Proteomic analysis using 2-D liquid separations of intact proteins from whole-cell lysates.
This unit describes procedures for 2-D liquid separations of proteins from whole-cell lysates. Protocols for protein isoelectric point (pI) fractionation in the first dimension include the use of liquid isoelectric focusing (IEF) and chromatofocusing. The liquid IEF provides a pI-based fractionation using a batch-phase electrophoretic method, while chromatofocusing uses a column-based chromatographic method to generate the pH gradient. Using either method, a second-dimension fractionation is provided in the liquid phase using nonporous silica-based reversed-phase HPLC (NPS-RP-HPLC) to generate a 2-D liquid map of the protein content of the cell. The eluate of the 2-D liquid fractionation is directly coupled to a mass spectrometer for on-line detection of the intact molecular weights of proteins. As a result, a multidimensional map of protein expression is obtained that characterizes cellular proteins by pI, hydrophobicity, and intact molecular weight. Such expression maps are useful for differential proteomic comparison between different cell samples.
Zhu
Kan
K
The University of Michigan, Ann Arbor, Michigan.
Yan
Fang
F
The University of Michigan, Ann Arbor, Michigan.
O'Neil
Kimberly A
KA
The University of Michigan, Ann Arbor, Michigan.
Hamler
Rick
R
The University of Michigan, Ann Arbor, Michigan.
Lubman
David M
DM
The University of Michigan, Ann Arbor, Michigan.
Lin
Linda
L
Eprogen, Inc, Darien, Illinois.
Barder
Timothy J
TJ
Eprogen, Inc, Darien, Illinois.
D000818:Animals; D002021:Buffers; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000295:instrumentation / Q000379:methods; D007526:Isoelectric Point; D008970:Molecular Weight; D011506:Proteins / Q000032:analysis*; D020543:Proteome / Q000032:analysis*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods
D002021:Buffers; D011506:Proteins; D020543:Proteome