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Keywords:

  • Bioinformatics;
  • Differential Analysis;
  • LCn-MS;
  • Mass Spectrometry;
  • Multidimensional Separation;
  • Proteomics

Abstract

Multidimensional chromatography coupled to mass spectrometry (LCn-MS) provides more separation power and an extended measured dynamic concentration range to analyse complex proteomics samples than one dimensional liquid chromatography coupled to mass spectrometry (1D-LC-MS). This review gives an overview of the most important aspects of LCn-MS with respect to optimizing peak capacity and evaluate orthogonality. We review recent developments in LCn-MS to analyse proteomics samples from the analyst point of view and give an overview over methods and future developments to process LCn-MS data for comprehensive differential protein expression profiling. Examples from our research, such as combining protein fractionation using high temperature reverse phase (RP) columns followed by analysis of the trypsin-digested fractions by RP LC-MS, serve to highlight possibilities and shortcomings of present-day approaches. Other LCn-MS systems that have been used to analyse highly complex shotgun proteomic samples, such as the combination of RP columns using low and high pH eluents or the combination of hydrophilic interaction liquid chromatography (HILIC) with RP-MS is discussed in detail.