Establishment of a PF2D‐MS/MS platform for rapid profiling and semiquantitative analysis of membrane protein biomarkers

Abstract

Current proteome profiling techniques have identified relatively few mammalian membrane proteins despite their numerous important functions. To establish a standard throughput-potential profiling platform for membrane proteins, Triton X-100-solubilized rat liver microsomal proteins were separated on a 2-D separation system (2-D liquid phase fractionation (PF2D)) in two different pH ranges (4.0-8.5 and 7.0-10.5). This system produced 182 proteins with more than two transmembrane domain (TMD), including 16 TMDs with high confidence. Comparative 2-D liquid maps with high resolution and reproducibility have been constructed for liver microsome from the phenobarbital (PB) treated rats. PF2D was also found to be useful for the semiquantification of some representative cytochrome P450 family proteins (e.g., cytochrome P450 2B2) that were induced by PB treatment compared with untreated controls. Thus, the combination of both high-detection capacity and rapid preliminary semiquantification in a PF2D platform could become a standard system for the routine analysis of membrane proteins.

Rapid T cell–based identification of human tumor tissue antigens by automated two-dimensional protein fractionation

“…immunogenic epitope in more detail, we synthesized overlapping 20-meric peptides of the respective region ( Figure 6C) and identified S100A9 [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] as the immunogenic epitope ( Figure 6D). This epitope was recognized both by CD8 + and CD4 + T cells of patient NCH550 ( Figure 6E).…”

“…T cell activation by antigen-pulsed DCs is based on a complex and tightly regulated process involving multiple steps, including protein uptake, cleavage into small peptide fragments, their loading into the peptide-binding cleft of MHC I and II molecules, and their presentation at the cell surface to antigen-specific T cells. However, since PF2D technology has predominantly been used for identifying differentially expressed proteins (12)(13)(14)(15) and for validating protein arrays (16), so far, it is not known whether proteins subjected to PF2D fractionation under partially denaturing conditions maintain the features required for appropriate antigen processing by DCs. In addition, it is unclear whether concentrations of respective proteins in the eluates are sufficient for successful presentation to CD4 + and CD8 + T cells.…”

Current methods of cytochrome P450 analysis

“…В ряде работ идентификация цитохромов Р450 была получена в рамках общего протеомного анализа [24][25][26]. Так, в работе [24] при протеомном анализе эндоплазматического ретикулума пораженных опухолью, индуцированной афлатоксином В1, тканей печени крыс обнаружено уменьшение экспрессии цитохромов Р450 по сравнению с контрольными образцами.…”

“…Так, в работе [24] при протеомном анализе эндоплазматического ретикулума пораженных опухолью, индуцированной афлатоксином В1, тканей печени крыс обнаружено уменьшение экспрессии цитохромов Р450 по сравнению с контрольными образцами. В работах [25,26] методами 2D-LC исследован эффект увеличения экспрессии цитохромов Р450 в печени крысы после введения животным фенобарбитала. В работе [26] показано, что при общем увеличении содержания цитохромов наибольший эффект наблюдается для изоформы CYP 2В2.…”

“…В работах [25,26] методами 2D-LC исследован эффект увеличения экспрессии цитохромов Р450 в печени крысы после введения животным фенобарбитала. В работе [26] показано, что при общем увеличении содержания цитохромов наибольший эффект наблюдается для изоформы CYP 2В2. Протеомный подход RPLC-ESI-MS/MS Nisar с соавт.…”