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Hi3 Standards 1

[ CARE AND USE MANUAL ]

I. INTRODUCTION

The Hi3 Ecoli and Phos B standards are a specialized set of 6

synthetically prepared and highly purified peptides that provide

the top 6 ionizing peptides of both the E.coli ClpB and rabbit

Phosphorylase B proteins. These standards are quantitated via

AAA to equimolar ratios to provide greater accuracy to each

analysis. They are greater than 95% pure and have been verified

by mass identification. In addition, each vial is in a Waters Max

Recovery vial 186000327C which simplifies the sample

preparation process.

These standards are primarily intended for use with the Hi3

quantification method for MS

proteomics data processed with

ProteinLynx Global Server

™

. The E. coli standard is intended for

samples of animal origin, and the Phos B standard is intended for

samples of microbial origin. The standards may also be of use in the

evaluation and benchmarking of proteomic LC/MS systems comprised

of nanoACQUITY UPLC

and Synapt

and Xevo

time-of-flight

mass spectrometers.

Table 1: Hi3 Phos B Standard (P/N 186006011)

Peptide Sequence

Average

Molecular

Weight

Concentration

Phos B

Sequence 1

VLYPNDNFFEGK 1442.5913 1nmol/vial

Phos B

Sequence 2

TC*AYTNHTVLPEALER 1875.0931 1nmol/vial

Phos B

Sequence 3

IGEEYISDLDQLRK 1678.8602 1nmol/vial

Phos B

Sequence 4

LLSYVDDEAFIR 1440.6164 1nmol/vial

Phos B

Sequence 5

LITAIGDVVNHDPVVGDR 1890.1273 1nmol/vial

Phos B

Sequence 6

VFADYEEYVK 1262.3817 1nmol/vial

The cysteine in sequence 2 is carbamidomethylated.

Hi3 STANDARDS

Table 2: Hi3 EColi Standard (P/N: 186006012)

Peptide Sequence

Average

Molecular

Weight

Concentration

Ecoli ClpB

Sequence 1

VIGQNEAVDAVSNAIR 1655.8292 1nmol/vial

Ecoli ClpB

Sequence 2

NNPVLIGEPGVGK 1293.4863 1nmol/vial

Ecoli ClpB

Sequence 3

AIDLIDEAASSIR 1373.5266 1nmol/vial

Ecoli ClpB

Sequence 4

VTDAEIAEVLAR 1286.4484 1nmol/vial

Ecoli ClpB

Sequence 5

AIQQQIENPLAQQILSGELVPGK 2474.8412 1nmol/vial

Ecoli ClpB

Sequence 6

LPQVEGTGGDVQPSQDLVR 1995.1776 1nmol/vial

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 quantitative benefit.

III. RECONSTITUTION OF THE

Hi3 DIGESTION STANDARDS

The Hi3 Digestion Standards contain 1 nmol of each of the peptides

listed in Table 1 or 2. It is recommended that 100 µL of 0.5%

trifluoroacetic acid in 3% acetonitrile/water be added to a vial of

standard to obtain a 10 pmol/µL stock solution. Because peptides can

adhere to glass and other surfaces resulting in significant losses from

highly dilute solutions, further dilutions should be made just prior to

addition to samples and excess diluted standard should be discarded.

Unused stock solutions may be stored in a freezer for a brief time.

Analysis of small amounts of standard (ca. 50 fmol) should only be

carried out in the presence of a sample matrix such as E. coli digest

of a serum digest (60 ng/µL or greater).

1 2 3 4

Summary of Contents

Page 1 - Hi3 STANDARDS

Hi3 Standards 1[ CARE AND USE MANUAL ]I. INTRODUCTIONThe Hi3 Ecoli and Phos B standards are a specialized set of 6 synthetically prepared and highly p

Page 2

Hi3 Standards 2[ CARE AND USE MANUAL ]IV. USING THE Hi3 DIGESTION STANDARDS FOR QUANTIFICATIONFor a typical proteomic analysis carried out with 75

Page 3

Hi3 Standards 3[ CARE AND USE MANUAL ]Process and search the data in the normal fashion. Calculated quantities for each identified protein (in fmol an

Page 4

Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.comHi3 Standards 4[ CARE AND USE MANUAL ]©2

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