针对小鼠肝脏药物治疗进行高通量鉴定和蛋白差异表达分析

前言：

The use of a robotic workstation for spot handling, digestion, and sample target preparation gave the desired level of automation and throughput, enabling a seamless link to the mass spectrometric analysis of the digested protein samples. A two-tiered strategy of mass spectrometric analysis was considered for protein identification: 1) All samples were subject to peptide mass fingerprinting, using a MALDI-TOF mass spectrometer for identification of the majority of gel spots, then 2) tandem mass spectrometry, using nanoelectrospray on an ion trap mass spectrometer, guaranteed unambiguous and exhaustive identification of those samples not identified by peptide mass fingerprinting. The suggested experimental workflow and key techniques used in the study are illustrated in Figure 1.

结论：

This study describes the application of an advanced, highly automated proteomics workflow consisting of differential expression analysis, peptide mass fingerprinting, and tandem mass spectrometry to gain an insight into a complex biological problem.

The DIGE technology in combination with image analysis 2-D software was instrumental for highlighting the proteins that were significantly up- or down-regulated, while Ettan Spot Handling Workstation streamlined the sample handling for mass spectrometric analysis. Peptide mass fingerprinting with Ettan MALDI ToF provided speedy identification of the majority of proteins.

The capillary LC-MS/MS technique afforded the identification of samples containing more than one protein. Such samples cannot be successfully analyzed by peptide mass fingerprinting analysis, clearly demonstrate the benefit of the MS/MS approach. The speed and sensitivity associated with the LTQ linear ion trap mass spectrometer then allow short method times and ensure the maximum sequence coverage for confident protein identification in a high throughput setup.

In addition, a focused study on proteins obtained by sub-cellular fractionation could prove suitable to identify proteins involved in metabolism and toxicity in the liver following administration of drugs. Greater understanding of these processes should significantly improve the efficiency of ADME/Tox screening studies during drug discovery campaigns.