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 sta...

Full description

Saved in:
Bibliographic Details
Published in:Journal of proteome research 2007-01, Vol.6 (1), p.298-305
Main Authors: Vasilescu, Julian, Zweitzig, Daniel R, Denis, Nicholas J, Smith, Jeffrey C, Ethier, Martin, Haines, Dale S, Figeys, Daniel
Format: Article
Language:English
Subjects:
Adenosine Triphosphatases - chemistry
Cell Cycle Proteins - chemistry
Cell Line
Cell Line, Tumor
Chromatography, Liquid
Humans
Kinetics
Mass Spectrometry - methods
Microfluidic Analytical Techniques
Plasmids - metabolism
Proteasome Endopeptidase Complex - metabolism
Proteome
Proteomics - instrumentation
Proteomics - methods
Ubiquitin - chemistry
Valosin Containing Protein
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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
ISSN:1535-3893
1535-3907
DOI:10.1021/pr060438j