A new insight into the impact of different proteases on
SILAC quantitative proteome of the mouse liver
Supplementary Materials and Methods
Mouse Liver samples
All mice used in these studies were 10 weeks male C57Bl/6, five to six mice
were treated in each experiment, all of the animal experiments were approved by the
institutional animal care and use committee and conformed to relevant guidelines and
laws. The mice were fasted for about 16 h before were sacrificed. All procedures were
conducted as previous described [1]. The 13C6-lysine labeled mouse liver is provided
by Silantes.
Digestion
Liver Tissues are disrupted by techniques such as grinding in a liquid
nitrogen-cooled mortar, sonication, shearing, or homogenization. Proteins are then
solubilized with repeated sonication in SDS sample buffer (1% (w/v), 100 mM
Tris-HCl (pH 9.5). After protein solubilization, samples were left at room temperature
for 5 minutes，prior to centrifugation in a table top eppendorf centrifuge at 14,000 rpm
and 4°C. The supernatant was carefully collected and the pellet resuspended, once
more, in an equal amount of lysis buffer. After spinning, the second supernatant was
collected and the pellet was resuspended again in an equal volume. Sonication was
used to increase protein solubility and the third supernatant was combined with the
first two. Bradford analysis was performed to establish protein concentrations.
Samples were aliquoted and stored at -80°C until further use.
For normal and SILAC labeled mouse liver, 30 μg protein from each were mixed
with an identical amount of the corresponding SILAC labeled standard. Reduction of
disulfide bridges was achieved by addition of DTT to a final concentration of 0.1 M
followed by incubation at 75 °C for 5 min. Protein was separated using 5–12%
SDS-PAGE. Dice each gel slice into small pieces (1mm2) and place into 0.65 mL
siliconized tube. Add ~100μL (or enough to cover) of 25mM NH4HCO3/50% ACN
and vortex for 10 min. Using gel loading pipet tip, remove the supernatant and discard.
Speed Vac the gel pieces completely dry (~ 20 min). Add 25 μL (or enough to cover)
10 mM DTT in 25 mM NH4HCO3 to dried gels. Allow reaction to proceed at 56°C for
30 min. Discard the supernatant, add 25 μl 55 mM iodoacetamide to the gel pieces.
Allow reaction to proceed in the dark for 45 min at room temperature. Discard
supernatant. Wash gels with ~100 μl NH4HCO3 vortex 10 min, and spin. Discard
supernatant. Dehydrate gels with ~100μL (or enough to cover) of 25 mM NH4HCO3
in 50% ACN, vortex 5 min, spin. Repeat one time [2-4].
1. Trypsin digestion
Dehydrate gels with ~100μL (or enough to cover) of 25 mM NH4HCO3 during
digest. 10 μg trypsin (Promega) was added to the protein sample (protein to trypsin
ratio = 50:1) and digestion was carried out at 37°C 15 hours. Digestion was stopped
by addition of 1% formic acid.
2. LysC digestion
Dehydrate gels with ~100μL (or enough to cover) of 25 mM NH4HCO3 during
digest. 5 μg LysC (Wako, Richmond, VA) was added to the protein sample (protein to
LysC ratio = 20:1) and digestion was carried out at 37°C 15 hours. Digestion was
stopped by addition of 1% formic acid.
3. Tandem LysC/trypsin digestion
Dehydrate gels with ~100μL (or enough to cover) of 25 mM NH4HCO3 during
digest. 5 μg LysC (Wako, Richmond, VA) was added to the protein sample, proteins
were digested for three hours with LysC at an enzyme/total protein ratio of 1:20 (w/w).
Proteins were then diluted 4 fold in water, and digested with trypsin at the same
enzyme/total protein ratio.and digestion was carried out at 37°C 15 hours. Digestion
was stopped by addition of 1% formic acid.
Organic solvent was removed in a SpeedVac concentrator. Obtained peptides
were acidified with trifluoroacetic acid. Peptide mixtures were measured both directly
and after fractionation into nine fractions via strong anion exchange chromatography.
Mass spectrometry
Samples were dissolved with loading buffer and then separated by a C18 column
(75 μm inner-diameter, 360 μm outer-diameter × 10 cm, 3 μm C18). Peptides were
separated using a linear gradient from 97% solvent A (0.1% formic acid in water
solution) 3% solvent B (0.1% formic acid in acetonitrile solution) to 28% solvent B
over 60 min at a flow rate of 350 nL/min was applied. The source was operated at 1.8
kV, with no sheath gas flow and with the ion transfer tube at 350 °C. The mass
spectrometer was programmed to acquire in a data dependent mode. The survey scan
was from m/z 375 to 1300 with resolution 60,000 at m/z 400. The 50 most intense
peaks with charge state 2 and above and intensity threshold of 500 were selected for
sequencing and fragmented in the ion trap by collision induced dissociation with
normalized collision energy of 35%, activation time of 5 ms and one microscan.
