[ CARE AND USE MANUAL ]
SILAC Hi3 Standards
CONTENTS
I. INTRODUCTION
II. STORAGE AND STABILIT Y
III. RECONSTITUTION OF THE
SILAC HI3 DIGESTION STANDARDS
IV. USING THE SILAC HI3 DIGESTION
STANDARDS FOR QUANTIFICATION
V. ORDERING INFORMATION
I. INTRODUCTION
The Hi3 stable isotope labeled (SIL) standards (Phos B and ClpB)
provides an isotopically unique exogenous standard to perform
Hi3 relative protein quantitation.
Minimize interferences with the endogenous peptides
Perform relative protein quantitation of mixed proteome samples
Add a second level of internal quality control (i.e., spike
Hi3 SILAC standards toward the lower limit of detection).
These standards, along with their unlabeled counterparts, 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 Maximum
Recovery Vial which simplifies the sample preparation process.
These standards are primarily intended for use with the Hi3
quantification method for MSE 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.
[ CARE AND USE MANUAL ]
Table 1. SILAC Hi3 Phos B Standard
Peptide Sequence Average Molecular Weight Concentration
SILAC Phos B Sequence 1 H2N-VLYPNDNFFEGK-OH 1449.7066 1 nmol/vial
SILAC Phos B Sequence 2 H2N-TC*AYTNHTVLPEALER-OH 1883.9227 1 nmol/vial
SILAC Phos B Sequence 3 H2N-IGEEYISDLDQLRK-OH 1685.8762 1 nmol /vial
SILAC Phos B Sequence 4 H2B-LLSYVDDEAFIR-OH 1449.7531 1 nmol/vial
SILAC Phos B Sequence 5 H2N-LITAIGDVVNHDPVVGDR-OH 1899.0242 1 nmol/vial
SILAC Phos B Sequence 1 H2N-VLYPNDNFFEGK-OH 1269.6055 1 nmol/vial
The cysteine in sequence 2 is carbamidomethylated.
Table 2. SILAC Hi3 E. coli Standard
Peptide Sequence Average Molecular Weight Concentration
SILAC E. coli ClpB Sequence 1 H2N-VIG QNE AVDAV SNA IR- OH 166 4.8 873 1 nmol/vial
SILAC E. coli ClpB Sequence 2 H2N-NNPVLIGEPGVGK-OH 130 0.7276 1 nmol/vial
SILAC E. coli ClpB Sequence 3 H2N-AIDLIDEAASSIR-OH 1382.7433 1 nmol/vial
SILAC E. coli ClpB Sequence 4 H2N-VTDAEIAEVLAR-OH 129 5 .7113 1 nmol/vial
SILAC E. coli ClpB Sequence 5 H2N-AIQQQIENPLAQQILSGELVPGK-OH 2482.3751 1 nmol/vial
SILAC E. coli ClpB Sequence 1 H2N-LPQVEGTGGDVQPSQDLVR-OH 2004.0304 1 nmol/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
SILAC HI3 DIGESTION STANDARDS
The SILAC Hi3 Standards are originally in a concentration of
1 nmol/vial of either the ClpB_ECOLI or the PYGM_RABIT.
The lyophilized peptides were re-suspended in 1000 µL of
3% acetonitrile w/ 0.1% Trifluoroacetic acid. The resulting
concentration was 1 pmol/µL. The SILAC Hi3 standards were then
diluted to 25 fmol/µL, i.e., 25 µL of each solution was diluted to
1000 µL. 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).
SILAC Hi3 Standards
2
[ CARE AND USE MANUAL ]
TAVVV
V
Y
Y
V
V
V
VTVQV
IV. USING THE SILAC HI3 DIGESTION STANDARDS FOR QUANTIFICATION
The following is an example of the standards run on a
Waters SYNAPT G2-S HDMS mass spectrometer and the
corresponding results.
