WO1992000526A1 - Procede de sequençage de peptides et reactifs utilises a cette fin - Google Patents

Procede de sequençage de peptides et reactifs utilises a cette fin Download PDF

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Publication number
WO1992000526A1
WO1992000526A1 PCT/US1991/004533 US9104533W WO9200526A1 WO 1992000526 A1 WO1992000526 A1 WO 1992000526A1 US 9104533 W US9104533 W US 9104533W WO 9200526 A1 WO9200526 A1 WO 9200526A1
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Prior art keywords
amino acid
terminal amino
polypeptide
complex
reagent
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PCT/US1991/004533
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English (en)
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Kathleen Cox Gariepy
Paul T. Matsudaira
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Kathleen Cox Gariepy
Matsudaira Paul T
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Application filed by Kathleen Cox Gariepy, Matsudaira Paul T filed Critical Kathleen Cox Gariepy
Publication of WO1992000526A1 publication Critical patent/WO1992000526A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/12—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by hydrolysis, i.e. solvolysis in general
    • C07K1/128—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by hydrolysis, i.e. solvolysis in general sequencing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6818—Sequencing of polypeptides
    • G01N33/6824—Sequencing of polypeptides involving N-terminal degradation, e.g. Edman degradation

Definitions

  • This invention relates to sequencing a polypeptide or protein molecule in order to determine its amino acid sequence. More particularly, the invention relates to a novel composition of matter and a method for the micro sequencing of very small amounts of proteins. In a further embodiment, this invention relates broadly to amino acid analysis, and in particular to chromatographic separation and the easier and more sensitive sequencing of amino acids via electrochemical detection.
  • the linear sequencing of amino acid units in proteins and peptides is of considerable interest as an aid to understanding their biological functions and ultimately synthesizing compounds performing the same functions. Although a variety of techniques have been used to determine the linear order of amino acids, probably the most successful is known as the Edman Process. Briefly summarized, a typical Edman Process, includes the steps of coupling, cleavage and conversion.
  • the peptide of N-amino acids in length is coupled to a coupling agent, phenylisothiocyanate (PITC) , in an alkaline environment to form a phenylthiocarbamyl (PTC) derivative of the peptide.
  • PITC phenylisothiocyanate
  • PTC phenylthiocarbamyl
  • the excess reagent (a 500 to 10,000 molar excess) is removed by liquid-liquid extraction (usually in multiple steps) and the solvent is removed.
  • the PTC peptide derivative is subsequently treated with a cleavage reagent, strong anhydrous acid, to form the unstable anilinothiazolinone (ATZ) derivative of the amino-terminal amino acid and a free peptide or amino acid of N-1 length which is the original peptide with the terminal amino acid removed.
  • ATZ unstable anilinothiazolinone
  • the ATZ amino acids are treated with aqueous acid to convert them into the more stable phenylthiohydantoin amino acids (PTH-amino acids) for chromatographic identification.
  • PTH-amino acids phenylthiohydantoin amino acids
  • the shortened peptide or protein is employed as a starting material for another cycle of the Edman Process. Since many physiologically active proteins are present in organisms at such extremely small concentrations, only very small amounts of proteins are normally obtained for sequencing analysis. For example, most chemical sequencing methods are done with an amount of protein in the 100 picomole to 100 nanomole (10 ⁇ 10 - 10 ⁇ 8 mole) range.
  • LC liquid chromatography
  • liquid chromatography In liquid chromatography an unknown sample is injected into a column having a liquid therein consisting of one or more liquid solvents or a so-called mobile phase.
  • a process of differential retention separates the various compounds contained in a sample mixture so that they appear or elute from the bottom of the column at different times.
  • the detector at the base of the column detects the presence of the solute constituents as they appear or elute from the column.
  • Two general types of LC detectors are used: 1. bulk property or general detectors measure a change in some overall physical property of the mobile phase plus the solute; 2. solute property or specific detectors which are sensitive only to some property of the solute (e.g. UV absorption) .
  • detectors which have so far found application in LC include: 1. colorimeters combined with color-forming reactions of separated sample components; 2. refractive index detectors; 3. UV-visible and fluorescent spectrophotometric -detectors; 4. amperometric (electrochemical) detectors.
  • a plot of detector output as a function of time, known as a chromatogra is used by the chromatographer for his analysis of the unknown sample.
  • electrochemical (EC) detectors now rival the widely-used fluorescence types for detecting small quantities of proteins and peptides.
  • EC detectors are sensitive to a relatively wide variety of compounds, as illustrated in Table 1.
  • the compounds in this list include those that also generally can be detected by UV absorption.
