Automated 2D peptide separation on a 1D nano-LC-MS system.
Given the complexity of the mammalian proteome, high-resolution separation technologies are required to achieve comprehensive proteome coverage and to enhance the detection of low-abundance proteins. Among several technologies, Multidimensional Protein Identification Technology (MudPIT) enables the on-line separation of highly complex peptide mixtures directly coupled with mass spectrometry-based identification. Here, we present a variation of the traditional MudPIT protocol, combining highly sensitive chromatography using a nanoflow liquid chromatography system (nano-LC) with a two-dimensional precolumn in a vented column setup. When compared to the traditional MudPIT approach, this nanoflow variation demonstrated better first-phase separation leading to more proteins being characterized while using rather simple instrumentation and a protocol that requires less time and very little technical expertise to perform.
Taylor
Paul
P
Program in Molecular Structure and Function, Hospital for Sick Children, Toronto, Ontario, M5G 1X8, Canada.
Nielsen
Peter A
PA
Trelle
Morten B
MB
Hørning
Ole B
OB
Andersen
Michael B
MB
Vorm
Ole
O
Moran
Michael F
MF
Kislinger
Thomas
T
D000818:Animals; D002478:Cells, Cultured; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D051379:Mice; D032383:Myocytes, Cardiac / Q000378:metabolism*; D036103:Nanotechnology; D020543:Proteome / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D020543:Proteome