We report a technique for isolation and solubilization of intermediate filament (IF)
proteins from colonic biopsies compatible with both gel electrophoresis and liquid chromatography "shotgun" proteomics using mass spectrometry (MS). This is important because changes in the IF
proteome, particularly in
keratin expression and modification, are noted in colonic mucosa of patients with
colorectal cancer. Though
keratins have traditionally been dissolved in high concentration of
urea, the latter
solvent precludes efficient proteolytic digestion by
trypsin prior to gel-free LC-MS/MS approaches. The extraction of
cytoskeletal proteins was initially evaluated using MCF-7
cancer cell lines using a published, differential
detergent solubilization protocol. IF
proteins were extracted from colonic biopsies using a combination of homogenization and sonication. Since comparable efficiency of solubilization was noted on the extracted IF from cell lines between
urea and
guanidine hydrochloride (GuHCl) in
triethylammonium bicarbonate buffer, isolated
proteins from endoscopic biopsies were solubilized in GuHCl. Using immunoblotting techniques, we successfully demonstrated isolation of
keratins and preservation of posttranslational modifications (phosphorylation, acetylation). Dissolved
proteins were tryptically digested and
peptides analyzed by MS, showing the functionality of the workflow in shotgun proteomic applications, specifically compatibility of the workflow for isobaric tagging relative and absolute quantification based quantitation approaches.