Supporting Information for Proteomics
Huifeng Wu, Chenglong Ji, Lei Wei and Jianmin Zhao
Evaluation of protein extraction protocols for 2-DE in marine
ecotoxicoproteomics
1
SUPPORTING INFORMATION
Materials and methods
Animals
Three marine animal specimens, mussel Mytilus galloprovincialis, flounder
Paralichthys olivaceus and polychaete Nereis diversicolor, were studied for quality
assessment of protein extraction protocols including straightforward lysis buffer
solution, TCA-acetone and TRIzol reagent. One individual flounder P. olivaceus
(approx. 500 g) were purchased from a local culturing farm in Yantai, China. The
blue mussel M. galloprovincialis and polychaete N. diversicolor were collected from
the local coast along the Yellow Sea near Yantai. Before dissection, all the
experimental animals were acclimatized in fresh seawater for three days without
feeding. After acclimatization, the gill tissue of M. galloprovincialis, liver of P.
olivaceus and whole soft tissue of N. diversicolor were sampled from single
individuals of these specimens and snap-frozen in liquid nitrogen. All the samples
were stored at –80oC prior to protein extraction.
Extraction protocols
Each biological sample was ground into homogeneous powder under liquid nitrogen
and divided into 12 aliquots for protein extraction. For each extraction method, four
replicate samples were used for protein extraction. The details of protein extraction
using different methods were described below.
Trizol reagent extraction
Total protein extraction was conducted based on the work of Butt et al [1] and Lee et
al [2] with some modifications. Briefly, the ground sample was re-suspended in 1 ml
Trizol reagent and centrifuged at 12 000 g for 5 min at 4 oC. The supernatant was
added with 200 μl chloroform before being shaken vigorously for 3 min and
precipitated at room temperature for 3 min. This mixture was then centrifuged at 12
000 g for 15 min at 4 oC and the upper aqueous phase was discarded. To the lower
organic phase, 300 μl of absolute ethyl alcohol was added and the mixture was
allowed to stand for 3 min at room temperature before being centrifuged at 2 000 g for
5 min at 4 oC. The phenol/ethanol supernatant was precipitated for at least 30 min at
2
room temperature by the addition of 750 μl isopropanol prior to centrifugation at 14
000 g for 10 min at 4 oC. The supernatant was removed, and the pellet obtained was
washed twice using 1 ml ethanol (v/v 95%) and centrifuged at 14 000 g for 10 min at
4 oC. The pellets were solubilized in the lysis buffer (7 M urea, 2 M thiourea, 4% m/V
CHAPS, 65 mM DTT and 0.2% W/V Bio-lyte buffer) and then incubated for 3 h at
room temperature [3]. The homogenate was centrifuged at 12 000 g for 10 min and
the supernatant was applied to electrophoresis.
TCA-acetone extraction
Two most frequently used extraction procotols with TCA-acetone system were used
for protein extraction from experimental animal samples [4-7]. The fundamental
procedures of TCA-acetone extraction were based on a previously published method
with some modifications [4-7].
TCA-Acetone-based protocol A:
The ground sample was re-suspended in cold buffer (10% w/v TCA in acetone
contained 0.07% w/v DTT). Then the sample was precipitated at –20 oC overnight and
centrifuged at 15 000 g for 30 min at 4 oC, the supernatant was discarded. The pellet
was re-suspended three times with cold acetone containing 0.07% w/v DTT kept at –
20 oC for 1 h and centrifuged at 15 000 g for 30 min at 4 oC. The pellet was
solubilized in the lysis buffer (7 M urea, 2 M thiourea, 4% m/V CHAPS, 65 mM DTT
and 0.2% W/V Bio-lyte buffer) and then incubated for 3 h at room temperature [3].
The homogenate was centrifuged at 12 000 g for 10 min and the supernatant was
applied to electrophoresis.
TCA-Acetone-based protocol B:
The powdered sample was then suspended in a four-fold dilution of 40 mM Tris
buffer including 10% AEBSF and Phosphatase Inhibitor Cocktail Ⅱ(Sangon CO. Ltd,
Shanghai, China). After centrifugation at 15 000 g for 30 min at 4 oC, the supernatant
was collected and three times volume of cold buffer (10 % w/v TCA in acetone
contained 0.07 % w/v DTT) was added. Then the sample was precipitated at –20 oC
overnight and centrifuged at 15 000 g for 30 min at 4 oC, the supernatant was
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discarded. The following procedures were completely identical with those in protocol
A.
Straightforward lysis buffer extraction
Three most frequently used extraction procotols with lysis buffer system were used
for protein extraction from experimental animal samples [8-10]. The fundamental
straightforward lysis procedures were based on a previously published method with
some modifications [8-10].