Protein identification and quantification
All raw files were converted to mzXML and MGF files using the Msconvert
module [5] in Trans-Proteomic Pipeline (TPP v4.5.2) [6] The MS/MS peak lists were
searched using the local Mascot v2.3.2 server [7] against the database containing
sequences of all mouse proteins from Refseq database (30,103 protein entries,
ftp.ncbi.nih.gov/refseq/M_musculus/) and common contaminant protein sequences
(115 proteins, ftp.thegpm.org/fasta/cRAP). The Decoy checkbox were chosen to
perform an automatic decoy database search by Mascot. The monoisotopic mass was
used for both peptide and fragment ions, taking Carbamidomethylation on cysteine as
fixed modification and
13
C6-Lysine and Oxidation on methionine as variable
modifications. Specific cleavages were selected for Trypsin and LysC precedence
trypsin (Lys and Arg) as well as for LysC (Lys), and up to 2 missed cleavage sites
were allowed. The precursor and fragment ion mass tolerance is 20 ppm and 0.5 Da
for this hybrid linear trap high-accuracy data. PepDistiller [8] was used for quality
control to facilitate the validation of Mascot search results. Peptides length shorter
than seven amino acids were removed and all peptide-spectra matches were filtered
by the probability value of PepDistiller to keep the FDR measured by the decoy hits
lower than 1%.
Using the in-house developed software, a normalized quantification method
based on the extracted ion chromatograms (XICs) was applied to all confidently
identified peptides and proteins [9, 10]. In brief, the extracted ion chromatograms
(XICs) of the highly confident peptides after quality control were constructed utilizing
the mono-isotope peak intensity. The goodness of least squares fitting between the
experimental isotope pattern and its corresponding theoretical pattern was computed
as a cutoff during the XIC construction. What's more, the signal-to-noise (S/N) ratio
of the XIC was calculated as the peptide-level cutoff. Then, the XIC area was
determined and the peptide ratio was defined as the ratio of the non-labeled XIC area
and its corresponding SILAC-labeled XIC area. And for protein quantification, the
weighted average ratio of all unique peptides for each protein was computed as the
protein ratio.
Reference
[1] Liu, W., Hou, Y., Chen, H., Wei, H., et al., Sample preparation method for
isolation of single-cell types from mouse liver for proteomic studies. Proteomics 2011,
11, 3556-3564.
[2] Michalski, A., Damoc, E., Hauschild, J. P., Lange, O., et al., Mass
spectrometry-based proteomics using Q Exactive, a high-performance benchtop
quadrupole Orbitrap mass spectrometer. Mol. Cell. Proteomics 2011, 10,
M111.011015.
[3] Geiger, T., Madden, S. F., Gallagher, W. M., Cox, J., Mann, M., Proteomic
portrait of human breast cancer progression identifies novel prognostic markers.
Cancer Res. 2012, 72, 2428-2439.
[4] Michalski, A., Damoc, E., Lange, O., Denisov, E., et al., Ultra high resolution
linear ion trap Orbitrap mass spectrometer (Orbitrap Elite) facilitates top down LC
MS/MS and versatile peptide fragmentation modes. Mol. Cell. Proteomics 2012, 11,
O111.013698.
[5] Kessner, D., Chambers, M., Burke, R., Agus, D., Mallick, P., ProteoWizard:
open source software for rapid proteomics tools development. Bioinformatics 2008,
24, 2534-2536.
[6] Deutsch, E. W., Mendoza, L., Shteynberg, D., Farrah, T., et al., A guided tour
of the Trans - Proteomic Pipeline. Proteomics 2010, 10, 1150-1159.
[7] Perkins, D. N., Pappin, D. J., Creasy, D. M., Cottrell, J. S., Probability-based
protein identification by searching sequence databases using mass spectrometry data.
Electrophoresis 1999, 20, 3551-3567.
[8] Li, N., Wu, S., Zhang, C., Chang, C., et al., PepDistiller: A quality control
tool to improve the sensitivity and accuracy of peptide identifications in shotgun
proteomics. Proteomics 2012, 12, 1720-1725.
[9] Han, D. K., Eng, J., Zhou, H., Aebersold, R., Quantitative profiling of
differentiation-induced microsomal proteins using isotope-coded affinity tags and
mass spectrometry. Nat. Biotechnol. 2001, 19, 946-951.
[10] Park, S. K., Venable, J. D., Xu, T., Yates, J. R., A quantitative analysis
software tool for mass spectrometry-based proteomics. Nat. Method 2008, 5, 319-322.