Conditions:
Solvent A: 0.1% formic acid in water
Solvent B: 0.1% formic acid in acetonitrile
Gradient Profile:
Time
Flow Rate
(µ L /min)
Initial 0.500 95.0 5.0 6
54.00 0.500 60.0 40.0 6
55.80 0.500 15.0 85.0 6
%A %B Curve
Weak wash: 3% ACN w/ 0.1% TFA
Trapping: 10 µL/min for 3 min, 10 µL loop
Trap: ACQUITY UPLC® PST C18 nanoACQUITY® Trap,
5 μm, 180 μm x 20 mm
4
1
1
4
3
3
5
5
2
2
59.40 0.500 15.0 85.0 6
61.20 0.500 95.0 5.0 6
72.00 0.500 95.0 5.0 6
Table 3. Base Peak Intensity Chromatograms: 1D MSE – 25 fmol
SILAC Hi3 ClpB E. coli.
Label Peptide Sequence [M+H]+
Label Peptide Sequence [M+H]+
NNPVLIGEPGVGK
NNPVLIGEPGVGK
1
1
LPQVEGTGGDVQPSQDVLR
LPQVEGTGGDVQPSQDVLR
2
2
VIGQNEADAVSNAIR
VIGQNEADAVSNAIR
3
3
VTDAEIAEVLAR
4
44
5
5
6
6
6
6
DAVVTDAEIAEVLAR
AIDLIDEAASSIR
AIDLIDEAASSIR
AIQQQIENPLAQQILSGELVPGK
AIQQQIENPLAQQILSGELVPGK
1300.723
0
2004.016
2004.016
1664.873
6681664.873
1295.696
2
1295.696
1382.727
221382.727
2481.369
4
2481.369
231300.723
Figure 1. Base Peak Intensity Chromatograms: 1D MSE – 25 fmol Hi3 SILAC ClpB E. coli.
Label Peptide Sequence [M+H]+
3
3
5
5
2
2
4
4
1
1
Label Peptide Sequence [M+H]+
TCamAYTNHTVLPEALER
A
V
TCamAYTNHTVLPEALER
1
1
CV 6.57%
CV 6.57%
2
2
3
3
4
4
5
5
m
VFADYEEYVK
AYVVFADYEEYVK
LITAIGDVVNHDPVVGDR
LITAIGDVVNHDPVVGDR
LYPNDNFFGK
LYPNDNFFGK
LLSYVDDEAFIR
LLSYVDDEAFIR
A
A
1884.9167
1884.9167
1270.6094
1270.6094
1900.0193
00931900.0193
1450.7104
4
1470.7469
47
1470.7469
41450.7104
Figure 2. Base Peak Intensity Chromatograms: 1D MSE – 25 fmol Hi3 SILAC PhosB.
SILAC Hi3 Standards
3
[ CARE AND USE MANUAL ]
V. ORDERING INFORMATION
Product Description Qty Part Number
SILAC Hi3 Phos B Standard 1/pk 186007083
SILAC Hi3 E. coli Standard 1/pk 186007084
Hi3 Phos B Standard 1/pk 186006011
Hi3 E. coli Standard 1/pk 186006012
MassPREP™ Digestion Standard,
Phosphorylase B
1/pk 186002326
MassPREP E. coli Digestion Standard 1/pk 1860 0 3196
ACQUITY UPLC PST C
5 μm, 180 μm x 20 mm
LCGC Certified Clear Glass, 12 x 32 mm,
Screw Neck, Max Recovery Vial
nanoACQUIT Y Trap,
18
1860 06527
100/pk 186000327C
Waters, The Science of W hat’s Possible, ACQUITY U PLC,
nanoACQUITY, nanoACQUITY UPLC, SYNAP T, and Xevo are
registered trademarks of Waters Corporation. ProteinLynx GLOBAL
SERV ER and MassPR EP are trademarks of Waters Corporation.
All other trademarks are t he property of their respective owners.
©2013 Waters Corporation. Produced in the U.S.A.
October 2013
20004785EN LM-PDF
Waters Corporation
34 Maple Street
Milford, MA 01757 U.S.A.
T: 1 508 478 2000
F: 1 508 872 1990
www.waters.com
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