Tissue sample
Cancer tissues and paracancerous tissues of 60 patients with HCC, including48 male cases and 12 female cases, were from Changhai Hospital, the affiliated hospital of Naval Medical University in Shanghai. (Table 1)
Table 1
Patients Information and the Protein Expression
|
Male(n)
|
Female(n)
|
p
|
Age
|
|
|
|
≤ 40
|
6
|
2
|
|
41–60
|
28
|
3
|
0.29
|
≥ 60
|
14
|
7
|
|
Differentiation
|
|
|
|
High
|
4
|
0
|
|
Middle
|
41
|
11
|
0.385
|
Low
|
3
|
1
|
|
Size
|
|
|
|
≤ 3cm
|
8
|
2
|
|
3-6cm
|
15
|
7
|
0.156
|
≥ 6cm
|
25
|
3
|
|
Stage
|
|
|
|
Ⅰ/Ⅱ
|
25
|
8
|
0.081
|
Ⅲ/Ⅳ
|
23
|
4
|
|
Lymph node metastasis
|
|
|
|
Yes
|
3
|
1
|
0.795
|
None
|
45
|
11
|
|
Hepatitis B history
|
15
|
3
|
0.024
|
Smoke history
|
20
|
0
|
0.006
|
CYP8B1
|
8
|
3
|
0.073
|
MTHFD2
|
36
|
5
|
0.034
|
NNMT
|
27
|
4
|
0.085
|
SHMT2
|
37
|
2
|
0.167
|
SLC27A5
|
10
|
3
|
0.102
|
Protein preparation
Hepatocellular carcinoma cells from 30 patients, 60 samples totally, were suspended in lysis buffer (7 M Urea, 2 M Thiourea, 4% CHAPS, 40 mM Tris-HCL, pH 8.5, 1mM PMSF, 2mM EDTA) and sonicated in ice. The proteins were reduced with 10 mM DTT (final concentration) at 56°C for 1 hour and then alkylated by 55 mM IAM (final concentration) in the darkroom for 1 hour. The reduced and alkylated protein mixtures were precipitated by adding 4 volume times of chilled acetone at -20°C overnight. After centrifugation at 4°C, 30 000g, the pellet was dissolved in 0.5 M TEAB (Applied Biosystems, Milan, Italy) and sonicated in ice. After centrifuging at 30,000g at 4°C, an aliquot of the supernatant was taken for determination of protein concentration by Bradford [20]. The proteins in the supernatant were kept at -80°C for further analysis.
iTRAQ Labeling and SCX fractionation
Total protein (100µg) was taken out of each sample solution and then the protein was digested with Trypsin Gold (Promega, Madison, WI, USA) with the ratio of protein: trypsin = 30:1 at 37°C for 16 hours. After trypsin digestion, peptides were dried by vacuum centrifugation. peptides were reconstituted in 0.5M TEAB and processed according to the manufacture’s protocol for 8-plex iTRAQ reagent (Applied Biosystems). Briefly, one unit of iTRAQ reagent was thawed and reconstituted in 24 µL isopropanol. Samples were labeled with the iTRAQ tags as follow format. The peptides were labeled with the isobaric tags, incubated at room temperature for 2h. The labeled peptide mixtures 28/30were then pooled and dried by vacuum centrifugation. SCX chromatography was performed with a LC-20AB HPLC Pump system (Shimadzu, Kyoto, Japan). The iTRAQ labeled peptide mixtures were reconstituted with 4 mL buffer A (25 mM NaH2PO4 in 25% ACN, pH 2.7) and loaded onto a 4.6×250 mm Ultremex SCX column containing 5-µm particles (Phenomenex). The peptides were eluted at a flow rate of 1mL/min with a gradient of buffer A for 10 min, 5–60% buffer B (25mM NaH2PO4, 1M KCl in 25% ACN, pH 2.7) for 27 min, 60–100% buffer B for 1 min. The system was then maintained at 100% buffer B for 1 min before equilibrating with buffer A for 10 min prior to the next injection. Elution was monitored by measuring the absorbance at 214 nm, and fractions were collected every 1 min. The eluted peptides were pooled into 20 fractions, desalted with a StrataXC18 column (Phenomenex) and vacuum dried.
