Chemistry: analytical and immunological testing – Peptide – protein or amino acid – Amino acid or sequencing procedure
Patent
1997-04-11
1998-09-15
Redding, David A.
Chemistry: analytical and immunological testing
Peptide, protein or amino acid
Amino acid or sequencing procedure
436172, 436173, 530341, 530410, 5483335, 5483341, G01N 3368, C07D23504
Patent
active
058077480
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to the sequential degradation of proteins and peptides from the N-terminus. More particularly, the invention relates to the sequential N-terminal degradation of small peptide samples. Sequencing sensitivity is enhanced by removal of the peptide N-terminal amino acid as a thiazolinone derivative which is derivatized by a nucleophile containing a fluorescent group or a functional group appropriate for a particular method of detection.
BACKGROUND OF THE INVENTION
One of the primary goals of protein chemists is to relate the function of a protein to its structure. With this goal in mind, an early step in the structural characterization of proteins is the determination of primary structure or sequence. Currently, primary structural determination can be accomplished either by sequencing the protein on an automated sequencer using the Edman chemistry for successive degradation or by sequencing the gene for that protein using established DNA sequencing methodology. Although protein sequencing can be considered to be more difficult and slower than DNA sequencing, it often provides information not obtainable by the latter method. Protein sequencing can provide information concerning posttranslational modifications which are not predictable from the gene sequence, such as location of proteolytic cleavage sites. Furthermore, it is a key method for the determination of protein sequence information which can be used for the design of oligonucleotide probes complementary to predicted gene sequences. In many cases, these oligonucleotide probes, obtained from protein sequence analysis, have been the only route to the cloning of a particular gene.
Currently, protein sequence analysis is primarily accomplished with the use of an automated sequencer using chemistry developed by Edman over 40 years ago (1).sup.1 (FIG. 1). Since that time improvement in the instrumentation (2,3) has resulted in the ability to sequence smaller and smaller sample quantities (mmole to pmol), although the original chemistry has remained essentially unchanged. Current automated instrumentation permits 10-20 cycles of sequence determination on 10-50 pmol of sample.
Advances in protein isolation methodology have recently made it possible to isolate proteins of biological interest which are present in tissues in sub-picomole quantities. Techniques such as 1- and 2-dimensional electrophoresis (with electroblotting to membranes), microcolumn liquid chromatography, and capillary electrophoresis have allowed protein and peptide purification down to the 10-100 femtomole level. Many of these proteins have been shown to have key roles in the development and treatment of human disease. Improved methods of protein sequencing requiring less sample quantity would make it possible to obtain the necessary sequence information in order to clone and express these proteins, thereby making it possible to study the structure function aspects of these important proteins. It is generally anticipated that this information could set the stage for advances in the treatment of human diseases through rationalized drug design and gene therapy.
A major limitation to increasing the sensitivity of protein sequencing down to the femtomolar level involves the intrinsic detectability of the released phenylthiohydantoin (PTH) amino acids. The PTH amino acids, which are detected by absorption at 269 nm, have relatively low extinction coefficients. Intrinsic background noise associated with absorbance measurements at this wavelength and chemical background from the reagents used in sequencing also contribute to the limit of detection. Although a recently published method involving the use of absorbance detection with capillary electrophoresis rather than HPLC for separation of the PTH amino acids has shown femtomolar detection (4), this technique requires subnanoliter injection volumes. Current automated sequencer technologies dissolve the PTH amino acids in a 50-200 .mu.l volume for injection. The required use of only a small fraction o
REFERENCES:
patent: 5008205 (1991-04-01), Horn
patent: 5246865 (1993-09-01), Stolowitz
City of Hope
Redding David A.
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