Edgar Nägele

2D-LC/MS techniques for the identification of proteins in highly complex mixtures.

Today, 2D online or offline liquid chromatography/mass spectrometry is state of the art for the identification of proteins from complex proteome samples in many laboratories. Both 2D liquid chromatography methods use two orthogonal liquid chromatography separation techniques. The most commonly used techniques are strong cation exchange chromatography for the first dimension and reversed phase separation for the second dimension. In order to improve sensitivity the reversed phase separation is usually performed in the nanoflow scale and mass spectrometry is used as the final detection method. The high-performance liquid chromatography techniques complement the 2D-gel techniques supporting their weaknesses. This is especially true for the gel separation of hydrophobic membrane proteins, which play an important role in living cells as well as being important targets for future pharmaceutical drugs.

Two-dimensional nano-liquid chromatography-mass spectrometry system for applications in proteomics.

This work demonstrates the development of a method for the analysis of complex proteome samples by two-dimensional nano-liquid chromatography-mass spectrometry. This approach includes strong cation-exchange, sample enrichment, reversed-phase chromatography and nanospray ion trap mass spectroscopy with data dependent tandem mass spectrometry spectra acquisition, and subsequent database search. The new methodology was first evaluated using standard protein digest samples. Finally, data for the analysis of a total Escherichia coli proteome are provided.