Waters Quantitative Peptide Retention Standard User Manual

[ CARE AND USE MANUAL ]

Quantitative Peptide Retention Standard

TABLE OF CONTENTS:

I. INTRODUCTION

II. STORAGE AND STABILITY

III. RECONSTITUTION OF THE PEPTIDE

RETENTION STANDARD

IV. EXAMPLE OF USING THE PEPTIDE

RETENTION STANDARD ON THE
ACQUIT Y U PLC

V. ORDERING INFORMATION

I. INT RO DUC TION

This standard was specifically designed and formulated as a simple peptide
mixture that provides a broad retention window for a variety for a variety of
peptide separation applications. It is an ideal reference material since the
peptides were designed to be stable and to not be prone to modification such
as oxidation.

This standard complements it’s qualitative counterpart, the MassPREP™
Peptide Standard which offers a versatility of various peptides but it not
quantitative. Each peptide in the Quantitative Peptide Retention Standard
is quantitated via amino acid analysis to provide greater accuracy to each
analysis, has been verified by mass identification and is highly pure (more
than 95% pure). Each standard is provided in a Waters TruView™ Maximum
Recovery Vial which simplifies the sample preparation process and allows
for direct injection once solubilized.

The standard consists of four neutral peptides with a single A280
chromophore and varying hydrophobicities and is formulated such that
the peptide components are present in equal abundance. See Table 1 for
specific peptide information.

Table 1: Peptide Mixture Components and Example Data Generated

from Figure 1

Peptide Retention Standard

Peak Area

Peptide MWmono RT (min)* (A

DGYGK
DTVGYGVK 837.4232 10.5 2572 25
DFVGYGVK 883.4440 13 2801 27
DFVGYGVKDFVGVGVK

280 nm chromophore (Y) highlighted
* See Figure 1 for retention time examples.

Applications of the standard can include, but are not limited to: evaluation
of columns and LC systems dedicated to peptide analysis as well as

monitoring performance and recovery across romatographic gradients.

538.2387

1684.8 825

2.6 2446 24

16.4 2437 24

) % Area

280

[ CARE AND USE MANUAL ]

II. STORAGE AND STABILITY

Lyophilized peptides generally have excellent stabilities, often showing little or no degradation after a few years at -20 °C. Long-term
storage (> 1 year) should be at -80 °C desiccated, medium-term storage (1–12 months) should be at -20 °C desiccated, short-term
storage (< 1 month) may be at 4 °C. Once reconstituted the solution should be used immediately to avoid degradation of peptides which
would compromise the benefit of the standard having been quantitatively formulated.

III. RECONSTITUTION OF THE QUANTITATIVE PEPTIDE RETENTION STANDARD

This standard contains 20 nmoles of each peptide lyophilized. Reconstitute with 133.3 µL of either 0.1% aqueous formic acid or 0.1%
aqueous trifluoroacetic acid to produce a sample concentration of 150 pmol/µL (per peptide). Suggested injection volumes and sample
loads are outlined below.

Table 2: Recommended Mass Load for LC Column Evaluation

Reconstitution Volume Column ID Injection Volume Quantity of Peptide Loaded (each species)

133.3 µ L 4.6 mm 19.2 µL 2.9 nmol

133.3 µ L 2.1 mm 4.0 µL 600 pmol

133.3 µ L 1.0 mm 0.9 µL 140 pmol

IV. EXAMPLE OF USING THE QUANTITATIVE PEPTIDE RETENTION STANDARD

Below is a reference chromatogram with conditions to help provide an example of using the Quantitative Peptide Retention Standard.
Please note retention times are dependent on LC conditions, column and LC system. If the column size is different, the gradient/
injection volume can be scaled accordingly

System: ACQUITY UPLC® H-Class Bio
Column: ACQUITY UPLC CSH130 C18, 130Å, 1.7 µm, 2.1 x 150 mm
Mobile Phase A: Water with 0.1% FA (v/v)
Mobile Phase B: ACN with 0.1% FA (v/v)
Gradient: 0.5% B for 1 min, then to 50% B over 30 min
Flow Rate: 0.3 mL/min
Column Temp. 40 °C
Detector: UV @ 280 nm , 10 Hz
Software: Monoisotopic masses calculated with MassLynx® 4.1

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Quantitative Peptide Retention Standard