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Peptide separation with immobilized p I strips is an attractive alternative to in‐gel protein digestion for proteome analysis
Author(s) -
Hubner Nina C.,
Ren Shubin,
Mann Matthias
Publication year - 2008
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200800351
Subject(s) - proteome , peptide , chromatography , proteomics , analyte , fractionation , bottom up proteomics , digestion (alchemy) , chemistry , peptide mass fingerprinting , sample preparation , mass spectrometry , biochemistry , tandem mass spectrometry , protein mass spectrometry , gene
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 μg 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 μg) 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.