The Development of a Facility to Analyze and Synthesize Genes and Proteins

Hood, Leroy; Kent, Stephen B. H.; Smith, L. C.; Aebersold, Ruedi; Teplow, David B.; Kaiser, Robert; Clark‐Lewis, Ian; Woo, D.; Hines, Wirt A; Sanders, Jane Z.

doi:10.1007/978-1-59259-480-1_2

Methods in Protein Sequence Analysis · 1986 1987

DOI: 10.1007/978-1-59259-480-1_2

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The Development of a Facility to Analyze and Synthesize Genes and Proteins

Leroy Hood

Stephen B. H. Kent

L. C. Smith

et al.

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Cited by 2 publications

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“…Amino acid sequence data can aid in gene isolation either through the use of synthetic oligonucleotides (1) or through the use of antisera of predetermined specificity (2). Sequence data can also be used to correlate the gene structure with the expressed polypeptide chain, to map active sites and domain boundaries, to define posttranslational modifications, or to establish evolutionary relationships between proteins (3).…”

mentioning

confidence: 99%

Internal amino acid sequence analysis of proteins separated by one- or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose.

Aebersold

Leavitt

Saavedra

et al. 1987

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

783

339

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We have developed a general two-step method for obtaining peptide fragments for sequence analysis from picomole quantities of proteins separated by gel electrophoresis. After separation by one-or two-dimensional polyacrylamide gel electrophoresis, proteins are electrophoretically transferred (electroblotted) onto nitrocellulose, the protein-containing regions are detected by reversible staining and are cut out, and each protein is digested in situ by proteolytic enzymes such as trypsin or staphylococcal V-8 protease. The resulting peptide fragments are separated by narrow-bore reverse-phase HPLC, collected, and sequenced in a gas-phase sequenator. Excellent peptide recoveries and the absence of extraneous contaminants in the separation of the peptide fragment mixture allow the generation of extensive internal sequence information from picomole amounts of protein. The method thus overcomes the problem of obtaining amino acid sequence data from Nterminally blocked proteins and provides multiple, independent stretches of sequence that can be used to generate oligonucleotide probes for molecular cloning and/or used to search sequence data bases for related proteins. This method has been successfully applied to the routine amino acid sequence analysis of a wide range of proteins isolated from oneand two-dimensional polyacrylamide gels.Protein sequence analysis is central to modern biological research. Amino acid sequence data can aid in gene isolation either through the use of synthetic oligonucleotides (1) or through the use of antisera of predetermined specificity (2). Sequence data can also be used to correlate the gene structure with the expressed polypeptide chain, to map active sites and domain boundaries, to define posttranslational modifications, or to establish evolutionary relationships between proteins (3).Currently, 20 pmol amounts of protein can give limited N-terminal sequence information using optimized isolation methods (4) and a commercially available gas-phase sequenator (5). It is desirable to develop a method of comparable sensitivity for internal amino acid sequence analysis of proteins for the following reasons:(i) Many proteins are not susceptible to the Edman degradation. This is assumed to be due to artefactual or biosynthetic blocking of the a-amino groups of the proteins. The nature of the modified N terminus is not easily determined and only a few of the modifications can be chemically or enzymatically reversed (6). Therefore, the most general way to obtain sequence information from a protein with a blocked a-amino group is to sequence peptide fragments generated by cleavage of the polypeptide chain.(ii) In the molecular cloning of genes through the use of synthetic oligodeoxynucleotides reverse translated from the protein sequence, it is advantageous to screen appropriate cDNA libraries with either a set of multiple probes or a single probe with minimum degeneracy to reduce the incidence of "false positives." Furthermore, the use of an oligodeoxynucleotide probe derived from the N...

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mentioning

confidence: 99%

Internal amino acid sequence analysis of proteins separated by one- or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose.

Aebersold

Leavitt

Saavedra

et al. 1987

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

783

339

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“…Most sequences are determined by the stepwise degradation of proteins and peptides as initially proposed by Edman (1 950). In spite of dramatic improvements and refinements in sequencing methods and instrumentation achieved during the last decade (Hood et al, 1986), current chemical protein sequencing capabilities are limited at a number of levels. First, at cycle times typically in the range of 35-60 min, stepwise sequence analysis is slow.…”

mentioning

confidence: 99%

Design, synthesis, and characterization of a protein sequencing reagent yielding amino acid derivatives with enhanced detectability by mass spectrometry

et al. 1992

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We report the design, chemical synthesis, and structural and functional characterization of a novel reagent for protein sequence analysis by the Edman degradation, yielding amino acid derivatives rapidly detectable at high sensitivity by ion-evaporation mass spectrometry. We demonstrate that the reagent 3-[4'(ethylene-N, N, N-trimethylamino)phenyl]-2-isothiocyanate is chemically stable and shows coupling and cyclization/cleavage yields comparable to phenylisothiocyanate, the standard reagent in chemical sequence analysis, under conditions typically encountered in manud or automated sequence analysis. Amino acid derivatives generated with this reagent were detectable by ion-evaporation mass spectrometry at the subfemtomole sensitivity level at a pace of one sample per minute. Furthermore, derivatives were identified by their mass, thus permitting the rapid and highly sensitive determination of the molecular nature of modified amino acids. Derivatives of amino acids with acidic, basic, polar, or hydrophobic side chains were reproducibly detectable at comparable sensitivities. The polar nature of the reagent required covalent immobilization of polypeptides prior to automated sequence analysis. This reagent, used in automated sequence analysis, has the potential for overcoming the limitations in sensitivity, speed, and the ability to characterize modified amino acid residues inherent in the chemical sequencing methods that are currently used.

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The Development of a Facility to Analyze and Synthesize Genes and Proteins

Cited by 2 publications

References 12 publications

Internal amino acid sequence analysis of proteins separated by one- or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose.

Internal amino acid sequence analysis of proteins separated by one- or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose.

Design, synthesis, and characterization of a protein sequencing reagent yielding amino acid derivatives with enhanced detectability by mass spectrometry

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