Mass Spectrometry-Based Proteomics to Define Intracellular Collagen Interactomes.
Methods Mol Biol
; 1944: 95-114, 2019.
Article
en En
| MEDLINE
| ID: mdl-30840237
ABSTRACT
We present the development, optimization, and application of constructs, cell lines, covalent cross-linking methods, and immunoprecipitation strategies that enable robust and accurate determination of collagen interactomes via mass spectrometry-based proteomics. Using collagen type-I as an example, protocols for working with large, repetitive, and GC-rich collagen genes are described, followed by strategies for engineering cells that stably and inducibly express antibody epitope-tagged collagen-I. Detailed steps to optimize collagen interactome cross-linking and perform immunoprecipitations are then presented. We conclude with a discussion of methods to elute collagen interactomes and prepare samples for mass spectrometry-mediated identification of interactors. Throughout, caveats and potential problems researchers may encounter when working with collagen are discussed. We note that the protocols presented herein may be readily adapted to define interactomes of other collagen types, as well as to determine comparative interactomes of normal and disease-causing collagen variants using quantitative isotopic labeling (SILAC)- or isobaric mass tags (iTRAQ or TMT)-based mass spectrometry analysis.
Palabras clave
Texto completo:
1
Bases de datos:
MEDLINE
Asunto principal:
Espectrometría de Masas
/
Colágeno
/
Mapeo de Interacción de Proteínas
/
Proteómica
/
Espacio Intracelular
/
Marcaje Isotópico
Límite:
Humans
Idioma:
En
Revista:
Methods Mol Biol
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2019
Tipo del documento:
Article
País de afiliación:
Estados Unidos