Mass Spectrometry-Based Proteomics to Define Intracellular Collagen Interactomes

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.

Keywords: Co-immunoprecipitation; Collagen proteostasis network; Cross-linking; Mass spectrometry; Proteomics.

Figure 1:

Schematic representation of collagen interactomics workflow.

Figure 2:

(a) Generalized schematic for collagen-I expression constructs. Either the endogenous collagen signal sequence or the preprotrypsin signal sequence can be employed. The tag can be HA, FLAG, c-MYC, or any of a number of other short antibody epitope tags. (b) Structure of dithiobis(succinimidyl propionate) (DSP). (c) Optimizing DSP cross-linker concentration by evaluating the coimmunoprecipitation of known collagen-I interactors in the HT-1080 cell system. The control sample represents HT-1080 cells that do not express HA-tagged collagen-I upon induction. This figure was adapted from DiChiara et al [15].