  • compound types e.g. mercaptans, hydroperoxides, etc.
  • TABLE 1 Some compound types sensed by the electrochemical detector.
  • Derivative formation of sample components prior to the LC separation can be useful for several reasons: (a) to improve the initial extraction or prepurification of compounds of interest from the sample matrix, (b) to improve the subsequent LC separation by reducing the polarity of selected compounds, (c) to decrease the relative detector response of compounds of no interest in the sample so that compounds of interest can be detected and quantitated, and (d) to increase the detector response for certain compounds in the sample.
  • the last goal is of major interest; and this area receives primary emphasis in the following discussion.
  • There have been numerous compounds used in precolumn derivatization of amino acids mostly electrophiles capable of reacting with a free alpha-amino group.
  • reagents include 5-dimethylaminonapthalene-l-sulfonyl (dansyl) chloride, the dansyl analog 4-dimethylaminoazoenzene-4'-sulfonyl (dansyl) chloride, 2,4-dinitrofluorobenzene, 4-chloro-7-nitrobenzo-2-oxa- 1,3-diazole (NBD-C1) and compounds related to NBD-C1.
  • NBD-C1 4-chloro-7-nitrobenzo-2-oxa- 1,3-diazole
  • OPA o- phthaladehyde
  • Potential derivatization reagents include fluorescent tags such as 9-anthryldiazomethane and broraomethoxycoumarin, the former reagent allowing low or even subpicomole detection. See, e.g., T. Yoshida, et al, J. Chromatoger. 348, pp. 425-429 (1985). However, this approach is unpopular most probably due to the poor reactivity of the alpha-carboxyl group, lack of selectivity of the reagents, and commercial unavailability of the reagents.
  • a procedure for amino acid analysis based on precolumn derivatization with phenylisothiocyanate (PITC) has been gaining popularity for the past several years. See, e.g.
  • a PTC derivative can be detected at the 1-pmol level via UV detection. See, e.g. K. Muramoto et al, Agr. Biol. Chem. 4_2, pp. 1559-1563 (1978).
  • This level of performance provided the first realistic alternative to ion-exchange analysis employing ninhydrins while retaining desirable features associated with the traditional methods of liquid chromatography.
  • the phenylisothiocyanate degradation process has been a valuable tool in amino acid sequence determination, one of its inadequacies which remains, however, is the unsatisfactory sensitivity when identifying low amounts of the PTH-amino acids with UV detectors.
  • a new type of reagent polypeptide complex has been prepared which contains functional groups designed to contribute in providing electrochemical activity.
  • the reagent polypeptide complex comprises the formula A-B-C-D wherein: A is a polypeptide which contains an N-terminal amino acid unit B; C is an aromatic ring which contains a functional group which can react with and bind to the N- terminal amino acid of the polypeptide and can result in unit derivatization of the terminal amino acid; and D comprise those moieties attached to the aromatic ring that contribute to the electrochemical activity of the derivatized N-terminal amino acid and are selected from those functional groups that do not interfere with the reactivity of the functional groups of the C moiety in a derivatization process.
  • the functional groups on the aromatic C moiety which can react with and bind to the N-terminal amino acid are selected from the group consisting of:
  • the D moieties comprise groups that are attached to the aromatic ring and are selected from, the group consisting of -OH, -OCH3, -Cl and -Br.
  • the D moieties may also comprise the structure -OX, wherein X does not interfere with the electrochemical activity of the adjacent oxygen atom nor with the derivatization process.
  • Table 2 provides information with respect to the limits of electrochemical detection for a given chromatographic system when m-hydroxy phenylisothiocyanate (m-PITC) , the preferred aromatic isothiocyanate, is employed for amino acid analysis, and is codependant upon electroactivity and retention time.
  • m-PITC m-hydroxy phenylisothiocyanate
  • a method for determining the identity of an N-terminal amino acid of the reagent polypeptide complex comprises: (a) providing a reagent polypeptide complex of the formula A-B-C-D as defined above; (b) cleaving the terminal amino acid from the polypeptide complex to form an aggregate of the cleaved N-terminal amino acid complex B-C-D and the cleaved polypeptide which then contains an N-terminal amino acid unit B' ; (c) separating the N- terminal amino acid complex B-C-D from the cleaved polypeptide by, e.g.
  • inventive process further comprises sequencing the polypeptide by repeating the steps (a) - (e) on the cleaved polypeptide from step (c).
  • FIG. 1 illustrates the HPLC chromatograms for amino acids derivatized with phenylisothiocyanate to give the phenylthiohydantoin derivative detected by UV absorbance (top) and by electrochemical detection (bottom).
  • FIG. 2 illustrates the HPLC chromatograms for amino acid derivatization with m-hydroxy phenylisothiocyanate to give meta-hydroxy phenylthiohydantoin derviatives when both UV and EC detectors are used in series.
  • the top chromatogram represents the output employing a conventional UV detector.
  • the bottom one represents electrochemical detection.
  • FIG. 3 illustrates the - ⁇ H-NMR of the compound m- hydroxy phenylisothiocyanate.
