Peptide separation with immobilized pI strips is an attractive alternative to in-gel protein digestion for proteome analysis.
Complex protein mixtures have traditionally been separated by 2-DE. Görg introduced IPGs as the first dimension of protein separation. In recent years, MS-based proteomics has increasingly become the method of choice for identifying and quantifying large number of proteins. In that technology, to decrease analyte complexity, proteins are often separated by 1-D SDS-gel electrophoresis before online MS analysis. Here, we investigate a recently introduced device for peptide separation with IPGs (Agilent OFFGEL). Loading capacity for optimal peptide focusing is below 100 microg and--similar to 2-D gels--IEF is more efficient in the acidic than the basic pH region. The 24-well fractionation format resulted in about 40% additional peptide identifications but less than 20% additional protein identifications than the 12-well format. Compared to in-gel digestion, peptide IEF consistently identified a third more proteins with equal number of fractions. Low protein starting amounts (10 microg) still resulted in deep proteome coverage. Advantages of the in-gel format include better reliability and robustness. Considering its superior performance, diminished sample and work-up requirements, peptide IEF will become a method of choice for sample preparation in proteomics.
Hubner
Nina C
NC
Department of Proteomics and Signal Transduction, Max-Planck-Institute for Biochemistry, Martinsried, Germany.
Ren
Shubin
S
Mann
Matthias
M
D002021:Buffers; D005591:Chemical Fractionation; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005782:Gels; D006367:HeLa Cells; D006801:Humans; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D007553:Isotope Labeling; D013058:Mass Spectrometry; D010455:Peptides / Q000302:isolation & purification*; D011506:Proteins / Q000378:metabolism*; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D011934:Reagent Strips; D012441:Saccharomyces cerevisiae / Q000378:metabolism
D002021:Buffers; D005782:Gels; D010455:Peptides; D011506:Proteins; D020543:Proteome; D011934:Reagent Strips