Straightforward lysis-based protocol A:
Proteins were extracted by suspending powdered samples in lysis buffer (7 M urea; 2
M thiourea; 40 mM Tris; 4% w/v CHAPS; 65 mM DTT; 0.2% w/v Bio-lyte buffer).
After being vigorously shaken at room temperature for 2 h, the mixture was
centrifuged at 14 000 g for 30 min and the supernatant was used for electrophoresis.
Straightforward lysis-based protocol B:
Proteins were extracted by suspending powdered samples in lysis buffer (7 M urea; 2
M thiourea; 40 mM Tris; 4% w/v CHAPS; 65 mM DTT; 0.2% w/v Bio-lyte buffer; 40
mM 10% AEBSF and 10% v/v Phosphatase Inhibitor Cocktail Ⅱ ). After being
vigorously shaken at room temperature for 2 h, the mixture was centrifuged at 14 000
g for 30 min and the supernatant was used for electrophoresis.
Straightforward lysis-based protocol C:
Proteins were extracted by suspending powdered samples in lysis buffer (7 M urea; 2
M thiourea; 40 mM Tris; 4% w/v CHAPS; 65 mM DTT; 0.2% w/v Bio-lyte buffer; 40
mM 10% AEBSF and 10% v/v Phosphatase Inhibitor Cocktail Ⅱ ). After
ultrasonication on ice for 10 min, the mixture was centrifuged at 14 000 g for 30 min
and the supernatant was used for electrophoresis.
Two-dimensional gel electrophoresis
The concentration of protein was determined by Protein Assay Kit of TianGen. IPG
strips (24 cm pH 3-10; GE Healthcare Biosciences Immobiline DryStrips) were
rehydrated with 450 μl IEF buffer (7 M urea, 2 M thiourea, 4% m/v CHAPS, 65 mM
4
DTT, 0.001% m/v Bromophenol blue and 0.2% W/V Bio-lyte buffer) containing 150
μg protein and were focused using an Ettan IPGphor3 system at 20 oC, applying the
following program: 30 V for 12 h, followed by 100 V for 5 h, 500 V for 1 h, 1000 V
for 1 h, and a linear increase of voltage to 8000 V for 11 h.
After the first dimension, strips were placed in equilibration buffer (0.05 M TrisHCl, pH 8.8, 6 M urea, 30% glycerol, 2% w/v SDS, containing 1% w/v DTT) for 15
min. Subsequently, the strips were incubated for another 15 min in the same
equilibration buffer with 2.5% (w/v) iodoacetamide without DTT. The second
dimension was performed on 12.5% SDS-PAGE gels using the Ettan DALTsix
system. The gels were silver stained after electrophoresis. Images were captured by
ImageScanner Ⅲ and spots were quantitatively analyzed by using ImageMaster 2D
Platinum 7.0. The abundance of each protein spot was estimated by the percentage
volume (% vol).
In gel digestion and MS analysis
To test the compatibility between the proteins extracted using TRIzol reagent and
mass spectrometry, twenty protein spots from the gill tissue of M. galloprovincialis
with different abundances in silver stained 2-DE gels were selected for further
identification using MS/MS and databases. The protein spots were washed three times
with ultrapure water, de-stained with 25 mmol/L NH4HCO3 in 50% (v/v) acetonitrile
at room temperature for 30 min. The gels were dried using 50% acetonitrile for 30
min and 100% acetonitrile for another 30 min. The samples were rehydrated in 10 μl
cover solution (0.02 g/l w/v trypsin, 25 mmol/L NH4HCO3 and 10% acetonitrile) for
30 min, and then covered with the same solution but without trypsin for digestion
overnight at 37 oC. The supernatants were extracted with 5% TFA in 67% acetonitrile
at 37 oC for 30 min, then was centrifuged at 5 000 g for 5 min, so the peptide extracts
and the supernatant of the gel spot were combined.
After being completely dried the samples were re-suspended with 5 µL 0.1% TFA
followed by mixing in 1:1 ratio with a saturated solution of α-cyano-4-hydroxy-transcinnamic acid in 50% acetonitrile. One microliter of the mixture was analyzed by an
ABI 4800 MALDI-TOF/TOF Plus mass spectrometer (Applied Biosystems, Foster
City, USA), data were acquired in a positive MS reflector using a CalMix5 standard
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to calibrate the instrument (ABI4800 Calibration Mixture). Both the MS and MS/MS
data were integrated and processed by using the GPS Explorer V3.6 software
(Applied Biosystems, USA) with default parameters. Proteins were successfully
identified based on 95% or higher confidence interval of their scores in the MASCOT
V2.1 search engine (Matrix Science Ltd., London, U.K.). The following parameters
were used in the search: NCBInr Metazoa (Animals) (2861494 sequences) database;
trypsin as the digestion enzyme; one missed cleavage site; partial modifications of
cysteine carbamidomethylation and methionine oxidization; no fixed modifications;
0.15 Da for precursor ion tolerance and 0.25 Da for fragment ion tolerance. Individual
ions scores >40 indicate identity or extensive homology (p<0.05).
