Quantitative Proteome Analysis of HGTs vs. PBTs

The differential proteome between HGTs and PBTs were identified by LC-MS/MS, which was described in detail in our papers [20 (link)–24 (link)]. Generally, 30 μg proteins from Leu-d₃-labeling cells were respectively mixed with 30 μg HGT or PBT proteins to separate on 12% SDS-PAGE. Gels were excised to in-gel digest and extract peptides, peptides were identified by LC-nanospray-tandem MS (MS/MS) on a QSTAR XL mass spectrometer (Applied Biosystems, USA). The parameters for database searching were mainly followed as our previous approaches [22 (link), 23 (link), 25 (link)]. The relative tissue protein expression level (SILAC ratio) was quantified by tracking pairs of labeling and unlabeling peptides from MS spectra. The differential expression protein was defined with its change ratio above 2 or below 0.5 times as a significantly up-regulated or down-regulated one between HGTs and PBTs, which was performed following bioinformatics analysis [23 (link), 24 (link)].