Electronic Western blot of matrix-assisted laser desorption/ionization mass spectrometric-identified polypeptides from parallel processed gel-separated proteins.
Identification of proteins previously separated by one-dimensional (1-D) or two-dimensional gel electrophoresis requires significant manipulations to digest the proteins into their respective peptides and to extract them from the gel prior to mass analysis. This article describes the simultaneous transfer and digestion of proteins directly from 1-D gels onto a membrane ready for matrix-assisted laser desorption/ionization (MALDI) mass spectrometric (MS) analysis. Protein transfer and digestion efficiencies are estimated to be more than 95%. The effectiveness of this procedure is demonstrated by identifying 110 unique proteins derived from a lysate of Escherichia coli and 149 proteins derived from a mouse liver homogenate separated by 1-D sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Using crude mouse liver homogenates, four distinct glutathione S-transferase classes, ranging from 23 to 27 kDa, are identified from a separating gel, indicating the discriminating potential for this method. A Visual Basic program allowed visualization of the identified proteins according to their respective positions on the 1-D gels. In many cases, two or more proteins could be identified within a single band of the SDS gel. The "digital" images generated resemble Western blots without the use of antibodies or signal amplification techniques.
Nadler
Timothy K
TK
Applied Biosystems, Advanced Research & Technology, 500 Old Connecticut Path, Framingham, MA 01701, USA. nadlertk@appliedbiosystems.com
Wagenfeld
Barrie G
BG
Huang
Yulin
Y
Lotti
Robert J
RJ
Parker
Kenneth C
KC
Vella
George J
GJ
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western / Q000379:methods*; D002457:Cell Extracts / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000378:metabolism; D004926:Escherichia coli; D008099:Liver / Q000737:chemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
D002457:Cell Extracts; D004800:Enzymes, Immobilized; D010455:Peptides; D014357:Trypsin