1. Evaluation and Refinement of Sample Preparation Methods for Extracellular Matrix Proteome Coverage
- Author
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Monika Dzieciatkowska, Lauren R. Schmitt, Toin H. van Kuppevelt, Willeke F. Daamen, Mark Maslanka, Ryan C. Hill, Maxwell C. McCabe, Kirk C. Hansen, and Danique J. Hof
- Subjects
collagen ,glycoprotein ,K2CO3, potassium carbonate ,Male ,Proteomics ,Proteome ,PSM, peptide spectral match ,Gnd-HCl, guanidine hydrochloride ,VitC, ascorbic acid ,GAG, glycosaminoglycan ,R/A, reduction and alkylation ,Biochemistry ,Analytical Chemistry ,Extracellular matrix ,NaCl, sodium chloride ,Protein purification ,NP-40, Nonidet-P40 ,Sample preparation ,ABC, ammonium bicarbonate ,FA, formic acid ,0303 health sciences ,Extracellular Matrix Proteins ,Decellularization ,Chemistry ,030302 biochemistry & molecular biology ,Technological Innovation and Resources ,MgCl2, magnesium chloride ,CAISU, chaotrope-assisted in-solution digest with ultrasonication ,ECM, extracellular matrix ,Extracellular Matrix ,WMP, whole mouse powder ,Reconstructive and regenerative medicine Radboud Institute for Molecular Life Sciences [Radboudumc 10] ,Tris-HCl, Tris(hydroxymethyl)aminomethane hydrochloride ,EUP, exclusive unique peptides ,IAM, iodoacetamide ,SDS, sodium dodecyl sulfate ,FDR, false discovery rate ,Pipes, piperazine-N,N′-bis(2-ethanesulfonic acid) ,CA, caffeic acid ,Computational biology ,CAIS, chaotrope-assisted in-solution digest ,CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate ,PI, protease inhibitor ,TFA, trifluoroacetic acid ,03 medical and health sciences ,Animals ,Molecular Biology ,030304 developmental biology ,proteoglycan ,matrisome ,EDTA, ethylenediaminetetraacetic acid ,Extraction (chemistry) ,LC-MS/MS, liquid chromatography tandem mass spectrometry ,SPEED, sample preparation by easy extraction and digestion ,HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ,sample preparation methods ,GA, gallic acid ,ACN, acetonitrile ,Ascorbic acid ,timsTOF, trapped ion mobility spectroscopy time-of-flight ,Mice, Inbred C57BL ,MS, mass spectrometry ,DTT, dithiothreitol ,NaOV, sodium orthovanadate ,Extraction methods ,CV, coefficient of variance ,HA, hydroxylamine hydrochloride ,SCAD, surfactant and chaotropic agent-assisted sequential extraction/on-pellet digestion ,DOC, sodium deoxycholate ,KCl, potassium chloride - Abstract
The extracellular matrix is a key component of tissues, yet it is underrepresented 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 with a standard in-solution digest, the optimized method yielded a fourfold improvement in unique ECM peptide identifications., Graphical abstract, Highlights • Decellularization drastically improves ECM coverage. • ECM coverage is the highest with a dual chemical/enzymatic digestion method. • SPEED is the best tested single-shot method for ECM identification. • A fourfold improvement in ECM peptide IDs was achieved compared with standard methods., Due to their insolubility, many extracellular matrix (ECM) proteins are resistant to typical extraction and require optimized extraction methods for proteomic analysis. A wide variety of decellularization and ECM extraction methods have been published, but it remains unclear how these methods perform compared with one another on a complex, ECM-rich sample. A direct comparison of both cell and ECM extraction methods on a whole organism sample and four additional organs serves as a reference for future ECM proteomics.
- Published
- 2021