Multidimensional HPLC of proteins Although two

Multidimensional HPLC of proteins
Schmidt O, Meyer HE & Marcus K.
Medical Proteom-Center, Ruhr-University Bochum
Zentrum für klinische Forschung (ZKF)
Universitätsstraße 150, 44780 Bochum, Germany
Although two-dimensional polyacrylamide gel electrophoresis (2DPAGE) has been used as a standard approach. Its drawbacks have
limited its widespread use and applications in proteomics. In the
meantime, various liquid-phase based separation techniques have
been explored. The ability to characterise a complex protein sample
(e.g. the brain proteome) by mass spectrometry (MS) depends on the
power and sensitivity of the separation technique employed prior to
the MS analysis. The high complexity of such samples and the large
dynamic range involved in analysing protein mixtures, make a multidimensional separation necessary.
Because the separation of complex samples on peptide basis is
insufficient for comprehensive protein identification, new techniques
based on protein separation should be established.
In our approach we have tried to separated the Proteins of the Serva
Proteome Standard (a mixture of 8 proteins) with 2D HPLC using
Chromatofocusing in the first and reversed phase chromatography in
the second dimension. The proteins were identified by nano-LC-ESIMS/MS after tryptic digestion of the RP fractionated Proteome
Standard.
Additionally we separated the Serva Proteome Standard with an
other 2D HPLC using ion exchange chromatography (SCX) in the
first and reversed phase chromatography in the second dimension.
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