Waters Reverse Peptide Kit User Manual

[ 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