METHOD FOR SEQUENCING PEPTIDES OR PROTEINS

Abstract: 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.

Description: Method for sequencing peptides or proteins 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 . NH2-CH-C0-(NH-CH-C0)n-NH-C,H-COOH R R Where 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. It is not uncommon that the N-terminus is acylated for example with a formyl (HCO-) or an acetyl (CH 3 C0-) group. 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. Almost universal in use is the chemical N-terminal method using phenylisothiocyanate (PITC) or derivatives thereof. It is usual in the cases of proteins to prepare fragments (peptides) and apply the methods to these constituent peptides. 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. For each peptide 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. With the exception of mass spectrometry, in which the amino acid sequence is established by the fragmentation pattern of the peptide in the spectrometer, most current methods utilise stepwise removal of an amino acid from either the N- or C-terminus. Removal of the terminal residue produces a free amino acid or derivative and a shortened peptide with a new terminal residue which can be subjected to a subsequent degradation cycle. 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 Alternative stepwise N-terminal chemical methods have been devised, e.g. thioacetyl thioglycollic acid, but they are not as successful. C-Terminal chemical methods: Chemistries that mimic the N-terminal degradations, based for example on thiocyanate or oxazoline chemistries, have been proposed. None have come into general use. 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. C-Terminal enzymatic methods: The use of carboxypeptidases is analogous to the use of aminopeptidases and suffers from the same drawback. All the above methods are characterised in that a terminal amino acid residue is cleaved by chemical or enzymatic means. Peptides and proteins in which the terminal residue is further derivatised are not suitable for these processes. For example a significant number of proteins occur naturally with an acylated (often acetylated or formylated) amino terminus. It is not possible to directly analyse these proteins by the chemical or enzymatic N-terminal methods described. We have considered the difficulties encountered in sequencing proteins or peptides in which the terminal residue is blocked (e.g. N-acylated proteins) . 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. Upon hydrolysis 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. When 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. In one embodiment of the invention 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. The purification of an enzyme preparation having the desired activity is described in the below experimental section. Only the acetylated terminal residue is removed and the shortened substrate peptide has an unmodified N-terminus. Therefore this peptide can be reacetylated and the next residue removed. Thus the sequential stepwise removal and identification of amino acids can be achieved. This is a general method and is applicable to solution phase, solid phase and vapour phase sequencing technologies. Peptides and proteins that are naturally acetylated are directly amenable to sequencing by this method. The use of isotopically labelled acetic anhydride in the first cycle would clearly establish if the acetyl group was naturally occuring or if it arose from the sequencing reactions. Experiments For a test case an 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. Example 1 20 micromoles of the peptide Met-Asp-Ser-Pro-Ser-Asp were dissolved in 0.25 ml of 0.1 M KH^PO./NaOH buffer pH 7.5. 0.2 ml of a solution of 40 mCi/ml (3H) - acetic anhydride (7.78 Ci/mmole) in 10 mM KH 2 P0 4 / aOH buffer pH 4.7 at 0 °C were added and the mixture incubated for 1 hour at 0 °C. 4 microlitres (40 micromoles) of a solution of unlabelled acetic anhydride were then added and the mixture incubated for a further 1 hour at 0 °C. The acetylated peptide g (10 dpm/20 micromoles) was recovered by reverse phase HPLC and then freeze dried. The acetylated peptide (200,000 dpm/sample) was incubated with ovine kidney enzyme fraction at 37 C for 30 minutes in 0.1 ml of 50 mM KH PO /NaOH buffer pH 7.2. 