Department of Molecular Pharmacology
National Cerebral and Cardiovascular Center Research Institute
Methods for proteome analysis of aortic aneurysmal tissue
I. Sample preparation and liquid chromatography-tandem mass spectrometric analysis
Instruments, softwares, and materials
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AB SCIEX Triple TOF 5600 System (AB SCIEX, Massachusetts, USA)
Nano-Frontier nLC (Hitachi High Technologies, Tokyo, Japan)
Monolith Trap column C18-50-150 (Hitachi High Technologies, Tokyo, Japan)
MonoCap for Fast-flow analytical column (5020-10101, GL Sciences, Tokyo, Japan）
Shake Master Neo Ver.1.0 (BMS-M10N21, BMS, Tokyo, Japan)
Savant SpeedVac (SPD111V, Thermo Scientific, Yokohama, Japan)
Lab Staff centrifuge (C1301, Nikkyo Technos, Tokyo, Japan)
Himac Compact Centrifuge (CF16RXII, Hitachi, Tokyo, Japan)
SPE C-TIP (NTCR-KT200-C18, Nikkyo Technos, Tokyo, Japan)
Lysyl endopeptidase (125-05061, Wako Pure Industries, Osaka, Japan)
Trypsin (V5111, Promega, Wisconsin, USA)
Qubit 2.0 Fluorometer (Q32866, Life Technologies, California, USA)
Mascot Ver. 2.4 (Matrix Science, London, UK) (http://www.matrixscience.com)
2DICAL2 Ver. 1.3.16 (Mitsui Knowledge Industry, Tokyo, Japan)
Procedures
1. Trypsin digestion of aortic medial tissue
1.1. Aortic medial tissue (~2 mg) was pulverized by Shake Master Neo, and suspended in
methanol.
1.2. To the pulverized tissue (~2 mg wet weight), sodium deoxycholate solution (SDC, 2%,
25 μL) was added, and the resulting suspension was heated at 95°C for 10 min.
1.3. Urea (5 M, 10 μL), ammonium hydrogen carbonate (NH4HCO3, 1M, 2.5 μL), and
deionized water (3.9 μL) were added and vortexed.
1.4. The suspension was digested with lysyl endopeptidase (1 μg) at 37°C for 3 h, and
then with trypsin (3.3 μg) at 37°C for 17-19 h.
1.5. To the digests (50 μL), 10 μL of 5% formic acid were added to precipitate the SDC.
1.6. After centrifugation, the resulting supernatant was extracted with ethyl acetate (60
μL), and the water layer was evaporated to dryness by a SpeedVac concentrator.
2. Peptide enrichment and desalting
2.1. SPE C-Tip were washed with methanol (50 μL), a washing buffer (30 μL, 80%
acetonitrile in 0.1% trifluoroacetic acid (TFA)), and twice with a loading buffer (20 μL,
2% acetonitrile in 0.1% TFA) by flowing them through by centrifugation with a Lab
Staff centrifuge.
2.2. The tryptic digest solution (11 μL in the loading buffer) was loaded onto the C-Tip
and centrifuged at 3,000 rpm for 2 min with a Himac compact centrifuge RXII.
2.3. The passed through fraction was re-loaded on the C-Tip and centrifuged, and the tip
was washed twice with the loading buffer (30 μL) by centrifugation.
2.4. Adsorbed peptides were eluted with an elution buffer (40 μL, 40% acetonitrile in 0.1%
TFA) by centrifugation and dried in a concentrator.
2.5. The tryptic peptides were dissolved in 20 μL of 0.1% formic acid, and the
concentration was determined by a Qubit fluorometer.
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Department of Molecular Pharmacology
National Cerebral and Cardiovascular Center Research Institute
3. LC-MS/MS analysis and peptide identification
3.1. Peptide identification was performed with the liquid chromatography coupled with
tandem mass spectrometric analysis (LC-MS/MS) by the aid of Mascot software.
Quantitative estimation of peptides was carried out using 2DICAL2 software.
