The Proteomic Reactor Facilitates the Analysis of Affinity-Purified Proteins by Mass Spectrometry: Application for Identifying Ubiquitinated Proteins in Human Cells

Mass spectrometry (MS) coupled to affinity purification is a powerful approach for identifying protein−protein interactions and for mapping post-translational modifications. Prior to MS analysis, affinity-purified proteins are typically separated by gel electrophoresis, visualized with a protein stain, excised,
and subjected to in-gel digestion. An inherent limitation of this series of steps is the loss of protein
sample that occurs during gel processing. Although methods employing in-solution digestion have
been reported, they generally suffer from poor reaction kinetics. In the present study, we demonstrate
an application of a microfluidic processing device, termed the Proteomic Reactor, for enzymatic digestion
of affinity-purified proteins for liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis.
Use of the Proteomic Reactor enabled the identification of numerous ubiquitinated proteins in a human
cell line expressing reduced amounts of the ubiquitin-dependent chaperone, valosin-containing protein
(VCP). The Proteomic Reactor is a novel technology that facilitates the analysis of affinity-purified
proteins and has the potential to aid future biological studies.
Keywords: Proteomic reactor • affinity purification • mass spectrometry • ubiquitination • H1299 cells • VCP