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.
Vasilescu
Julian
J
Ottawa Institute of Systems Biology, 451 Smyth Road, Ottawa, Ontario, K1H 8M5, Canada.
Zweitzig
Daniel R
DR
Denis
Nicholas J
NJ
Smith
Jeffrey C
JC
Ethier
Martin
M
Haines
Dale S
DS
Figeys
Daniel
D
D000251:Adenosine Triphosphatases / Q000737:chemistry; D018797:Cell Cycle Proteins / Q000737:chemistry; D002460:Cell Line; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods*; D046210:Microfluidic Analytical Techniques; D010957:Plasmids / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D020543:Proteome; D040901:Proteomics / Q000295:instrumentation* / Q000379:methods*; D025801:Ubiquitin / Q000737:chemistry*; D000074405:Valosin Containing Protein
D018797:Cell Cycle Proteins; D020543:Proteome; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex; D000251:Adenosine Triphosphatases; C000614178:VCP protein, human; D000074405:Valosin Containing Protein