Electronic Supplementary Material 1
Title: Protein-protein interaction network of the marine microalga Tetraselmis subcordiformis:
prediction and application for starch metabolism analysis
Journal: Journal of Industrial Microbiology & Biotechnology
Authors: Chaofan Ji , Xupeng Cao, Changhong Yao, Song Xue*, Zhilong Xiu*
The first two authors contributed equally to this paper.
* Corresponding authors:
Song Xue: Marine Bioproducts Engineering Group, Dalian Institute of Chemical Physics, Chinese
Academy of Sciences, Dalian 116023, China. Tel./fax: +86 411 84379069, e-mail address:
xuesong@dicp.ac.cn
Zhilong xiu: School of Life Sciences and Biotechnology, Dalian University of Technology, Dalian
116023, China. Tel./fax: +86 411 84706369, e-mail address: zhlxiu@dlut.edu.cn
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1. Protein extraction
The algal cells (2-4×107) were lysed ultrasonically (400W, 5s, 150 times on ice) in 1 ml lysis
buffer [8M Urea, 150 mM NaCl, 1 mM EDTA, 50 mM Tris (pH 7.4), 65 mM DTT, 1 mM EDTA,
1 mM EGTA, 1 mM PMSF, cocktail inhibitor]. Lysates were centrifuged at 13,000 g at 4 oC for 15
min. The sample proteins within the supernatant were precipitated by 5 mL acidic acetone
(acetone: ethanol: acetic acid=50:50:1) at -20oC overnight. Then the pellets were resuspended in 1
mL denaturing buffer containing 50 mM Tris-HCl (pH 7.4) and 8 M urea and the protein
concentration was determined by Bradford assay.
2. Proteomic analysis processes
2.1 Sample Digestion and Dimethyl Labeling Reaction
Each of 2 mg protein samples was reduced by DTT at 37oC for 2 h and alkylated by
iodoacetamide in dark at room temperature for 40 min. Then, the sample solution was diluted to 2
M urea by 50 mM Tris-HCl (pH 8.2), and lysine C was added with weight ratio of enzyme to
protein at 1/50, incubating at 37oC for 20 h.
Every protein digest was loaded onto a homemade C18 solid phase extraction (SPE) column
and labeled by 5 mL of light (0.2% CH2O and 30 mM NaBH3CN), medium (0.2% CD2O and 30
mM NaBH3CN), or heavy (0.2% 13CD2O and 30 mM NaBD3CN) dimethylation reagents [1].
After a wash with 1 mL 0.1% trifluoroacetic acid (TFA) aqueous solution, the isotopic labeled
peptides were eluted from the C18 SPE column using 1 mL 80% ACN containing 0.1% TFA.
Then, the comparative samples were mixed together with the ratio of
CON/EXP1/EXP2=1:1:1(v/v/v) for group one and CON/EXP3 = 1:1 (v/v) for group two, then
kept at -20oC.
2.2 2D nano LC–MS/MS analysis
The 2D nano LC–MS/MS system is composed of the Surveyor HPLC system and the
LTQ-Orbitrap mass spectrometer equipped with a nano-spray source (Thermo, San Jose, CA,
USA). The 2D nano LC–MS/MS analysis was carried out according to a published procedure [4],
with a few modifications. The labeled peptides were loaded onto the SCX trap column (150 μm ×
7 cm). After equilibrated with 5% acetonitrile (0.1% acetic acid), the trap column was directly
connected to a reverse-phase (RP) column in tandem. The peptides were eluted onto the RP
column using an ammonium acetate step gradient (pH 2.7), which concentration was 50, 100, 150,
200, 250, 300, 350, 400, 500 and 1000 mM, respectively. After each elution step, a subsequent
RPLC–MS/MS was executed. The RP buffer solution was 0.1% formic acid aqueous solution
(buffer A) and ACN with 0.1% formic acid (buffer B); one separation cycle lasted 120 min (from
0% to 10% buffer B for 2 min, 10% to 35% buffer B for 90 min, 35% to 80% buffer B for 5 min,
80% buffer B for 10 min, after which the whole system was re-equilibrated by buffer A for 10
min). MS parameter was set as follows: the temperature of the ion transfer capillary was set to 200
oC and the spray voltage was 1.8 kV. One full-range mass-scan was followed by the collection of
10 data-dependent tandem mass spectra.
2.3 Data analysis
All acquired raw files were converted to *.mgf files using DTASupercharge (v2.0a7). Then the
*.mgf files were searched against the transcriptome database of T. subcordiformis, obtained from
GenBank (http://www.ncbi.nlm.nih.gov/genbank/, accession number: GANN01000000). The false
discovery rate (FDR) was evaluated by Mascot Version 2.1 (Matrix Science) in decoy searching
mode. Cysteine residues were searched as static modification of +57.0215 Da, methionine
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residues as variable modification of +15.9949 Da. The isotope labeled peptides amino termini and
lysine residues were set as variable modification as Dimethyl: +28.0313 Da, Dimethyl:2H(4):
+32.0564 Da, and Dimethyl:2H(6)13C(2): +36.0757 Da for both K and N-term. Peptides were
searched using fully tryptic cleavage constraints and up to two missed cleavages sites were
allowed for tryptic digestion. The mass tolerances were 10 ppm for parent masses and 0.8 Da for
fragment masses. In this work the identified peptides with Mascot score≥25 (rank 1, P-value≤0.05)
were reserved for quantitative analysis. Protein quantification was performed using a
dimethyl-adapted version of MSQuant (v2.0a81). Peptide ratios were obtained by calculating the
extracted ion chromatograms (XIC) of the light, medium and heavy forms of the peptide using the
monoisotopic peaks only and protein ratios were calculated from the average of the all quantified
peptides. Then, all the MSQuant outputs of the same online multidimensional separation were
imported in StatQuant (v1.2.2) and the quantified proteins were normalized against the log2 of the
median ratio of all peptides quantified [3,2].
1. Boersema PJ, Raijmakers R, Lemeer S, Mohammed S, Heck AJR (2009) Multiplex peptide stable
isotope dimethyl labeling for quantitative proteomics. Nat Protocols 4 (4):484-494.
doi:10.1038/nprot.2009.21
2. Lemeer S, Jopling C, Gouw J, Mohammed S, Heck AJR, Slijper M, den Hertog J (2008)
Comparative phosphoproteomics of zebrafish Fyn/Yes morpholino knockdown embryos. Mol
Cell Proteomics 7:2176-2187. doi:10.1074/mcp.M800081-MCP200
3. van Breukelen B, van den Toorn HWP, Drugan MM, Heck AJR (2009) StatQuant: a
post-quantification analysis toolbox for improving quantitative mass spectrometry.
Bioinformatics 25:1472-1473. doi:10.1093/bioinformatics/btp181
4. Wang F, Dong J, Jiang X, Ye M, Zou H (2007) Capillary trap column with strong cation-exchange
monolith for automated shotgun proteome analysis. Analytical Chemistry 79:6599-6606.
doi:10.1021/ac070736f
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