Waters Hi3 Standards - Manual

Hi3 Standards 1
[ CARE AND USE MANUAL ]
I. INT RODUCTION
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
E 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).