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SILAC Hi3 E. Coli and Phos B Standards
Product Features■■ Perform relative protein
quantitation of mixed
proteome samples.
■■ Minimize interferences
with the endogenous
peptides.
■■ Add a second level of
internal quality control
(i.e., spike SILAC Hi3
standards toward
the lower limit of
detection).
Mass spectrometry (MS)-based proteomics is increasingly applied in a quantitative format,
often based on labeling of samples with stable isotopes. The more robust and accurate
quantification methods uses these stable isotopes. SILAC (Stable Isotope Labeling by
Amino Acids in Cell Culture) is used for comparative, quantitative proteome analysis.
It is a technique based on mass spectrometry that detects differences in protein abundance
among samples using non-radioactive isotopic labeling. SILAC has emerged as a very
powerful method to study cell signaling, post translational modifications, protein-protein
interactions, and regulation of gene expression.1
SILAC external standards are produced to have a heavy labeled reference and are typically
labeled on the c-terminal lysine (K) or arginine (R).
Some applications can include adding in the spiked internal standard to the proteome
samples to be processed with MS after cell lyses and before protein digestion. Applications
range from absolute quantification of single proteins to the quantification of whole proteomes.1
General Information
The Hi3 E. coli and Phosphorylase B peptide line of standards provides both a labeled and
unlabeled version of the top 6 ionizing peptides represented in each proteome. The Hi3
stable isotope labeled (SIL) standards (Phos B and ClpB) provides an isotopically unique
exogenous standard to perform Hi3 relative protein quantitation.2
The E. coli standard is intended for samples of animal origin, and the Phos B standard is
intended for samples of microbial origin in order to minimize interferences between the
standard and the protein sample. The standards may also be of use in the evaluation
and benchmarking of proteomic LC/MS systems comprised of nanoACQUITY UPLC,®
SYNAPT,® and Xevo® time-of-flight mass spectrometers.
2
Component Information
Table 1. Hi3 Phos B Standard
Peptide SequenceAverage
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 SequenceAverage
Molecular Weight
Concentration
SILAC E. coli ClpB Sequence 1
H2N-VIGQNEAVDAVSNAIR-OH 1664.8873 1 nmol/vial
SILAC E. coli ClpB Sequence 2
H2N-NNPVLIGEPGVGK-OH 1300.7276 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 1295.7113 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
Utilization of Standards
A. The following is an example of the standards run on a
Waters SYNAPT G2-S HDMS mass spectrometer and the
corresponding results.
Sample Preparation
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 1,000 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.
Conditions
Solvent A: 0.1% formic acid in water
Solvent B: 0.1% formic acid in acetonitrile
Weak Wash: 3% acetonitrile w/ 0.1% TFA
Trapping: 10 µL/min for 3 min, 10 µL loop
Column: nanoACQUITY UPLC HSS T3 C18, 1.8 µm,
75 µm x 150 mm (P/N 186005776)
Trap: ACQUITY UPLC® PST C18 nanoACQUITY Trap,
5 µm, 180 µm x 20 mm (P/N 186006527)
Gradient Profile
Time Flow rate (µL/min) %A %B Curve
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
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
3
Figure 1. Base Peak Intensity Chromatograms: 1D MSE – 25 fmol SILAC Hi3 E. coli.
La be l P ept ide S equence [ M+ H] +
1 NNPVLIGEPGVGK 1300.723
2 LPQVEGTGGDVQPSQDVLR 2004.016
3 VIGQNEADAVSNAIR 1664.873
4 VTDAEIAEVLAR 1295.696
5 AIDLIDEAASSIR 1382.727
6 AIQQQIENPLAQQILSGELVPGK 2481.369
La be l P ept ide S equence [ M+ H] +
11 NNPVLIGEPGVGKNNPVLIGEPGVGK 1300.7231300.723
22 LPQVEGTGGDVQPSQDVLRLPQVEGTGGDVQPSQDVLR 2004.0162004.016
33 VIGQNEADAVSNAIRVIGQNEADAVSNAIR 1664.8731664.873
44 VTDAEIAEVLARVTDAEIAEVLAR 1295.6961295.696
55 AIDLIDEAASSIRAIDLIDEAASSIR 1382.7271382.727
66 AIQQQIENPLAQQILSGELVPGKAIQQQIENPLAQQILSGELVPGK 2481.3692481.369
11
22
33
44
55
66
Figure 3. The background proteome for this experiment was a E. coli lysate (P/N 186003196) prepared at 50 ng/µL with the SILAC Hi3 E. coli standard at 5 fmol/µL. A total of 50 ng of E. coli lysate was injected on column.
Figure 2. Base Peak Intensity Chromatograms: 1D MSE – 25 fmol SILAC Hi3 Phos B.
