Systematic analysis of the total proteins of a mammalian organism: Principles, problems and implications for sequencing the human genome

Klose, Joachim

doi:10.1002/elps.1150100208

Cited by 41 publications

(16 citation statements)

References 75 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Analyses of two-dimensional gels allowed estimation of 6000-10000 different protein species being present in a lymphoid cell (Klose, 1989). The number of copies of each of these proteins may vary from 1 to 10' or more, and their turnover rates may change considerably.…”

Section: Discussionmentioning

confidence: 99%

Peptidylproline cis‐trans‐isomerases: immunophilins

Gałat

1993

European Journal of Biochemistry

338

184

View full text Add to dashboard Cite

Two sequence-unrelated families of proteins possess peptidylproline cis-trans-isomerase activities (PPIase). PPIases are highly sequence conserved and multifunctional proteins which are present in many types of cells with a considerably divergent phylogenetic distribution. On the cellular level, PPIases occur in every compartment, both as free species and anchored to membranes. Diverse posttranslational modifications such as glycosylation, N-terminal modifications and phosphorylation constitute the additional functional features of PPIases. Folding, assembly and trafficking of proteins in the cellular milieu are regulated by PPIases. These enzymes accelerate the rate of in-vitro protein folding and they have the ability to bind proteins and act as chaperones. Some PPIases are coregulatory subunits of molecular complexes including heat-shock proteins, glucocorticoid receptors and ion channels. Secreted forms of PPIases are inflammatory and chemotactic agents for monocytes, eosinophils and basophils. The potent and clinically useful immunosuppressants CsA, FK.506 or rapamycin bind with high affinities to PPIases (immunophilins). The binding criterion allows us to sort the PPIases for the following two superfamilies of proteins : the cyclophilins (CsA-binding proteins) and the FKBP (FK.506/rapamycin-binding proteins).Although none of PPIases appeared to be essential for the viability of haploid yeast cells some of the immunophilidimmunosuppressant complexes are toxic both for yeast and mammalian cells. At least seven unlinked genes of cyclophilins and four unlinked genes of FKBP exist in human genomic DNA. Selected immunophilins regulate two different signalling pathways in lymphoid cells, namely the secretion of growth factors by stimulated T-cells and interleukin-2-induced T-cell proliferation. Moreover, selected FKBP mediate the cytotoxic effects of rapamycin in non-lymphoid cells. Accounts of the discovery of PPIases (immunophilins) and their functions are given in this review. A larger spectrum of proteins is analysed in relation to various signal-transduction pathways in lymphoid cells which involve immunophilins or their complexes with the immunosuppressants CsA, FK.506 or rapamycin.

show abstract

Section: Discussionmentioning

confidence: 99%

Peptidylproline cis‐trans‐isomerases: immunophilins

Gałat

1993

European Journal of Biochemistry

338

184

View full text Add to dashboard Cite

show abstract

“…This includes the resolution of all the different mouse proteins by 2-DE and the characterization of these proteins by classification [164]. In collaboration with a European project (The European Collaborative Interspecific Backcross, EUCIB) established to map the mouse genome by using DNA polymorphisms, we map coding genes on the basis of protein polymorphisms [215, 2171.…”

Section: Genome Analysesmentioning

confidence: 99%

Two‐dimensional electrophoresis of proteins: An updated protocol and implications for a functional analysis of the genome

1995

View full text Add to dashboard Cite

The two-dimensional electrophoresis (2-DE) technique developed by Klose in 1975 (Humangenetik 1975, 26, 211-234), independently of the technique developed by O'Farrell (J. Biol. Chem. 1975, 250, 4007-4021), has been revised in our laboratory and an updated protocol is presented. This protocol is the result of our experience in using this method since its introduction. Many modifications and suggestions found in the literature were also tested and then integrated into our original method if advantageous. Gel and buffer composition, size of gels, use of stacking gels or not, necessity of isoelectric focusing (IEF) gel incubation, freezing of IEF gels or immediate use, carrier ampholytes versus Immobilines, regulation of electric current, conditions for staining and drying the gels - these and other problems were the subject of our concern. Among the technical details and special equipment which constitute our 2-DE method presented here, a few features are of particular significance: (i) sample loading onto the acid side of the IEF gel with the result that both acidic and basic proteins are well resolved in the same gel; (ii) use of large (46 x 30 cm) gels to achieve high resolution, but without the need of unusually large, flat gel equipment; (iii) preparation of ready-made gel solutions which can be stored frozen, a prerequisite, among others, for high reproducibility. Using the 2-DE method described we demonstrate that protein patterns revealing more than 10 000 polypeptide spots can be obtained from mouse tissues. This is by far the highest resolution so far reported in the literature for 2-DE of complex protein mixtures. The 2-DE patterns were of high quality with regard to spot shape and background. The reproducibility of the protein patterns is demonstrated and shown to be thoroughly satisfactory. An example is given to show how effectively 2-DE of high resolution and reproducibility can be used to study the genetic variability of proteins in an interspecific mouse backcross (Mus musculus x Mus spretus) established by the European Backcross Collaborative Group for mapping the mouse genome. We outline our opinion that the structural analysis of the human genome, currently pursued most intensively on a worldwide scale, should be accompanied by a functional analysis of the genome that starts from the proteins of the organism.

show abstract

“…Two-dimensional polyacrylamide gel electrophoresis (2D PAGE), coupled with microsequencing techniques and the establishment of protein data bases, has proven to be a powerful and sensitive strategy for the detection, purification, and identificatidn of individual proteins from complex mixtures of cellular proteins. In fact, the application of these methods is viewed as both essential and complementary to efforts directed toward mapping and sequencing the entire human genome (1)(2)(3). As an analytical technique, 2D PAGE is unequaled in its ability to simultaneously resolve hundreds of proteins from cellular extracts and provide data on their isoelectric points, putative molecular weights, relative abundance, and electrophoretic pattern (4,5).…”

mentioning

confidence: 99%

Mass spectrometric and Edman sequencing of lipocortin I isolated by two-dimensional SDS/PAGE of human melanoma lysates.

Hall

Smith

Masiarz

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

We have integrated preparative twodimensional polyacrylamide gel electrophoresis with highperformance tandem mass spectrometry and Edman degradation. By using this approach, we have isolated and identified, by partial sequencing, a human melanoma protein (34 kDa, pI 6.4) as lipocortin I. To our knowledge, this protein was not previously known to be associated with melanoma cells. The identity of the protein was confirmed by two-dimensional immunoblot analysis. High-energy colsion-induced dissociation analysis revealed the sequence and acetylation of the N-terminal tryptic peptide and an acrylamide-modifled cysteine in another trptic peptide. Thus, knowledge concerning both the primary structure and covalent modifications of proteins isolated from two-dimensional gels can be obtained directly by this approach, which is applicable to a broad range of biological problems.

show abstract

Systematic analysis of the total proteins of a mammalian organism: Principles, problems and implications for sequencing the human genome

Cited by 41 publications

References 75 publications

Peptidylproline cis‐trans‐isomerases: immunophilins

Peptidylproline cis‐trans‐isomerases: immunophilins

Two‐dimensional electrophoresis of proteins: An updated protocol and implications for a functional analysis of the genome

Mass spectrometric and Edman sequencing of lipocortin I isolated by two-dimensional SDS/PAGE of human melanoma lysates.

Contact Info

Product

Resources

About