Comprehensive two‐dimensional gel protein databases offer a global approach to the analysis of human cells: The transformed amnion cells (AMA) master database and its link to genome DNA sequence data

Abstract

A total of 3430 polypeptides (2592 cellular; 838 secreted) from transformed human amnion cells (AMA) labeled with [35S]methionine were separated and recorded using computer-aided two-dimensional (2-D) gel electrophoresis. A master 2-D gel database of cellular protein information that includes both qualitative and quantitative annotations has been established. The protein numbers in this database differ from those reported in an earlier version (Celis et al. Leukemia 1988, 2,561-602) as a result of changes in the scanning hardware. The reported information includes: percentage of total radioactivity recovered from the gels (based on quantitations of polypeptides labeled with a mixture of 16 14C-amino acids), protein name (including credit to investigators that aided identification), antibody against protein, cellular localization, (nuclear, 40S hnRNP, 20S snRNP U5, proteasomes, endoplasmic reticulum, mitochondria, Golgi, ribosomes, intermediate filaments, microfilaments and microtubules), levels in fetal human tissues, partial protein sequences (containing information on 48 human proteins microsequenced so far), cell cycle-regulated proteins, proteins sensitive to interferons alpha, beta, and gamma, heat shock proteins, annexins and phosphorylated proteins. The results presented should be considered as the initial phase of a joint effort between our laboratories to undertake a general and systematic analysis of human proteins. Using this integrated approach it will be possible to identify phenotype-specific proteins, to microsequence them and store the information in the database, to identify the corresponding genes, to search for homology with previously characterized proteins and to study the function of groups of proteins (pathways, organelles, etc.) that exhibit interesting regulatory properties. In particular, the 2-D gel protein database may become increasingly important in view of the concerted effort to map and sequence the entire human genome.

The N-terminal domain of a rab protein is involved in membrane-membrane recognition and/or fusion.

“…We used the high resolution two-dimensional gel electrophoresis system established by Celis and his collaborators (Celis et al, 1990), as in our previous studies (Thomas et al, 1992;Emans et al, 1993). Protein determination was according to Bradford (1976).…”

Recombinant human casein kinase II

“…Two-dimensional gel electrophoresis was carried out after methanol precipitation of the recombinant CKII subunits according to the method described by Celis et al (1990). In the first dimension isoelectric focusing was applied to the CKIIP subunit and non-equilibrium pH-gradient electrophoresis to the CKIIa and CKIIa' subunits, respectively.…”

Degradation of cellular mRNA during influenza virus infection: its possible role in protein synthesis shutoff

“…The proteins present in total cell extracts were resolved by two-dimensional gel electrophoresis and the identification of the spots corresponding to flactin, ~-and fl-tubulin and vimentin was carried out by using some of the two-dimensional gel protein databases available (Celis et al, 1990). The radioactive label associated with such spots was quantified as described in Methods.…”