  • the present invention is a general system for the sequencing or analysis of peptides or proteins starting from the N-terminal end thereof and is effective for very small quantities of starting material.
  • the N-terminal amino acid can be identified by electrochemical detection techniques from a reagent polypeptide complex comprising the formula A-B-C- D, wherein: A is a polypeptide which contains an N-terminal amino acid unit B; C is an aromatic ring which contains a functional group which can react with and bind to the N- terminal amino acid of the polypeptide and can result in unit derivatization of the terminal amino acid; and D comprise those moieties attached to the aromatic ring that contribute to the electrochemical activity of the derivatized N-terminal amino acid and are selected from those functional groups that do not interfere with the reactivity of the functional groups of the C moiety in a derivatization process.
  • the functional groups on the aromatic C moiety which can react with and bind to the N-terminal amino acid are selected from the group consisting of:
  • the D moieties comprise groups that are attached to the aromatic ring.
  • the D moieties are selected from the group consisting of -OH, -OCH3, -Cl and -Br.
  • the D moieties have the structure -OX, wherein X does not interfere with the electrochemical activity of the adjacent oxygen atom nor with the derivatization process.
  • the reagent polypeptide complex as described above can then be converted, via the Edman Process, to a unit derivatized N-terminal amino acid which comprises an anilinothiazolinone, phenylthiocarbonyl, or phenylthiohydantoin type derivative. Accordingly, an electrochemically active PTH amino acid derivative of the following structure is obtained:
  • R can be any aliphatic, aromatic, or mixed aliphatic/aromatic group and the moieties X, Y and Z are selected from those groups that combine to contribute to the electrochemical activity of the amino acid derivative.
  • the moieties X, Y and Z are selected from those groups that have an electron-donating effect on the aromatic ring.
  • the moieties X, Y and Z are selected from the group consisting of -OH, -OCH3 , -Cl and -Br.
  • the remaining moieties can also be selected from the above group.
  • the reagent polypeptide complex is prepared from meta-hydroxy phenylisothiocyanate.
  • the - ⁇ H-NMR of this compound is shown in FIG. 3.
  • This compound is now commercially available from ESA, Inc. of Bedford, Massachusetts.
  • aromatic isothiocyanates provide for electrochemical activity when employed for amino acid analysis and sequencing of peptides or proteins according to the Edman degradation process as well as for electrochemical analysis of traditionally non-electroactive primary and secondary aliphatic amines.
  • Figure 1 compares amino acids reacted with phenylisothiocyanate to form the phenylthiohydantoin derviatives and are separated by reverse phase HPLC.
  • the method for determining the identity of an N-terminal amino acid of a polypeptide comprises: (a) providing a reagent polypeptide complex of the formula A-B-C-D as defined above; (b) cleaving the terminal amino acid from the polypeptide complex to form an aggregate of the cleaved N-terminal amino acid complex B-C-D and the cleaved polypeptide which then contains an N-terminal amino acid unit B'; (c) separating the N- terminal amino acid complex B-C-D from the cleaved polypeptide by, e.g.
  • the inventive process further comprises sequencing the polypeptide by repeating the steps (a) - (e) on the cleaved polypeptide from step (c) for a given number of cycles.
  • the identification of the stable compound in step (d) comprises passing the stable compound reaction mixture, minus the polypeptide N-1, through a chromatographic column. The effluent is monitored electrochemically such that the stable compound is detected, and identified according to its position on the chromatogram.
  • the N-terminal amino acid complex is cleaved from the polypeptide by treatment with acid to form an anilinothiazolinone type N-terminal amino acid complex.
  • the anilinothiazolinone type N- terminal amino acid complex is then separated from the cleaved polypeptide and this can be achieved by extraction with an organic solvent in a wash step. It can then be converted to a stable m-hydroxy PTH amino acid compound and identified by passing the phenylthiohydantoin type amino acid compound through a chromatographic column such that the effluent containing the m-PTH amino acid compound is detected electrochemically and identified according to its position on the chromatogram.
  • the sample is introduced into a reverse phase column. Column temperature is maintained at 50°C.
  • Said mobile phase is a combination of both A and B whereby the gradient from the perspective of B is varied from 0-30% over the first minute, 30-55% over 1 to 4 minutes, and 55% for 4-12 minutes. Regulation of this mobile solution phase includes a variation in the percent of NH4-acetate and CH3C , the pH, elution time and column temperature.
  • a Model 5100A Coulochem Multielectrode Electrochemical Detector is employed for electrochemical detection.
  • a base deactivated column was employed with the following separation method: Mobile Phase: 0.03M NaOAc (sodium acetate) pH 4.91 with H3P0 4 in 24% acetonitrile Column: Supelco pKblOO, 25 cm, 4.6 mm ID Temperature: ambient Flow: 1.0 ml/minute
  • the method comprises the following steps when employed for the manual sequencing of ⁇ -lactogobulin:

