Evaluation and refinement of sample preparation methods for extracellular matrix proteome coverage

• Published in: bioRxiv
• Main Authors: Mccabe, Maxwell C, Schmitt, Lauren R, Hill, Ryan C, Dzieciatkowska, Monika, Maslanka, Mark, Daamen, Willeke F, Van Kuppevelt, Toin H, Hof, Danique J, Hansen, Kirk C
• Format: Paper
• Language: English
• Published: Cold Spring Harbor Cold Spring Harbor Laboratory Press 25.11.2020
• Subjects: Acetic acid; Acetonitrile; Acids; Alkylation; Ammonium; Ascorbic acid; Bicarbonates; Caffeic acid; Dithiothreitol; Extracellular matrix; Formic acid; Gallic acid; Glycosaminoglycans; Guanidine hydrochloride; Hydroxylamine; Liquid chromatography; Magnesium chloride; Mass spectrometry; Mass spectroscopy; Orthovanadate; Peptides; Piperazine; Potassium carbonate; Potassium chloride; Proteinase inhibitors; Proteomes; Scientific imaging; Sodium chloride
• DOI: 10.1101/2020.11.25.391946

ABSTRACT The extracellular matrix is a key component of tissues, yet it is under-represented in proteomic datasets. Identification and evaluation of proteins in the extracellular matrix (ECM) has proved challenging due to the insolubility of many ECM proteins in traditional protein extraction buffers. Here we separate the decellularization and ECM extraction steps of several prominent methods for evaluation under real-world conditions. The results are used to optimize a two-fraction ECM extraction method. Approximately one dozen additional parameters are tested and recommendations for analysis based on overall ECM coverage or specific ECM classes are given. Compared to a standard in-solution digest, the optimized method yielded a 4-fold improvement in unique ECM peptide identifications. Figure1 Figure1 * Download figure * Open in new tab Competing Interest Statement The authors have declared no competing interest. * ABBREVIATIONS ABC Ammonium bicarbonate ACN Acetonitrile CA Caffeic acid CAIS Chaotrope-assisted in-solution digest CAISU Chaotrope-assisted in-solution digest with ultrasonication CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate CV Coefficient of variance DOC Sodium deoxycholate DTT Dithiothreitol ECM Extracellular matrix EDTA Ethylenediaminetetraacetic acid FA Formic acid FDR False discovery rate GA Gallic acid GAG Glycosaminoglycan Gnd-HCl Guanidine hydrochloride HA Hydroxylamine hydrochloride HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid IAM Iodoacetamide K2CO3 Potassium carbonate KCl Potassium chloride LC-MS/MS Liquid chromatography tandem mass spectrometry MgCl2 Magnesium chloride MS Mass spectrometry NaCl Sodium chloride NaOV Sodium orthovanadate NP-40 Nonidet-P40 PI Protease Inhibitor Pipes Piperazine-N,N′-bis(2-ethanesulfonic acid) PSM Peptide spectral match R/A Reduction and alkylation SCAD Surfactant and chaotropic agent assisted sequential extraction/on-pellet digestion SDS Sodium dodecyl sulfate SPEED Sample preparation by easy extraction and digestion TFA Trifluoroacetic acid Tris-HCl Tris(hydroxymethyl)aminomethane hydrochloride VitC Ascorbic acid WMP Whole mouse powder