10 microlitres of 100 % (w/v) triσhloroacetic acid were added and the mixture centrifuged at 10,000 x g for 5 minutes. Supernatant (0.1 ml) was diluted with water (0.5 ml) and separated by reverse phase HPLC. (Column = RP-18, OD-032, SPHERI-5, 30 x 2.1 mm, 5 micron). A concave gradient, 0.1 % trifluoroacetic acid (TFA) to 100 % acetonitrile/0.1 % TFA, at a flow rate of 1 ml/min was used with on line radioactivity detection (Radiomatic detector using 4 ml/min Pico-Fluor 40) . Example 2 The use of cycles of acetylation and hydrolysis to determine the amino acid sequence of a peptide was demonstrated using the peptide acetyl-Met-Ala-Ser. 300 nmoles of peptide were treated with 0.0125 units of acylamino acid releasing enzyme (Takara Biomedical, Japan) in 0.1 ml 0.1 M ammonium acetate pH 8/1 mM 2-mercaptoethanol for 1 hour at 37 C and was then freeze dried. A portion was analysed by mass spectrometry before and after hydrolysis. The 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) . A second cycle of acetylation and hydrolysis was then performed. The remaining portion was acetylated for 1 hour at room temperature in 0.2 ml of acetic acid/acetic anhydride (1:1 v/v) . 0.5 ml water were added and after 5 minutes the mixture was centrifuged (10,000 x g, 5 minutes) and freeze dried. The residue was redissolved in water and the acetylated peptide isolated by reverse phase chromatography (C18, 220 x 2.1 mm in 0.1 % TFA) , freeze dried and a portion was examined by mass spectrometry. The 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-merσaptoethanol for 1 hour at 37 C. After freeze drying a portion was examined by mass spectrometry. The spectra prior to hydrolysis and after hydrolysis were compared. In the absence of enzyme acetyl-Ala-Ser (MH+ 219 amu) is clearly visible but declines after hydrolysis. Acetyl-Ala (MH+ 132 amu) and Ser (MH+ 106 amu) appear after hydrolysis. This indicates a sequence acetyl-Ala-Ser for the peptide recovered from cycle one and therefore the original peptide must have had the structure acetyl-Met-Ala-Ser. References: Gade, W. & Brown, J.L. (1978) Purification and partial characterisation of alpha-N acylpeptide hydrolase from bovine liver. J. Biol. Chem. 253:5012-5018 Jones, W.M. & Manning, J.M. (1985) Acylpeptide hydrolase activity from erythrocytes. Biochem, Biophys. Res. Commun. 128:933-940 Kobayashi, K. & Smith, J.A. (1987) Acyl-peptide hydrolase from rat liver. Characterisation of enzyme reaction. J. Biol. Chem. 262:1143-1145 Radhakrishma, G. & Wold, F. (1986) Rabbit muscle extracts catalyze the specific removal of N-acetylmethionine from acetylated peptides. J. Biol. Chem. 261:9572-9575 Thomas, D.W. et al (1968) Biochem Biophys Res Commun 33:519-521 Tsunasawe, S. & Narita, K. (1976) Acylamino acid-releasing enzyme from rat liver. Meth. Enzymol. XLV 552-561. Tsunasawe, S. & Narita, K. (1982) Meth. Enzymol. 46:852-861 Wold, F. (1984) Acetylated N-terminals in proteins - a perennial enigma. Trends in Biochemical Sciences 9:256-257

Claims: Claims 1. A method of sequencing peptides or proteins by stepwise removal of modified terminal amino acid residues and subsequent analysis of the released amino acid, c h a r a c t e r i z e d in that the N-terminal amino acid is acylated and then removed by enzymatic cleavage. 2. A method according to claim 1, c h a r a c t e r i z e d in that the N-terminal amino acid is acetylated. 3. A method according to claim l or 2, c h a r a c t e r i z e by the use of an acylamino hydrolase. 4. A method according to claim 3, c h a r a c t e r i z e d in that the enzyme has been purified from ovine kidney.

Inventors: AITKEN ALASTAIR, AUFFRET ANTHONY DAVID, FARRIES TIMOTHY CHARLES, AITKEN, ALASTAIR, AUFFRET, ANTHONY, DAVID, FARRIES, TIMOTHY, CHARLES
Applicants: PHARMACIA AB
Application year: 1989
Main classification: C12Q1
IPC codes: C12Q1, G01N33