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Impact of prefractionation using Gradiflow™ on two‐dimensional gel electrophoresis and protein identification by matrix assisted laser desorption/ionization‐time of flight‐mass spectrometry
Author(s) -
Pang Lisa,
Fryksdale Beth G.,
Chow Nicole,
Wong David L.,
Gaertner Alfred L.,
Miller Brian S.
Publication year - 2003
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200305515
Subject(s) - mass spectrometry , chromatography , chemistry , fractionation , proteome , sample preparation , matrix assisted laser desorption/ionization , electrophoresis , time of flight mass spectrometry , surface enhanced laser desorption/ionization , two dimensional gel electrophoresis , peptide mass fingerprinting , sample preparation in mass spectrometry , analytical chemistry (journal) , electrospray ionization , ionization , proteomics , desorption , biochemistry , ion , organic chemistry , adsorption , gene
Prefractionation of protein samples prior to two‐dimensional electrophoresis (2‐DE) has the potential to increase the dynamic detection range for proteomic analysis. We evaluated a membrane‐based electrophoretic separation technique (Gradiflow™) for its ability to fractionate an exoproteome sample from the filamentous fungus Trichoderma reesei. The sample was separated on the basis of size and charge. Buffer optimization was found to be necessary for successful size fractionation. Fractionation by charge was used to resolve the sample into four fractions that were subjected to analysis by two‐dimensional electrophoresis (2‐DE). Enhanced detection of low‐abundance proteins with selective removal of high‐abundance species was achieved. Fractionated and unfractionated samples were examined for differences in the ability to identify proteins following 2‐DE using trypsin in‐gel digestion followed by peptide mass fingerprinting using matrix assisted laser desorption/ionization‐time of flight‐mass spectrometry (MALDI‐TOF‐MS). Fractionated samples showed marked improvement in protein identification ability and sequence coverage. This study demonstrates the utility of the Gradiflow™ for fractionation, resulting in an enhancement of resolution and characterization of a moderately complex proteome.