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Electronic Western blot of matrix-assisted laser desorption/ionization mass spectrometric-identified polypeptides from parallel processed gel-separated proteins
- Source :
- Analytical Biochemistry. 332:337-348
- Publication Year :
- 2004
- Publisher :
- Elsevier BV, 2004.
-
Abstract
- 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.
- Subjects :
- Cell Extracts
Blotting, Western
Molecular Sequence Data
Biophysics
Biochemistry
Mice
Peptide mass fingerprinting
Western blot
Enzyme Stability
Escherichia coli
medicine
Animals
Trypsin
Amino Acid Sequence
Molecular Biology
Polyacrylamide gel electrophoresis
Electroblotting
Gel electrophoresis
Chromatography
medicine.diagnostic_test
Chemistry
Reproducibility of Results
Cell Biology
Enzymes, Immobilized
Mice, Inbred C57BL
Blot
Matrix-assisted laser desorption/ionization
Liver
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Electrophoresis, Polyacrylamide Gel
Bottom-up proteomics
Peptides
Subjects
Details
- ISSN :
- 00032697
- Volume :
- 332
- Database :
- OpenAIRE
- Journal :
- Analytical Biochemistry
- Accession number :
- edsair.doi.dedup.....77ff79928ccffad810bba591dbdbff86