21 22 33 investigating the role of the matrisome during complex tissue development. 34 35 Keywords: mouse; extracellular matrix; matrisome; limb development; murine morphogenesis and growth; 36 mass spectrometry; bioorthogonal non-canonical amino acid tagging; osteogenesis imperfecta; brain 37 38 102 These methods can be easily extended to investigating the role of the matrisome in other systems during 103 morphogenesis and growth. 104 105 6 Results and Discussion 106 Fractionation of whole embryonic tissue increased ECM protein identification and enabled temporal 107 analysis of the matrisome 108We previously demonstrated that fractionation of E15.5 murine embryos facilitated the analysis of the 109 matrisome via LC-MS/MS (Saleh, et al., 2019a). To confirm that fractionation provides the same benefits 110 for earlier embryonic timepoints wherein the ECM may be less cross-linked, E12.5 wild-type (WT) 111 embryos were homogenized then fractionated using buffers designed to selectively extract cytosolic (C), 112 nuclear (N), membrane (M) and cytoskeletal (CS) proteins, leaving behind an ECM-rich insoluble (IN) 113 pellet (Naba, et al., 2015) (Figure S1A). All samples were processed for LC-MS/MS, raw protein intensities 114 were determined by MaxQuant (Cox and Mann, 2008) and proteins were categorized into cellular 115 compartments (Saleh, et al., 2019a). Within the homogenate, 20 ECM proteins were identified; however, 116 these proteins only represented 0.8% ± 0.3% of the total raw intensity (Figure S1B, Table S1). In contrast, 117 the percentage of matrisome in the IN pellet was 91.5% ± 3.5%, indicating tissue fractionation significantly 118 increased the identification of ECM proteins in E12.5 embryos ( Table S1). The amount of ECM protein in 119 the C, N and M fractions contributed to less than 1.0% ± 0.9% of the total raw intensity. In comparison, 120 ECM proteins contributed 2.3% ± 1.1% of the total raw intensity of the CS fractions. For subsequent studies, 121 the CS and IN fractions were analyzed via LC-MS/MS separately, while the C, N and M fractions were 122 combined into one 'CNM' fraction. 123To investigate whether fractionation was amenable for temporal analysis of the embryonic matrisome, 124 the CNM, CS and IN fractions of E11.5-E14.5 WT embryos were analyzed by LC-MS/MS (Figure 1A). 125Across all fractions and timepoints, 3227 proteins were identified, 122 of which were part of the matrisome 126 (Table S2). Less than 1% and 5% of the raw intensity in CNM and CS fractions, respectively, were 127 attributed to ECM proteins, consistent with our previous study (Saleh, et al., 2019a) (Figures 1B, S1). 128Importantly, the matrisome was enriched in the IN fraction, where at least 30% of the raw intensity was 129 from ECM proteins. 130 7 GO analysis was conducted on the 50 most abundant proteins in each fraction for E14.5 embryos. 131"Cellular Component" terms indicated proteins extracted in the C, N and M buffers were from intracellular 132 compartments, and the most significant "Biological Process" ter...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.