**2.5 The preliminary experiment of SILAC-labeled protein samples**

The "heavy"- and "light"-SILAC-labeled proteins were mixed and loaded onto 1X SDS-PAGE to check the quality. SDS-PAGE-separated proteins were further analyzed with MS/MS as a preliminary experiment to check the labeling efficiency. (i) The loading sample preparation: According to the 1:1 ratio, the "heavy"- and "light"-SILAC-labeled proteins were mixed in a 5X loading buffer. (ii) Electrophoresis: The mixed SILAClabeled proteins were loaded onto SDS-PAGE gel (gel concentration: 12.5%) with the

*The Use of Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) to Study… DOI: http://dx.doi.org/10.5772/intechopen.102092*

amount of 20 μg/lane by constant current (14 mA, 90 min). (iii) Coomassie brilliant blue staining: Prepare Coomassie brilliant blue stain and destain solutions. Filter the stain solution through Whatman 1 filter paper. (iv) MS/MS: Proteins were separated from SDS-PAGE bands, and then were reduced, alkylated, and trypsin-digested. The tryptic peptides were analyzed with MS/MS.

#### **2.6 Trypsin-digestion of SILAC-labeled proteins**

The main reagents and methods included: (i) Reducing agent: 100 mM DTT was added to SILAC-labeled protein sample. (ii) Uranyl acetate (UA) buffer: The UA buffer contained 8 M urea and 0.1 M Tris/HCL. The SILAC-labeled protein sample with DTT was filtered by a 10-kD ultrafiltration centrifuge tube for two times. (iii) Isolation mixture reacted: A total of 100 μL of 0.05 M iodoacetamide was added to the isolation mixture following centrifugalization (14,000 × g, 15 min). A total of 25 mM ammonium bicarbonate (NH4HCO3) was added to the mixture following centrifugalization (14,000 × g, 15 min). (iv) Trypsin buffer: 2 μg trypsin in 40 μL 100-mM NH4HCO3. (v) Tryptic peptide content: A volume of trypsin buffer (40 μL) was added to the mixture from last step and shaken by 600 rpm for 1 min. Enzymatic hydrolysis of the mixture was done for 16–18 h at 37°C. A volume (40 μL) of 25 mM NH4HCO3 was added to the mixture and that mixture was centrifuged (14,000 × g, 15 min); the filtrate was collected.

### **2.7 LC-MS/MS analysis**

The instrument and materials are as follows: (i) MS instrument, for example, Q Exactive mass spectrometer (Thermo Fisher Scientific); (ii) Easy nLC system, for example, Proxeon Biosystems (Thermo Fisher Scientific); (iii) Thermo scientific EASY column: Acclaim PepMap, 100 μm × 2 cm, nanoViper, 5 μm-C18; (iv) analytical column: Thermo scientific EASY column (75 μm \* 100 mm 3 μm-C18); (v) solvent A: 0.1% formic acid in H2O; and (vi) solvent B: 0.1% formic acid, 84% acetonitrile in H2O.

#### **2.8 Search protein database with MaxQuant software**

The main parameters are following [20]: Main search ppm: 6; missed cleavage: 2; MS/MS tolerance ppm: 20; de-isotopic: TRUE; enzyme: trypsin/P; database: uniprot\_Homo\_sapiens\_169753\_20190313; fixed modification: carbamidomethyl (C); lables: Lys(8), Arg(10); variable modification: oxidation (M), acetyl (protein N-term) decoy database; pattern: reverse; peptide FDR: 0.01; and protein FDR: 0.01.

#### **2.9 Bioinformatics analysis**

Several bioinformatics analyses were used, which are as follows: (i) The enrichment of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway was performed with R package clusterProfiler-KEGG (https://bioconductor.org/packages/release/ bioc/html/clusterProfiler.html). (ii) The enrichment of biological processes (BPs) was analyzed with Cytoscape ClueGO. (iii) The level of statistical significance was set as p < 0.05 and adjusted p value < 0.05. For KEGG and BPs enrichment analyses, a Benjamini-Hochberg multiple text was used to adjust p value.