B. The following experiment illustrates the ability to perform Hi3 relative quantitation using stable isotope labeled peptides derived
from the same organism.
CV 6.57%CV 6.57%
La be l P ept ide S equence [ M+ H] +
1 TCamAYTNHTVLPEALER 1884.9167
2 VFADYEEYVK 1270.6094
3 LITAIGDVVNHDPVVGDR 1900.0193
4 LYPNDNFFGK 1450.7104
5 LLSYVDDEAFIR 1470.7469
La be l P ept ide S equence [ M+ H] +
11 TCamAYTYY NHTVLPEALERTCamAYTNHTVLPEALER 1884.91671884.9167
22 VFADYEEYVKVFADYEEYVK 1270.60941270.6094
33 LITAIGDVVNHDPVVGDRLITAIGDVVNHDPVVGDR 1900.01931900.0193
44 LYLL PNDNFFGKLYPNDNFFGK 1450.71041450.7104
55 LLSYVDDEAFIRLLSYVDDEAFIR 1470.74691470.7469
11
44
33
2255
4
F
E
D
C
B
A
F
E
D
C
B
A
L a b e l P e p t i d e S e q u e n c e
A NNPVLIGEPGVGK
B LPQVEGTGGDVQPSQDLVR
C VIGQNEAVDAVSNAIR
D VTDAEIAEVLAR
E AIDLIDEAASSIR
F AIQQQIENPLAQQILSGELVPGK
L a b e l P e p t i d e S e q u e n c e
AA NNPVLIGEPGVGKNNPVLIGEPGVGK
BB LPQVEGTGGDVQPSQDLVRLPQVEGTGGDVQPSQDLVR
CC VIGQNEAVDAVSNAIRVIGQNEAVDAVSNAIR
DD VTDAEIAEVLARVTDAEIAEVLAR
EE AIDLIDEAASSIRAIDLIDEAASSIR
FF AIQQQIENPLAQQILSGELVPGKAIQQQIENPLAQQILSGELVPGK
Labe l P eptide S equence
A NNPVLIGEPGVGK
B LPQVEGTGGDVQPSQDLVR
C VIGQNEAVDAVSNAIR
D VTDAEIAEVLAR
E AIDLIDEAASSIR
F AIQQQIENPLAQQILSGELVPGK
Labe l P eptide S equence
AA NNPVLIGEPGVGKNNPVLIGEPGVGK
BB LPQVEGTGGDVQPSQDLVRLPQVEGTGGDVQPSQDLVR
CC VIGQNEAVDAVSNAIRVIGQNEAVDAVSNAIR
DD VTDAEIAEVLARVTDAEIAEVLAR
EE AIDLIDEAASSIRAIDLIDEAASSIR
FF AIQQQIENPLAQQILSGELVPGKAIQQQIENPLAQQILSGELVPGK
Figure 4. Extracted Ion Chromatograms: 1D MSE – 25 fmol/µL Hi3 E. coli.
Figure 5. Extracted Ion Chromatograms: 1D MSE – 50 ng E. coli w/ 5 fmol Hi3 E. coli.
5
Figure 7. SILAC Hi3 Phos B Heavy and Light Standards run together.
Figure 6. Elevated Energy Mass Spectra: VTDAEIAEVLAR (A) Neat Standard (B) Within E. coli Background.
C. Finally, a comparison between the SILAC Hi3 (Heavy) standards compared to their unlabeled counterparts which are the Hi3 Phos B
and Hi3 E. coli (Light) standards.
Neat Standard
Within E. coli background
HeavyMixture
Light Mixture
Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com
References
1. Silva et al. Mol. Cell Proteomics, 2006, 144-156
2. Ong Genome Medicine 2010, 2:49
3. Gethings, Tonge, Williamson, and Whetton. A Qualitative and Quantitative Ion Mobility MS-Enabled, Data-Independent SILAC Workflow, June 2013, Waters, The University of Manchester. Application Note 720004725EN.
Waters, T he Science of What’s Possible, ACQUITY UPLC, nanoACQUITY UPLC, SYNAPT, and Xevo and are registered trademarks of Waters Corporation. MassPREP is a trademark of Waters Corporation. All other trademarks are the property of their respective owners.
©2013 Waters Corporation. Produced in the U.S.A.October 2013 720004792EN LM-PDF
Ordering Information
Description Qty Part Number
SILAC Hi3 Phos B Standard 1 186007083
SILAC Hi3 E. coli Standard 1 186007084
Hi3 Phos B Standard 1 186006011
Hi3 E. coli Standard 1 186006012
MassPREP™ Digestion Standard, Phosphorylase B
1 186002326
MassPREP E. coli Digestion Standard
1 186003196
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