Statistical analysis
Principal components analysis (PCA), an unsupervised pattern recognition method,
has been proven as a powerful tool to discover the similarities and differences
between various sample groups and has been widely applied in system biology [11,
12]. In this study, PCA was conducted on the percentage volume (% vol) of common
protein spots (matched protein spots in all 2-DE gels) to compare the reproducibilities
of protein extraction methods using software PLS Toolbox (version 4.0, Eigenvector
Research, Manson, WA). The reproducibility of each protein extraction method was
determined from PC scores (Fig. 2) by multiplying standard deviation (SD) along PC1
by SD along PC2 for the replicates within each extraction method, where obviously
the smaller the value, the better the reproducibility. To facilitate the comparison of
reproducibilities of protein extraction methods, we normalized the reproducibility of
TRIzol method to 1 (Table 1). Since the protein extraction method using
straightforward lysis buffer could extract very few proteins resolved in 2-DE gels, it
was not analyzed in principal component analysis for both P. olivaceus and N.
diversicolor samples.
References
[1]
Butt, R. H., Pfeifer, T. A., Delaney, A., Grigliatti, T. A., Tetzlaff, W. G., Coorssen,
J. R., Enabling coupled quantitative genomics and proteomics analyses from rat
spinal cord samples. Mol. Cell Proteomics 2007, 6, 1574–1588.
6
[2]
Lee, F. W., Lo, S. C., The use of Trizol reagent (phenol/guanidine isothiocyanate)
for producing high quality two-dimensional gel electrophoretograms (2-DE) of
dinoflagellates. J. Microbiol. Method 2008, 73, 26–32.
[3]
Tullius, M. V., Phillips N. J., Scheffler, N. K., Samuels, N. M., Jr, R. S. M.,
Hansen, E. J., Stevens-Riley, M., Campagnari, A. A., Gibson, B. W.The lbgAB
gene cluster of Haemophilus ducreyi encodes α β-1, 4-galactosyltransferase and
an α-1,6-DD-heptosyltransferase involved in lipooligosaccharide biosynthesis.
Infect. Immun. 2002, 70, 2853–2861.
[4]
Jiang, H., Li, F., Xie, Y., Huang, B., Zhang, J., Zhang, J., Zhang, C., Li, S., Xiang,
J., Comparative proteomic profiles of the hepatopancreas in Fenneropenaeus
chinensis response to hypoxic stress. Proteomics 2009, 9, 3353–3367.
[5]
McDonagh, B., Sheehan, D., Effect of oxidative stress on protein thiols in the
blue mussel Mytilus edulis: Proteomic identification of target proteins.
Proteomics 2007, 7, 3395–3403.
[6]
Keyvanshokooh, P., Vaziri, B., Gharaei, A., Mahboudi, F., Esmaili-Sari, A.,
Shahriari-Moghadam, M., Proteome modifications of juvenile beluga (Huso
huso) brain as an effect of dietary methylmercury. Comp. Biochem. Physiol. Part
D. Genomics. Proteomics 2009, 4, 243–248.
[7]
Shrader, E., A., Henry, T. R., Greeley, M. S., Bradley, B. P., Proteomics in
Zebrafish exposed to endocrine disrupting chemicals. Ecotoxicology 2003, 12,
485–488.
[8]
Lopez, J. L., Marina, A., Vazquez, J., Alvarez, G., A proteomic approach to the
study of the marine mussels Mytilus edulis and M. galloprovincialis. Mar. Biol.
2002, 141, 217–223.
[9]
Zhu, B., Gao, K., Wang, K., Ke, C., Huang, H., Gonad differential proteins
revealed with proteomics in oyster (Saccostrea cucullata) using alga as food
contaminated with cadmium. Chemosphere 2012, 87, 397–403.
[10]
Chen, H. B., Huang, H. Q., Proteomic analysis of methyl parathion-responsive
proteins in Sparus latus liver. Fish Shellfish. Immunol.2011, 30, 800–806.
[11]
Santos, E., Ball, J. S., Williams, T. D., Wu, H., Orgeta, F., Van Aerle, R.,
Katsiadaki, I., Falciani, F., Viant, M. R., Chipman, J. K., Tyler, C. R., Identifying
health impacts of exposure to copper using transcriptomics and metabolomics in a
fish model. Environ. Sci. Technol. 2010, 44, 820–826.
