Images were recorded on a GE ImageScanner III
Images were recorded on a GE ImageScanner III system. The gels were analyzed with the ImageMaster 2D Platinum software, and automatic spot matching in conjunction with detailed manual checking of the spot finding, to identify proteins in both the NECs and TECs. The quality of the gels was verified by using the quality control of the software. Spot intensities were expressed as the percentage of the integrated spot density (volume) over the total density of all measured spots. Significantly over-abundant spots were detected at a significance level of 5% and a fold number of >1.5. After statistical analysis, 63 spots were identified in TECs, compared with NECs, and the histograms in Fig. 2 show the relative levels of signal intensity. The histograms contain information about spot ID, spot intensity, relative ratio, and statistical result of triplicate repeats. Spots that were differentially expressed between NECs and TECs were then isolated and identified using mass spectrometry as described below.
Gels were analyzed with the ImageMaster 2D Platinum software. The quality of the gels was verified by using the quality control of the software. Spot intensities were expressed as the percentage of the integrated spot density (volume) over the total density of all measured spots. Significantly over-abundant spots were detected at a significance level of 5% (p-value < 0.05%) and a fold number of >1.5.
Differentially expressed protein spots were picked manually and enzymatic GDC0199 in-gel was carried out according to the procedure of Zimmerman et al. with some modifications . Briefly, dried gel pieces were incubated with 10μL of 25μg/mL sequencing-grade trypsin (Promega) in 40mM ammonium bicarbonate for 30min at 4°C. Then another 20μL of 40mM ammonium bicarbonate was added to ensure complete cover of the pieces. Digestion was carried out at 37°C for 12h and peptides were recovered by sequencing extractions with 25mM ammonium bicarbonate, 50% ACN/0.1% TFA, and 100% ACN, and all steps were repeated once more.
Database searching MALDI-MS/MS data were obtained in an automated analysis loop using a 4800 Plus MALDI TOF/TOF™ Analyzer (Applied Biosystems, USA). Digested peptides were desalted using C18 ZipTips® (Millipore, USA). MS and MS/MS spectra were collected using the 4000 Series Explorer™ software and submitted to database search via GPS Explorer™ (Applied Biosystems). MASCOT Server version 2.2 (Matrix Science, London, UK) and the NCBI non-redundant database were used for protein identification. The search parameters were set as follows: taxonomy: Mouse; mass values, monoisotopic; precursor mass tolerance, ±1Da; fragment mass tolerance, ±0.3Da; enzyme, trypsin; maximum missed cleavage allowed, 1; modifications, carbamidomethyl Cys (permanent); methionine oxidation (variable); Ser, Thr, and Tyr phosphorylation. Results were scored using the probability-based MASCOT score. Among these identified proteins, many have been identified in previous cancer studies, including Tagln2, Hspd1, Pgam1, Dld, Cct2, Npm1, Arhgdia, Gdi2, Aprt, Park7, Acy1, Capzb, Ctsb, Hnrnpk, Vcp, Enol, Pkm2, Pcna, Pgk1, and Map2k1, which are list in Table 1. For these candidate biomarkers, our results are in agreement with published data.
Real-time PCR analysis of selected proteins On the basis of GO annotations (20 proteins, including in the top 10 GO BP terms) and protein–protein interaction analysis results (3 proteins), the mRNA levels of 23 differentially expressed proteins were analyzed by real-time RT-PCR. Total RNA was extracted using TRIzol. RT-PCR analysis was performed by using the SYBR® Green I RT-PCR Master Mix kit from Bio-Rad Laboratories, Inc. on a Rotor-Gene 3000 system. The relative mRNA levels of differentially expressed proteins were normalized to that of GAPDH, and NECs were used for calibration. Primers for selected proteins are listed in Table 2. Measurement of △Ct was performed in triplicate. RT-PCR data were analyzed for relative gene expression using the △△Ct method. The results of the RT-PCR analysis were mostly consistent with those obtained in the 2D-PAGE analysis (see Fig. 3A).