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Abstract

Procédé de séquençage d'une protéine utilisant une composition de matières comprenant un complexe polypeptide réactif A-B-C-D dans lequel A représente un polypeptide qui contient une unité B d'aminoacide à terminaison N, C représente un noyau aromatique qui contient un groupe fonctionnel pouvant réagir à, et se lier avec, l'acide aminoacide à terminaison N du polypeptide et pouvant provoquer une dérivation de l'unité de l'aminoacide de terminaison et D comprend les fragments fixés au noyau aromatique qui contribuent à l'activité électromagnétique de l'aminoacide de terminaison N dérivé et qui sont choisis dans les groupes fonctionnels qui ne perturbent pas la réactivité des groupes fonctionnels du fragment C dans le procédé de dérivation. Dans ce procédé, l'identité d'un aminoacide à terminaison N est établie au moyen de la dérivation avec ledit réactif A-B-C-D, suivie d'une analyse chromatographique et d'une détection électrochimique.
PCT/US1991/004533 1990-06-29 1991-06-25 Procede de sequençage de peptides et reactifs utilises a cette fin WO1992000526A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5780597A (en) * 1989-12-22 1998-07-14 Hoffmann-La Roche Inc. Monoclonal antibodies to cytotoxic lymphocyte maturation factor

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4548904A (en) * 1982-12-03 1985-10-22 Molecular Genetics Research & Development Protein sequencing method
US4665037A (en) * 1986-04-28 1987-05-12 Analytichem International, Inc. Method of sequencing peptides

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4548904A (en) * 1982-12-03 1985-10-22 Molecular Genetics Research & Development Protein sequencing method
US4665037A (en) * 1986-04-28 1987-05-12 Analytichem International, Inc. Method of sequencing peptides

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5780597A (en) * 1989-12-22 1998-07-14 Hoffmann-La Roche Inc. Monoclonal antibodies to cytotoxic lymphocyte maturation factor
US6683046B1 (en) 1989-12-22 2004-01-27 Hoffmann-La Roche Inc. Purification and characterization of cytotoxic lymphocyte maturation factor and monoclonal antibodies thereto

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