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.

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©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