EP0397813A1 - Sequenzierung von peptiden oder proteinen - Google Patents

Sequenzierung von peptiden oder proteinen

Info

Publication number
EP0397813A1
EP0397813A1 EP89911635A EP89911635A EP0397813A1 EP 0397813 A1 EP0397813 A1 EP 0397813A1 EP 89911635 A EP89911635 A EP 89911635A EP 89911635 A EP89911635 A EP 89911635A EP 0397813 A1 EP0397813 A1 EP 0397813A1
Authority
EP
European Patent Office
Prior art keywords
amino acid
peptide
terminal
proteins
acetylated
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP89911635A
Other languages
English (en)
French (fr)
Inventor
Anthony David Auffret
Alastair Aitken
Timothy Charles Farries
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Merck Biosciences AG
Original Assignee
Novabiochem AG
Pharmacia AB
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novabiochem AG, Pharmacia AB filed Critical Novabiochem AG
Publication of EP0397813A1 publication Critical patent/EP0397813A1/de
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • 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

  • the invention relates to a method for the determination of amino acid sequences in which the N-terminal amino acid is acylated and thereafter selectively cleaved off by an acylamino hydrolase for analysis. This cycle is repeated for each amino acid in the protein or peptide to be analyzed.
  • Proteins and peptides are composed of a linear combination of amino acid residues linked via peptide bonds with the general formula.
  • R is any one of a series of chemical side chains which differentiate individual amino acids and "n" is zero or an integer.
  • One end of the chain carries an amino group (NHA and is termed the “amino” or “N” terminus, the other end carries a carboxyl (COOH) group and is termed the “carboxyl” or “C” terminus.
  • NHA amino group
  • COOH carboxyl
  • C carboxyl
  • the N-terminus is acylated for example with a formyl (HCO-) or an acetyl (CH 3 C0-) group.
  • amino acid sequence The order of the amino acid residues in the peptide or protein chain is termed the amino acid sequence or primary structure. The determination of this amino acid sequence is often called "protein sequencing".
  • Methods for the determination of protein sequences usually involve stepwise degradation which may be chemical or enzymatic and proceed from the N- or from the C-terminus.
  • stepwise degradation which may be chemical or enzymatic and proceed from the N- or from the C-terminus.
  • PITC phenylisothiocyanate
  • the first protein sequence to be determined (insulin) , however, used a method that did not involve stepwise degra ⁇ dation.
  • the protein was fragmented and the component peptides isolated.
  • the N-terminal residue was identified by chemical labelling and the peptide amino acid composition determined. Exhaustive examination of sufficient peptides identified a unique sequence that fitted all the data.
  • N-terminal chemical methods Edman's PITC method is used almost exclusively. Many variants exist using alternative isothiocyanate derivatives but these are less popular. This method is uniquely successful because it separates reagent addition from cleavage of the terminal amino acid residue. Thus one can ensure complete reagent addition before per ⁇ mitting cleavage. Only the intermediate phenylthiocarbamyl terminal residue can cyclise and cleave and as this step is " done in the absence of the PITC reagent the newly exposed terminal residue cannot undergo the reaction. This chemical escapement mechanism ensures a controlled stepwise degra ⁇ dation in discrete one amino acid residue steps. 9 00586
  • N-Terminal enzymatic methods The use of aminopeptidase enzymes (e.g. leucine amino peptidase, L.A.P.) or even of amino dipeptidases have been documented. They have a universal drawback in that there is not "escapement" mechanism to ensure discrete stepwise degradation in the population of protein molecules. In an individual molecule when the N-terminal amino acid residue is removed the enzyme may immediately proceed to cleave the newly exposed N-terminal residue before the reaction has gone to completion in other molecules. Cleavages within the population simply get out of register.
  • aminopeptidase enzymes e.g. leucine amino peptidase, L.A.P.
  • C-Terminal enzymatic methods The use of carboxypeptidases is analogous to the use of aminopeptidases and suffers from the same drawback.
  • Enzymes that specifically remove acetylated N-terminal amino acid residues have been described in a number of mammalian tissues and are referred to as acylamino acid releasing enzymes or acylpeptide hydrolases, see for instance Gade W. (1978) , Jones W.M. (1985) , Radhakrishma (1986) , Tsunasawe (1976) , Wold F. (1984) and Kobayashi (1987) . The latter indicating the use of acylpeptide hydrolase for deblocking naturally occuring acetylated peptides. Enzymes of this class may be important in the cellular processing of immature proteins.
  • This invention describes a novel use of such enzymes as a means of determining protein amino acid sequences.
  • the invention is characterised in that unlike existing methods it concerns not the removal of amino acids but the removal of chemically or biochemically labelled amino acids in cycle for sequential analysis.
  • the terminal residue of the protein or peptide to be seq enced is chemically or enzymatically blocked by an acetylating agent and then enzymes active in the hydrolysis of blocked terminal amino acids are used.
  • the new terminal residue of the substrate peptide or protein is not cleaved as it contains a free terminal moiety. Thus a molecular escapement mechanism restricting hydrolysis is effected.
  • the hydrolysis reaction is completed the released amino acid derivative may be identified (particularly but not exclusively) by chromatographic or spectroscopic methods and the shortened substrate peptide or protein may be chemically or enzymatically blocked and the process repeated.
  • the invention accordingly concerns a method of sequencing peptides or proteins by stepwise removal of modified terminal amino acid residues and subsequent analysis of the released amino acid.
  • the cycle which is repeated for each amino acid comprises the following step: (i) acylation of the N-terminal amino acid, (ii) removal of the acylated amino acid and (iii) analysis of the released acylated amino acid.
  • the N-terminal amino acid is acetylated and the advantages of using the method of the invention is well demonstrated in this system.
  • Methods for acetylation are known from the literature, see for instance Thomas D.W. et al (1968) .
  • One example of a convenient method is the use of an acid anhydride, e.g. acetic acid anhydride.
  • the thus N-terminal acetylated residue is treated with an enzyme preparation having acyl-peptide hydrolase activity.
  • This enzyme preparation could be a commercially available enzyme like for instance Tsunasawa S. et al (1982) or be prepared from an appropriate source like for instance from mammalian kidney, e.g. ovine kidney.
  • acylamino acid hydrolase activity was purified from ovine kidney. Fresh kidney was homogenised in phosphate buffer and the extract was filtered and then cen- trifuged (100,000 x g) . The supernatant was fractionated on DEAE-Sephacel (Pharmacia) using a salt gradient in the same buffer. The crude enzyme preparation was concentrated and further purified either directly by gel filtration (Sepharose 6B-100) or by filtration on Mono-Q Sepharose (Pharmacia) followed by gel filtration on Sepharose 6B-100.
  • acetyl-Met-Ala-Ser peak after hydrolysis is reduced and 2 new peaks acetyl-Met (MH+ 192 amu) and Ala-Ser (MH+ 117 amu) confirm hydrolysis. This establishes the partial sequence acetyl-Met- (Ala, Ser) .
  • acetylated peptide residue was then incubated with 0.0125 units of acylamino acid releasing enzyme (Takara Biomedical, Japan) in 0.1 ml ammonium acetate pH 8/1 mM 2-mer ⁇ aptoethanol for 1 hour at 37 C. After freeze drying a portion was examined by mass spectrometry.
  • N-acetylmethionine from acetylated peptides N-acetylmethionine from acetylated peptides.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Hematology (AREA)
  • Biochemistry (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Cell Biology (AREA)
  • Wood Science & Technology (AREA)
  • Food Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
EP89911635A 1988-11-03 1989-10-24 Sequenzierung von peptiden oder proteinen Ceased EP0397813A1 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE19888803986A SE8803986D0 (sv) 1988-11-03 1988-11-03 Method for sequencing peptides or proteins
SE8803986 1988-11-03

