IDENTIFICATION OF STRESS PROTEINS IN LYSATES OF HUMAN CELL-LINES SEPARATED BY 2-DIMENSIONAL ELECTROPHORESIS AND ELECTROBLOTTED SIMULTANEOUSLY ONTO 2 DIFFERENT MEMBRANES

A protocol based on a combination of established methods for the chara cterization and identification of inducible stress proteins in human c ell lines is described. A particular protein spot, collected from seve ral micropreparative two-dimensional electrophoresis (2-DE) gels, is c oncentrated into a new gel prior to simultaneous electrotransfer onto a Cationic Durapore (CD) membrane and onto a polyvinylidene (PVDF) bac kup membrane. The protein blotted onto the PVDF support is subjected t o N-terminal sequence analysis. From the protein bound to the CD membr ane the peptide mass profile is obtained by proteolytic digestion of t he protein followed by the separation of the resulting peptides by hig h performance liquid chromatography (HPLC) and their detection by on-l ine electrospray mass spectrometry (LC/MS). Additional internal sequen ce information may be obtained by amino acid sequence analysis of pept ides collected in the HPLC effluent, The efficiency of this strategy i s demonstrated with two proteins extracted from 15 micropreparative 2- DE gels of an extract of a human liver cell line, The peptide mass fin gerprinting of a 60 kDa protein with a pI of 5.3 assigned 22 of 37 pep tides to the heat shock protein 60 (Hsp 60). The result was confirmed by the N-terminal sequence analysis of the undigested protein and of a n 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 cogn ate 71 kDa protein (Hsc 70).