tip)
[ CARE AND USE MANUAL ]
II. SAMPLE PREPARATION AND
HANDLING GUIDELINES
Waters recommends the following sample preparation materials
and procedures to ensure the purity, accurate measurement, and
stability of the reverse peptide standard solution.
Note: For detailed information on guidelines for the prevention
of system contamination, refer to the document Controlling
Contamination in Ultra Performance LC-MS and HPLC-MS Systems
(P/N 715001307) located in the Waters Knowledge Center.
a. Sample Preparation Guidelines
To maximize the accuracy, precision, and reliability your analytic
data, Waters recommends integrating the following practices into
your sample preparation protocols:
Thoroughly wash all glassware with a laboratory-cleaning
product and rinse with high-purity solvents before using.
Do not wash solvent containers or pipettes with detergents
or with other glassware.
Wear suitable powder-free gloves when installing sample
vials or performing system maintenance.
Avoid exposing samples to contaminants such as keratins
(e.g., dust, skin, hair) plasticizers, polymers).
Use appropriately sized pipettes to ensure accurate
volumes are dispensed.
Prepare samples in an ultrasonic liquid processor to ensure
they dissolve completely.
b. Storage and Handling
To maintain the quality and stability of samples, do not exceed
recommended temperatures or storage times.
Store unused portions of the reverse peptide stock
solutions and diluent in a freezer at -20 °C for no longer
than 2 weeks.
Aliquot solutions into Nalgene or PTFE containers before
freezing.
Refrigerate unused aliquots of the reverse peptide standard
solution at 4 °C for no longer than 1 week.
Label all samples with sample name, concentration and
composition of solvents, preparation date, and expiration
date before storage.
Allow all liquid samples to warm to room temperature
before using. Do not refreeze samples after thawing.
III. REVERSE PEPTIDE STANDARD
PREPARATION PROTOCOL
a. Diluent (50:50 Acetonitrile: H20 +0.1% Formic Acid)
Preparing the Diluent
Prepare diluent by mixing equal parts acetonitrile and water and
adding 0.1% formic acid. Refrigerate unused solution at 4 °C for
no longer than 2 weeks.
1. Measure 100 mL LC-MS-grade acetonitrile into a cylinder and
pour into a 250-mL Duran bottle.
2. Add 100 mL LC-MS-grade or deionized water to the bottle.
3. Pipette 0.1% formic acid into the bottle.
4. Sonicate the contents of the bottle for 10 minutes.
5. Label the bottle as follows: “50:50 acetonitrile:water
(w/ 0.1% formic acid).”
b. Reverse Peptide Stock Solutions
Preparing the Reverse Peptide Stock Solutions
Prepare reverse peptide stock solutions by mixing the
reconstituted samples in an ultrasonic liquid processor.
Preparing 1 mg/ml (1 µg/µl) reverse peptide stock solution
1. Pipette 1000 µL of the 50:50 acetonitrile:water (w/0.1%
formic acid) diluent into the 1 mg vial of Ser-Asp-Gly-Arg-Gly.
2. Sonicate mixture for 5 minutes.
3. Label as “1 mg/mL SDGAG w/50:50 acetonitrile:water
(w/ 0.1% formic acid).”
4. Repeat the procedure with the Gly-Arg-Gly-Asp-Ser sample.
5. Label as “1 mg/mL GAGAS w/50:50 acetonitrile:water
(w/ 0.1% formic acid).” Unused samples may be stored in a
freezer at -20 °C for no more than 1 month.
Reliant HPLC Columns
2
[ CARE AND USE MANUAL ]
Preparing 10 ng/µl reverse peptide stock solution
1. Add 10 mL of 50:50 acetonitrile:water (with 0.1% formic acid)
to a 20 mL volumetric flask.
2. Pipette 200 µL of the 1mg/ml reconstituted SDGAG sample
into a 20-mL volumetric flask.
