Preserved clinical material is a unique source for proteomic investigation of 
human disorders. Here we describe an optimized protocol allowing large scale 
quantitative analysis of formalin fixed and paraffin embedded (FFPE) tissue. The 
procedure comprises four distinct steps. The first one is the preparation of 
sections from the FFPE material and microdissection of cells of interest. In the 
second step the isolated cells are lysed and processed using 'filter aided 
sample preparation' (FASP) technique. In this step, proteins are depleted from 
reagents used for the sample lysis and are digested in two-steps using 
endoproteinase LysC and trypsin. After each digestion, the peptides are 
collected in separate fractions and their content is determined using a highly 
sensitive fluorescence measurement. Finally, the peptides are fractionated on 
'pipette-tip' microcolumns. The LysC-peptides are separated into 4 fractions 
whereas the tryptic peptides are separated into 2 fractions. In this way 
prepared samples allow analysis of proteomes from minute amounts of material to 
a depth of 10,000 proteins. Thus, the described workflow is a powerful technique 
for studying diseases in a system-wide-fashion as well as for identification of 
potential biomarkers and drug targets.