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[12]
Wu, H., Southam, A. D., Hines, A., Viant, M. R., High throughput tissue
extraction protocol for NMR and Mass spectrometry based metabolomics. Anal.
Biochem. 2008, 372, 204–212.
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Supplementary Table 1. Comparison of protein yields, total numbers of protein spots in 2-DE gels, numbers of matched protein spots in 4
replicate 2-DE gels and reproducibilities among protein extraction methods
Gill of M. galloprovincialis
Liver of P. olivaceus
Whole soft tissue of N. diversicolor
Extraction method
TRIzol
TCA-A
Lysis
TRIzol
TCA-A
Lysis
TRIzol
TCA-A
Lysis
Protein yield (mg/g fresh weight)
13.3 ± 2.3
30.5 ± 2.0
32.6 ± 1.5
7.8 ± 0.4
24.4 ± 1.1
47.8 ± 2.8
8.6 ± 0.8
25.3 ± 1.3
30.7 ± 1.2
Number of protein spots
1023 ± 65
979 ± 84
936 ± 85
982 ± 102
1102 ± 80
1199 ± 195
878 ± 113
1018 ± 95
340 ± 14
Number of matched protein spots
748
587
519
601
406
285
427
712
235
1.00
0.50
0.78
1.00
0.05
NE
1.00
2.81
NE
in 4 replicate 2-DE gels
Reproducibilitya
a
The reproducibility of each protein extraction method was determined from PC scores (Fig. 2) by multiplying standard deviation (SD) along PC1 by SD along
PC2 for the replicates within each extraction method. To facilitate the comparison of reproducibilities of protein extraction methods, we normalized the
reproducibility of TRIzol method to 1. NE: Not Evaluated.
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Supplementary Table 2. Selected protein spots from the gill of M. galloprovincialis identified by MALDI-TOF/TOF MS.
Spot
number
1
Protein name
Species
Accession number
MW/kDa
PI
5.24
Protein
score
348
Sequence
coverage
15%
40S ribosomal protein SA
Pinctada fucata
gi|229891605
33499
2
sulfotransferase family
Mytilus galloprovincialis
FL594282
3
Col protein
Mytilus galloprovincialis
4
5
signal sequence receptor betalike protein
apextrin-like protein
6
24486
6.07
128
11%
EH662975
26429
8.81
96
20%
Mytilus galloprovincialis
FL492377
24604
8.76
110
9%
Mytilus galloprovincialis
EH662598
33866
7.57
351
22%
heat shock protein 40A
Ruditapes philippinarum
gi|256549334
35715
8.24
99
4%
7
USP-like protein isoform 2
Mytilus galloprovincialis
FL490498
21388
7.85
72
7%
SGGLDVLAL
FTPGTFTNQIQAAFR
SGGLDVLALKEEDITK
FTPGTFTNQIQAAFREPR
HLSWEEFFEK
NWFTVAQNEIFDR
QNNNIDHR
YQLGVLDEFKK
GREPTWAPDYLDK
GGGANFLCLPKDPEWR
SRYDQPKPK
NILNQYLVEGR
TSYSGSGSWPR
SGVLPEGVYDR
SGCPSGWAEGWR
SHHFFGTFNSNTK
YQDNEDSNNINSVTPSR
AVYDQFGEEGLK
RAVYDQFGEEGLK
CAAE LNAAIIITGCR
8
tubulin alpha-8 chain
Mus musculus
gi|8394493
50704
4.97
83
3%
AVMVDLEPTVVDEVR
9
septin-6
Mytilus galloprovincialis
FL500031
24824
6.27
71
4%
10
voltage-dependent
anion
channel 2-like protein
myosin essential light chain
Mytilus californianus
ES397798
38194
9.46
96
6%
SLHNFHDTR
HPSCLYFIAPTGHSLK
VSDDLEAGVSLNWAAGSNATR
Mytilus galloprovincialis
FL593768
22202
4.51
85
14%
12
short-chain collagen C4-like,
partial
Mytilus galloprovincialis
FL497051
22255
5.55
197
16%
13
Hsp22
Mytilus californianus
ES737901.1
25707
5.94
160
11%
14
Sb:cb283 protein, partial
Mytilus californianus
ES400183
31048
7.01
65
13%
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Peptide sequence
DCATYEDFVEGLR
ISFEVFLPILHTVSK
QNNNIDHR
YQLGVLDEFKK
GREPTWAPDYLDK
SPWYNEDYVR
NDNFFVKPFESQFR
DYDDVRR
10
15
Histone H3.3
Ruditapes philippinarum
gi|164587973
23376
11.2
9
51
24%
LIFSPTGPLNR
KPLFVRPVDDNFVGFAGK
STELLIR
RVTIMPK
YRPGTVALR
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