Publications (1)

Publication Number Publication Date
EP0397813A1 true EP0397813A1 (de) 1990-11-22

Family

ID=20373849

Family Applications (1)

Application Number Title Priority Date Filing Date
EP89911635A Ceased EP0397813A1 (de) 1988-11-03 1989-10-24 Sequenzierung von peptiden oder proteinen

Country Status (5)

Country Link
EP (1) EP0397813A1 (de)
JP (1) JPH03502284A (de)
AU (1) AU4410689A (de)
SE (1) SE8803986D0 (de)
WO (1) WO1990005192A1 (de)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2026205A1 (en) * 1989-02-08 1990-08-09 Miro Rusnak Apparatus and method for the sequential performance of chemical processes
US5470753A (en) * 1992-09-03 1995-11-28 Selectide Corporation Peptide sequencing using mass spectrometry
JP3795534B2 (ja) * 1997-01-23 2006-07-12 イクスツィリオン ゲゼルシャフト ミット ベシュレンクテル ハフツング ウント コンパニー コマンディトゲゼルシャフト ポリペプチドの特性検査

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9005192A1 *

Also Published As

Publication number Publication date
JPH03502284A (ja) 1991-05-30
WO1990005192A1 (en) 1990-05-17
AU4410689A (en) 1990-05-28
SE8803986D0 (sv) 1988-11-03

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