2-D LC-MS/MS Analysis of secreted proteins from HepG2 cells: Combination with various sample preparation methods before in-solution trypsin digestion

Abstract

SUMMARYTwo-dimensional liquid chromatography coupled with tandem mass spectrometry (2-D LC-MS/MS) technique has high capability of resolving peptides, and is used for analysis of tryptic peptides derived from highly complex protein mixtures such as plasma. However, the detection of low-abundant proteins and low-molecular-weight proteins is often very difficult in this system, because major peptides from high-abundant proteins such as albumin mostly disturb the separation of minor peptides from low-abundant proteins. To resolve these problems, different methods of sample preparation for in-solution trypsin digestion were tested, and the optimized method was applied to the 2-D LC-MS/MS analysis of proteins secreted from human hepatoma cell line, HepG2. We could identify 247 and 142 proteins from the chemically and thermally denatured samples, respectively. Among them, 71 proteins were identified in common to both methods, while most of the proteins were identified using either of the two procedures. In addition to these denaturation methods, the molecular-mass cutoff via ultrafiltration improved the efficiency in identifying lowmolecular-weight proteins. Finally, we could identify 478 secreted proteins in total using the combination of these processes. These data indicate that in sample preparation the combination of various denaturation methods, as well as molecular-mass cutoff, are very critical for the identification of a wider range of low-abundant proteins via 2-D LC-MS/MS analysis.

Toxicoproteomic identification of TiO2nanoparticle-induced protein expression changes in mouse brain

“…The Xcorr criteria for peptide evaluation were !2.0 for singly charged ions, !2.5 for doubly charged ions, and !3.7 for triply charged ions. Only the best-matched peptides were adopted (Yamashita et al 2005).…”

“…The LCQ Deca XP automatically sets the collision energy in LC-MS/MS mode. After acquiring full-scan mass spectra, three LC-MS/MS scans were acquired for the next three most intense ions, using dynamic exclusion (Yamashita et al 2005).…”

Proteomic profiling of the differentially expressed proteins by TiO2 nanoparticles in mouse kidney

“…The criteria for peptide evaluation were Xcorr¤2.0 for singly charged ions, Xcorr¤2.5 for doubly charged ions, and Xcorr¤3.7 for triply charged ions. Only the bestmatched peptides were adopted 30 .…”

“…Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension was performed using 8-16% gradient polyacrylamide gels without stacking gels. Separation was carried out at 10� C for 2 h at 5 mA/gel and then at 18 mA/gel; the current was stopped when the dye front began to exit the gel 30 . The gels were visualized using the CBB-G250 (Coomassie brilliant blue G-250) staining method.…”

“…Two-dimensional HPLC was performed using a combination of SCX chromatography (Microtrap SCH, 1×8 mm× 12 μm; Michrom BioResources, Auburn, CA) with reversed-phase HPLC (MagicC18, 0.2×50 mm×5 μm; Michrom Bio Resources) 30 .…”