LC-ESI-MS/MS analysis based on Q EXACTIVE
Each fraction was resuspended in buffer A (2% ACN, 0.1% FA) and centrifuged at 20000g for 10 min, the final concentration of peptide was about 0.5 µg/µl on average. 10µl supernatant was loaded on a LC-20AD nano-HPLC (Shimadzu, Kyoto, Japan) by the autosampler onto a 2cm C18 trap column. Then, the peptides were eluted onto a 10cm analytical C18 column (inner diameter 75 µm) packed in- house. The samples were loaded at 8 µL/min for 4min, then the 44min gradient was run at 300 nL/min starting from 2 to 35% B (98%ACN, 0.1%FA), followed by 2 min linear gradient to 80%, and maintenance at 80% B for 4 min, and finally return to 5% in 1 min. The peptides were subjected to nano-electrospray ionization followed by tandem mass spectrometry (MS/MS) in an Q EXACTIVE (Thermo Fisher Scientific, San Jose, CA) coupled online to the HPLC. Intact peptides were detected in the Orbitrap at a resolution of 70 000. Peptides were selected for MS/MS using high-energy collision dissociation (HCD) operating mode with a normalized collision energy setting of 27.0; ion fragments were detected in the Orbitrap at a resolution of 17500. A data-dependent procedure that alternated between one MS scan followed by 15 MS/MS scans was applied for the 15 most abundant precursor ions above a threshold ion count of 20000 in the MS survey scan with a following dynamic exclusion duration of 15 s. The electrospray voltage applied was 1.6 kV. Automatic gain control (AGC) was used to optimize the spectra generated by the orbitrap. The AGC target for full MS was 3e6 and 1e5 for MS2. For MS scans, the m/z scan range was 350 to 2000 Da. For MS2 scans, the m/z scan range was 100–1800.
Data Analysis
The original data files acquired from the Orbitrap were converted into MGF files using Proteome Discoverer 1.2 (PD 1.2, Thermo), [5600 ms converter] and the MGF file were searched. Proteins identification were performed by using Mascot search engine (Matrix Science, London, UK; version 2.3.02) against database containing 200,000 sequences. For protein identification, a mass tolerance of 0.05Da (10 ppm) was permitted for intact peptide masses and 0.01Da for fragmented ions, with allowance for one missed cleavage in the trypsin digests. Gln->pyro-Glu (N-term Q), Oxidation (M), Deamidated (NQ) as the potential variable modifications, and Carbamidomethyl (C), iTRAQ8plex (N-term), iTRAQ8plex (K) as fixed modifications. The charge states of peptide were set to + 2 and + 3. Specifically, an automatic decoy database search was performed in Mascot by choosing the decoy checkbox in which a random sequence of database is generated and tested for raw spectra as well as the real database. To reduce the probability of false peptide identification, only peptide at the 95% confidence interval by a Mascot probability analysis greater than “identity” were counted as identified. And each confident protein identification involves at least one unique peptide. For protein quantitation, it was required that a protein contains at least two unique peptides. The quantitative protein ratios were weighted and normalized by the median ratio in Mascot. We only used ratios with p-values < 0.05, and only fold changes of > 1.2 were considered as significant.
Function annotation methods
Functional annotations of the identified proteins from our investigation were conducted via Blast2GO program against the non-redundant protein database. Kegg database (http://www.genome.jp/kegg/) and COG database (http://www.ncbi.nlm.nih.gov/COG/) were applied to sort those identified proteins into different categories. As we all known, Gene Ontology (GO) is an international standardization of gene function classification system. it provides a set of dynamic updating controlled vocabulary to describe genes and gene products attributes in organisms, which contains 3 main ontologies context, i.e., molecular function, cellular component, biological process respectively. Cluster of Orthologous Groups of proteins (COG) is the database for protein orthologous classification. Each protein in COG is derived from the same ancestor. KEGG PATHWAY comprises all the available pathway maps on the molecular interaction and reaction networks.
