An integrated workflow for extraction and solubilization of intermediate filaments from colorectal biopsies for proteomic analysis

Abstract

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.

Reduced keratin expression in colorectal neoplasia and associated fields is reversible by diet and resection

“…The insoluble fraction was processed to yield intermediate filament (IF)-enriched material. 26 The soluble fractions were pooled in fours (empirically determined to yield analysable acetyl-proteins by IP, data not shown). Samples from the highest and lowest butyrate pools from normal participants, and AD participants at each of the three biopsy sites (a total of eight pools) were immunoprecipitated to yield a pooled IP eluate (acetyl proteins), and a pooled flow-through (other soluble proteins).…”

“…A small number of studies have converged on keratins as hallmarks of colorectal mucosal health 7 we undertook a proteomic analysis of the insoluble fraction which is enriched for intermediate filament proteins, including keratins. 26 Samples grouped by diagnosis/biopsy location (normal MS, AD MS, CL and lesion AD) and were stratified by low (∼1mM) and high (∼14 mM) butyrate (see above and SOI for concentrations). The iTRAQ workflow identified 55 proteins were represented by ≥2 peptides (complete list: SOI, section 5).…”

“…Acetyl proteins were separated from other soluble proteins using antibody immobilised to a Pierce Seize matrix. The insoluble fraction, principally intermediate filaments, was prepared using our integrated workflow for iTRAQ-compatible analysis 26 which modified the high-salt extraction technique of Achstaetter 27 and Herrmann. 28 The insoluble and soluble fractions were analysed using iTRAQ workflows while acetyl proteins were analysed by 2D gel electrophoresis ( vide infra ).…”

Inflammation decreases keratin level in ulcerative colitis; inadequate restoration associates with increased risk of colitis-associated cancer

“…We followed our technique for isolation and solubilisation of IFs for mass spectrometry (MS). 30 The IF fraction from cultured MCF-7 cells used as controls in the western immunoblot and as internal standards for densitometry. 30 Experimental details are provided in full (see online supplementary data, sections 3 and 5).…”