3.2. The tryptic peptides (300 ng of peptides), prepared by Procedure 2, were separated by
LC using a linear gradient elution of 2% to 11.6% of acetonitrile for 5 min, then 11.6%
to 45.2% of acetonitrile for 60 min. Ionized peptides and its fragment ions generated
by the collision-induced dissociation were analyzed by a Triple-TOF 5600 mass
spectrometer.
3.3. Peptides were identified by MS/MS spectra using the Mascot software, and their
quantitative analysis was carried out using the 2DICAL2 software[1].
Data quality control and functional assessment of the liquid chromatography and mass
spectrometer
LC-MS/MS analysis was performed by the Trap-and-Elute method. Calibration of a
Triple-TOF 5600 mass spectrometer was performed every 10 samples using
trypsin-digested bovine serum albumin MS standard (TD-BSA-S). To confirm the
retention times and peak intensities, selected TD-BSA-S peptides were monitored.
Peak intensity of each peptide was determined as an average of duplicate
measurements.
II. Protein quantitation in the proteome analysis and its quality control
1. Criteria for identification of trypsin-digested peptides
1.1. The parameter sets for MS/MS ion search were shown below;
Database: SwissProt_2014.fasta
Enzyme: Trypsin
Miss cleavages allowed: Up to one
Variable modification: Methionine oxidation
Peptide tolerance: 20 ppm
MS/MS tolerance: 0.05 Da
1.2. The peptides identified with an expectation value p<0.05 were considered as reliable.
1.3. If multiple peptides with high sequence similarity were selected for the same peptide
peak, the peptide sequence is not determined unambiguously and is excluded from
the identified peptide list.
1.4. PeptideProphet analysis[2] was also performed and the results were used to select
peptide peaks for the protein quantification, as described in 3.2.
2. Quantitation of tryptic peptides by the non-labeling method
2.1. MS peak intensities did not always correlate with absolute amounts of peptides.
However, if samples of the same tissue were processed by the fixed method, the
peptide peak intensity ratio of the same peptide is expected to correlate with the
relative abundance of the peptides. This is the principle of the non-labeling
quantitative proteomic analysis method.
2.2. In the 2DICAL software, the MS peaks of the same peptides were adjusted by the
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Department of Molecular Pharmacology
National Cerebral and Cardiovascular Center Research Institute
specific program and superimposed, and the average peak intensity was calculated
and compared between the sample groups.
2.3. If the same peptide was identified as various charged forms (2+, 3+, 4+), the highest
peak is selected for the quantitative analysis.
3. Selection method of tryptic peptide peaks for protein quantification
3.1. The protein levels in the target tissue were evaluated by using multiple peptide peak
data from each protein.
3.2. The peptide peak data are filtered by the following rule;
a. If the weight value in PeptideProphet analysis was less than 0.5, the peptide is
excluded.
b. If multiple peptides showed nearly equal values in both m/z and retention time
(within 0.05Da and 0.6 min each), these peptides are excluded except the value of
number of sibling peptides (NSP)-adjusted probability in the PeptideProphet
analysis was more than 0.8.
c. The peptides including methionine are excluded because methionine residue is
easily oxidized during the sample preparation.
d. Peptides generated by miss cleavages of trypsin/lysyl endopeptidase are excluded.
e. Peptides with the low peak intensity (mean intensity of the group is less than 500)
are excluded.
f. Peptides of which peaks are detected in less than 90 % of samples are excluded.
4.
Protein quantification and statistical analysis
Protein quantification is performed by the Top3 method (established by Drs. Ono and
Yamada of National Cancer Center Research Institute), and unpaired t-test is used
for detecting statistical significance between groups.
References:
[1] Ono, M. et al. Label-free quantitative proteomics using large peptide data sets
generated by nanoflow liquid chromatography and mass spectrometry. Mol. Cell.
Proteomics. 5, 1338-1347 (2006)
[2] Keller, A. et al. Empirical statistical model to estimate the accuracy of peptide
identifications made by MS/MS and database search. Anal. Chem. 74, 5383-5392
(2002)
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