Public data source
The preprocessed level 3 RNAseq data and corresponding clinical information of LIHC were downloaded from TCGA data portal. The clinical samples from liver were selected. A total of 222 LIHC patients with detailed follow-up time were included for subsequent analysis. Except the progress (overall survival), Pearson statistics between genders and clinical pathological characteristic were analysed, such as the HBsAg, HBV DNA, drinking history, smoking history cirrhosis, tumor size, metastasis, tumor thrombus, alanine aminotransferase, aspartate aminotransferase, ALB (albumin of blood), TBIL (total bilirubin) GGT (Glutamyltransferase), independently.
Immunohistochemistry staining
The expressions of proteins in the specimens were detected by immunohistochemistry assay[13] with the monoclonal or polyclonal antibody against human according to the instructions of the invitrogen. For antibody control, one set of samples was incubated with non-immune rabbit IgG (1:150) instead of primary antibody. Evaluation of proteins staining was independently performed by two experienced pathologists. The intensity of proteins immunostaining was semiquantitatively estimated according to the signal intensity and distribution. Briefly, a mean percentage of positive tumor cells was determined in at least five areas 400 magnification and assigned to one of the five following categories: 0, 5%; 15–25%; 2, 25–50%; 3, 50–75%, and 4, 75%. The intensity of immunostaining was scored as follows: 1 weak; 2 moderate and 3 intense. For tumors that showed heterogeneous staining, the predominant pattern was taken into account for scoring. The percentage of positive tumor cells and the staining intensity were multiplied to produce a weighted score for each case. Tissues with immunohistochemical scoring 4 were considered as having low expression, and scores of 5 to 12 were considered high expression. The proteins tested and the their antibody were NNMT (Nicotine N-methyltransferase, Thermofisher Scientific.co, MA5-15738), CYP8B1(Cytochrome P450 Family 8 Subfamily B Member 1, Thermofisher Scientific.co, PA5-102021), SLC27A5(solute carrier family 27 member 5, Thermofisher Scientific.co, PA5-82292), SHMT2 (Serine Hydroxymethyltransferase 2, Thermofisher Scientific.co, MA5-27057), MTHFD2 (Methylenetetrahydrofolate Dehydrogenase, Thermofisher Scientific.co, PA5- 97986).
mRNA analysis
Expression on mRNA was determined by quantitative reverse-transcription (qRT- PCR) using the LightCycler system (Roche, Mannheim, Germany) 18S was used as an endogenous control to normalize for differences in the amount of total RNA in each sample. The primers used for PCR were as follows:
gene
|
Primer (sense)
|
Primer antisense
|
18S
|
-5' GGAGTATGGTTGCAAAGCTGA-3'
|
5'-ATCTGTCAATCCTGTCCGTGT-3’
|
CYP8B1
|
5’-GTACACATGGACCCCGACATC-3'
|
5'-CAGGGTTGAGGAACCGATTG-3’
|
MTHFD2
|
5’-GATCCTGGTTGGCGAGAATCC-3'
|
5'--TCTGGAAGAGGCAACTGAACA-3’
|
NNMT
|
5’-TCACCTTCGACGAGGAAGCT-3'
|
5'-GCTCTGCAGACTTCAGACCA-3’
|
SHMT2
|
5’-TTTGCTTCCCCAGTCTGAGT − 3'
|
5'-TTCTCTTTGTTTTGGGCGG-3’
|
SLC27A5
|
5’-CGGGGTACCATGGGTGTCAGG
CAACAGTTGGCCTTG-3'
|
5'-CCCAAGCTTTCAGAGCCTC
CAGGTTCCCTCACA-3’
|