ISSN:
0173-0835
Keywords:
Two-dimensional polyacrylamide gel electrophoresis
;
Electroblotting
;
Liquid chromatography
;
Mass spectrometry
;
Peptide mass fingerprinting
;
Amino acid sequence analysis
;
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
A protocol based on a combination of established methods for the characterization and identification of inducible stress proteins in human cell lines is described. A particular protein spot, collected from several micropreparative two-dimensional electrophoresis (2-DE) gels, is concentrated into a new gel prior to simultaneous electrotransfer onto a Cationic Durapore (CD) membrane and onto a polyvinylidene (PVDF) backup membrane. The protein blotted onto the PVDF support is subjected to N-terminal sequence analysis. From the protein bound to the CD membrane the peptide mass profile is obtained by proteolytic digestion of the protein followed by the separation of the resulting peptides by high performance liquid chromatography (HPLC) and their detection by on-line electrospray mass spectrometry (LC/MS). Additional internal sequence information may be obtained by amino acid sequence analysis of peptides collected in the HPLC effluent. The efficiency of this strategy is demonstrated with two proteins extracted from 15 micropreparative 2-DE gels of an extract of a human liver cell line. The peptide mass fingerprinting of a 60 kDa protein with a pI of 5.3 assigned 22 of 37 peptides to the heat shock protein 60 (Hsp 60). The result was confirmed by the N-terminal sequence analysis of the undigested protein and of an internal tryptic fragment. The second sample, a 40 kDa protein with a pI of 4.9, was identified as a processed form of the heat shock cognate 71 kDa protein (Hsc 70).
Additional Material:
6 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/elps.1150170510
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