Label-Free Proteomics of Serum

In this chapter we describe a method to analyze human serum with the goal of discovering disease-related changes in the serum proteome. The methodology is based on the removal of the six most abundant serum proteins by immunoaffinity chromatography. This step is followed by trypsin digestion and reversed-phase high-performance liquid chromatography (HPLC) coupled on-line to mass spectrometry (MS) using either a capillary HPLC or a microfluidics chip HPLC system. The obtained, highly complex data sets are processed and statistically analyzed to discover significant differences between groups of samples. The complete analytical procedure will be described with serum samples, to which a given amount of horse heart cytochrome c has been added as well as with serum samples from early stage cervical cancer patients prior to and after therapy. The use of reversed-phase HPLC to separate serum proteins at 80^ˆ C with subsequent analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) in order to lower the concentration sensitivity will also be briefly described.