Top–down analysis of low mass proteins in exosomes shed by murine myeloid-derived suppressor cells

• A workflow has been developed for successful top–down analysis of low mass proteins using HPLC-MS/MS.
• Multiple proteoforms are identified of the pro-inflammatory mediators S100 A8 and A9 in exosomes shed by MDSC cells that suppress the immune response to cancer.
• Histone proteoforms are found to constitute 56% of the protein cargo carried by these exosomes.
• Four histone variants are shown to carry a rare processing modification.

Top–down analysis is reported for a portion of the protein cargo of exosomes shed by myeloid-derived suppressor cells that participate in intracellular signaling in the tumor microenvironment. Instrument mass resolution limited the study to proteins of molecular masses below 30 kDa. A two-step fractionation strategy was used, including open tubular gel electrophoresis and C3 reverse phase high performance liquid chromatography. Twenty-one unique proteins were identified among more than 200 proteoforms, and comprising primarily two functionally important protein families: the S100 proinflammatory mediators and an abundance of histones. Fifty-six percent of the total protein in these exosomes was determined to comprise histones, of which H2B variants contribute 42%.

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