Most human
tumor tissues that are obtained for pathology and diagnostic purposes are
formalin-fixed and
paraffin-embedded (FFPE). To perform quantitative proteomics of FFPE samples,
paraffin has to be removed and
formalin-induced crosslinks have to be reversed prior to proteolytic digestion. A central component of almost all deparaffinization protocols is
xylene, a toxic and highly flammable
solvent that has been reported to negatively affect
protein extraction and quantitative
proteome analysis. Here, we present a 'green'
xylene-free protocol for accelerated sample preparation of FFPE tissues based on
paraffin-removal with hot water. Combined with tissue homogenization using disposable micropestles and a modified
protein aggregation capture (PAC) digestion protocol, our workflow enables streamlined and reproducible quantitative proteomic profiling of FFPE tissue. Label-free quantitation of FFPE cores from human ductal
breast carcinoma in situ (
DCIS) xenografts with a volume of only 0.79 mm3 showed a high correlation between replicates (r2 = 0.992) with a median %CV of 16.9%. Importantly, this small volume is already compatible with tissue micro array (TMA) cores and core needle biopsies, while our results and its ease-of-use indicate that further downsizing is feasible. Finally, our FFPE workflow does not require costly equipment and can be established in every standard clinical laboratory.