An integrated workflow for extraction and solubilization of intermediate filaments from colorectal biopsies for proteomic analysis.
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.
Molecular Gastroenterology Research Group, Academic Unit of Surgical Oncology, Department of Oncology, The Medical School, University of Sheffield, Sheffield, UK.
Debabrata
D
Majumdar
Ria
R
Rosser
Suzanne
S
Havard
Alan J
AJ
Lobo
Phillip C
PC
Wright
Caroline A
CA
Evans
Bernard M
BM
Corfe
D001706:Biopsy; D016022:Case-Control Studies; D045744:Cell Line, Tumor; D005591:Chemical Fractionation / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods; D003093:Colitis, Ulcerative / Q000378:metabolism / Q000473:pathology; D003106:Colon / Q000737:chemistry* / Q000473:pathology*; D005615:Freezing; D019791:Guanidine / Q000737:chemistry; D006801:Humans; D015151:Immunoblotting; D007381:Intermediate Filament Proteins / Q000032:analysis* / Q000302:isolation & purification*; D010449:Peptide Mapping / Q000379:methods; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D012107:Research Design; D012995:Solubility; D013010:Sonication; D053719:Tandem Mass Spectrometry / Q000379:methods
D007381:Intermediate Filament Proteins; D019791:Guanidine