posted on 2020-11-24, 20:14authored bySamantha
J. Knott, Kyle A. Brown, Harini Josyer, Austin Carr, David Inman, Song Jin, Andreas Friedl, Suzanne M. Ponik, Ying Ge
The
extracellular matrix (ECM) provides an architectural meshwork
that surrounds and supports cells. The dysregulation of heavily post-translationally
modified ECM proteins directly contributes to various diseases. Mass
spectrometry (MS)-based proteomics is an ideal tool to identify ECM
proteins and characterize their post-translational modifications,
but ECM proteomics remains challenging owing to the extremely low
solubility of the ECM. Herein, enabled by effective solubilization
of ECM proteins using our recently developed photocleavable surfactant,
Azo, we have developed a streamlined ECM proteomic strategy that allows
fast tissue decellularization, efficient extraction and enrichment
of ECM proteins, and rapid digestion prior to reversed-phase liquid
chromatography (RPLC)-MS analysis. A total of 173 and 225 unique ECM
proteins from mouse mammary tumors have been identified using 1D and
2D RPLC-MS/MS, respectively. Moreover, 87 (from 1DLC-MS/MS) and 229
(from 2DLC-MS/MS) post-translational modifications of ECM proteins,
including glycosylation, phosphorylation, and hydroxylation, were
identified and localized. This Azo-enabled ECM proteomics strategy
will streamline the analysis of ECM proteins and promote the study
of ECM biology.