3. Pipette 200 µL of the reconstituted GAGAS sample into the
flask and sonicate for 5 minutes.
4. Add enough of the 50:50 acetonitrile:water diluent to the
contents of the flask to make 20 mL of 10 ng/μL reverse
peptide stock solution standard solution.
5. Label as “10 ng/µL reverse peptide stock solution” as
appropriate. Store unused aliquots in freezer at -20 °C for no
longer than 1 week.
c. Reverse Peptide Standard Solution Working Concentration
Preparing 100 pg/µL Standard Solution
Prepare 100 pg/µL reverse peptide specification sample by
diluting the10 ng/µL peptide stock solution with 50:50
acetonitrile:water (with 0.1% formic acid). Store unused aliquots
20 mL standards solution at -20 °C for no more than 1 week.
IV. MOBILITY MODE COMMISSIONING TEST
a. Resolution Mode (Positive Ion)
The mobility separation of the m/z 246.1 ion species in nitrogen
gives two distinct arrival time peaks from which a mobility
resolution (Ω/ΔΩ) of >36 will be demonstrated using collision
cross-section (W values of 222.7 Å2 and 211.7 Å2 for the
ser-asp-gly-arg-gly and gly-arg-gly-asp-ser peptides
respectively. The high mass will be set to 600 Da.
This procedure requires the sample described in Table 3:
Table 3: Setup samples
Sample Reservoir Flow rate Sprayer
100 pg/μL each of
the peptides: serasp-gly-arg-gly and
gly-arg-gly-asp-ser
A
5 μL/min
(0.5 μL/min for
NanoFlow)
Sample
1. Pipette 200 µL of the 10-ng/µL reverse peptide stock solution
into a 20-mL volumetric flask.
2. Add enough of the 50:50 acetonitrile:water diluent to the
contents of the flask to make 20 mL of 100 pg/μL reverse
peptide standard solution.
3. Sonicate the contents of the flask for 5 minutes.
4. Label as “100 pg/µL reverse standard solution”.
5. To prepare samples for use with built-in fluidics systems,
transfer the standards solution from the 20-mL volumetric
flask into a 30 mL nalgene bottle, label as “100 pg/µL reverse
standard solution” and store at -20 °C for no longer than
1 week.
Figure 1. Example display of the non-storage TOF and mobility TOF windows.
The screen-shot shows expected TIC and Mobility TIC for the
reverse peptide mixture, the MS/MS set mass should be m/z
246.1. The screen-shot shows on the left, the expected TOF TIC
and on the right the expected Mobility ToF trace for the reverse
peptide mixture is shown.
Drift times for the two peptides (indicated by the two peaks)
should be as observed, tune for maximum separation using
the IMS, Transfer and Trap DC tabs, parameters required are
instrument model specific. Refer to instrument operations manual
or applications support for assistance if required.
Reliant HPLC Columns
3
[ CARE AND USE MANUAL ]
Figure 2. Chromatogram showing the mass of the peak.
Extracted ion chromatogram of m/z 246.1 created from combined scans of a 3.5 min
acquisition. Mobility separation of the two reverse peptides is evident in the two peaks on the
extracted ion chromatogram.
V. ORDERING INFORMATION
To order these products, contact your nearest subsidiary, or visit www. waters.com/order
Description Qty. Part number
Waters Reverse Peptide Kit 1 700005089
Description Part Number
Waters Reverse Peptide Kit (contains supplies listed below) 700005089
Ser-Asp-Gly-Arg-Gly
Gly-Arg-Gly-Asp-Ser
700005089-1
700005089-2
Sample Preparation document 715002238
Certificate of Analysis N/A
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©2014 Waters Corporation. Produced in the U.S.A. March 2014 Rev A 720004421EN KP-PDF
Waters Corporation
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Milford, MA 01757 U.S.A.
T: 1 508 478 2000
F: 1 508 872 1990
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