WO1992000526A1

WO1992000526A1 - Peptide sequencing method and reagents therefor

Info

Publication number: WO1992000526A1
Application number: PCT/US1991/004533
Authority: WO
Inventors: Kathleen Cox Gariepy; Paul T. Matsudaira
Original assignee: Kathleen Cox Gariepy; Matsudaira Paul T
Priority date: 1990-06-29
Filing date: 1991-06-25
Publication date: 1992-01-09

Abstract

A protein sequencing method utilizing a composition of matter comprising a reagent polypeptide complex 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 comprises 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. In its method form, the identity of an N-terminal amino acid is established through derivatization with said A-B-C-D reagent followed by chromatographic analysis and electrochemical detection.

Description

PEPTIDE SEQUENCING METHOD AND REAGENTS THEREFOR

Field of Invention

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. 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. In the final stage of the process, conversion, 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. 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. Other methods used in the micro sequencing of polypeptides involve radio labeling of the peptide or reagent, intrinsic radio labeling of the polypeptide, and enhanced UV detection of sequence degradation products, and others. See, e.g. U.S. Patent No. 4,548,904.

IDENTIFICATION OF PTH-AMINO ACIDS In the broad field of analytical instruments, chromatographs have been used to separate and measure constituents of complex mixtures at such low concentrations. Liquid chromatography (LC) is known to be ideally suited for the separation of macromolecules and ionic species of biomedical interest, labile natural products, and a wide variety of other high-molecular weight and/or less stable compounds such as proteins, nucleic acids, amino acids, dyes, polysaccharides, plant pigments, polar lipids, explosives, synthetic polymers, surfactants, pharmaceutical and plant and animal metabolites. 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) . Which type of detector should be used for a particular problem depends on the characteristics of the solute, the sensitivity and selectivity required, and the convenience and versatility desired. The specific 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. For a complete review of detector performance, see, for example, Introduction to Modern Liquid Chromatography, 2nd Edition, L.R. Snyder and J. J. Kirkland. pp. 126-167 (1979). Of these detectors, electrochemical (EC) detectors now rival the widely-used fluorescence types for detecting small quantities of proteins and peptides. Furthermore, 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. There are, however, several compound types (e.g. mercaptans, hydroperoxides, etc.) sensed by the EC detector that either cannot be detected at all by UV absorption or can be detected only with difficulty at low wavelengths. TABLE 1 Some compound types sensed by the electrochemical detector.

* Depending on structure. A major problem with respect to amino acid analysis, however, is one of sensitivity rather than resolution. Few of the protein amino acids have side chains with functional groups which permit direct sensitive detection by the detectors noted above, when in solution. Those that do— henylalanine, tyrosine and tryptophan—owe their various UV-absorbing, fluorescent or electrochemical activities to their substituted aromatic groups, and these also confer reasonable retention but primarily on a reversed-phase (RP) type column. Thus, aromatic amino acids and their biologically important metabolites can be determined in their free form by reversed phase high pressure liquid chromatography. Note that RP chromatography is so named because it behaves in the way opposite to normal phase chromatography. For a complete comparison of adsorption (normal phase or liquid-solid) , partition (liquid-liquid), ion exchange and reversed-phase chromatography see, e.g. HPLC of Small Molecules, A Practical Approach., C.K. Kim, Ed., pp. 1-12 (1986). For a general determination of protein amino acids, however (e.g., those without active aromatic functional groups), precolumn derivatization is universally employed for detection and quantification. 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. In most cases, and as noted earlier in the case of protein amino acids, 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. These 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. Until recently, the most widely reported reagent was o- phthaladehyde (OPA) , which in conjunction with a thiol reagent reacts with primary amine groups to form highly fluorescent isoindole products. Despite the convenience of automated derivatization, exquisite sensitivity and rapid analysis times associated with OPA-based amino acid analysis (AAA) , it has not achieved the high level of use initially predicted for this procedure. Lack of reactivity with secondary amino acids has been the predominant drawback. See, e.g. P. Lindroth et al, Anal. Chem. , ^, pp 1667-1674 (1979). Secondary amino acids can be rendered reactive by the addition of chloramine T, a highly reactive oxidizing agent prior to the addition of OPA. However, the poor reproducibility of this method has hindered its widespread acceptance. Even without the oxidation step, OPA analysis has also been reported to be less reproducible than desired by glycine and lysine, presumably due to the poor stability of their derivatives formed with beta- mercaptoethanol as the thiol. More recently, FM0C-C1 (9-fluoroenyl methyl chloroformate) , a reagent originally introduced as a blocking agent in peptide synthesis, has been employed for amino acid analysis. See, e.g. Einarsson et al, J. Chromatogr., 282, pp.609-618 (1983). This compound reacts rapidly with both primary and secondary amino acids to form highly fluorescent products. However, a major problem exists with histidine, which can form either a mono- or disubstituted derivative; recent reports indicate wide variability in the relative yields of the two derivatives. Also troublesome is the fact that the hydrolysis product with water, FMOC-OH, also fluoresces, and when FM0C-C1 is used in great excess (as with an unknown trace level sample), reagent-caused interference can be significant. Extraction of residual reagent with organic solvents slightly improves this problem, with single-step extraction efficiencies of approximately 70%. See, e.g. R. Cunico et al, BioChromatography 1., pp. 6-14 (1986). Derivatization of the carboxy group of amino acids is a technique which has been rarely employed. 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. Amino Acid Analysis utilizing Phenylisothiocyanate Derivatives, S. Cohen and D. Strydom, Anal. Biochem. 174(1) , pp. 1-16 (1988). The phenylthiocarbamyl (PTC) amino acids formed during this procedure can be detected with high sensitivity at 254 nm following separation of the derivatives by reverse phase chromatography. Formation of PTC derivatives in this fashion constitutes a step in the three-step sequential Edman degradation process described earlier, the major means of determining the primary structure of peptides and proteins. The three step Edman procedure and associated method of sequencing polypeptides utilizing liquid-solid affinity chromatography has been described in U.S. Pat. No. 4,665,037. There are several key features of the phenylthiocarbamylation chemistry that has encouraged further development of PTC-AAA. The specificity of PITC is well characterized, and there is no evidence for the formation of any disubstituted derivatives with Tyr or His. As expected, only cystine (C s2) and Lys react with two PITC molecules. Under mild conditions the reaction is essentially complete in less than 10 minutes and, because the reagent is volatile, a large excess can be used; this excess can be readily removed under reduced pressure. Thus, only small, easily separated reagent-related components remain, and reagent interference is minimal. Although not as sensitive some of the fluorescent reagents, if grafted to a fluorescent moiety, 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. Although 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. While the response to this problem, as noted above, has led to the preparation and investigation of fluorescent isothiocyanates, this approach has led to new and additional problems. First, the incorporation of large fluorescent moieties serves to sterically hinder and limit the reactivity of the isothiocyanate functionality. Furthermore, the fluorescent moieties generally have an electron effect which is also adverse to the isothiocyanate with respect to any subsequent reactivity with amino acids. Some workers have suggested that this reduction in reactivity accounts for only 25-50% of terminal amino groups becoming derivatized as the first coupling reaction. See, e.g. J.Y. Change et al, FEBS Lett., 9^(2) pp. 205-214 (1978). Such limitations have prevented the conventional Edman method from being a more practical micro-sequencing technique. Furthermore, although normal PTH derivatives have been suitable for UV detection, they have yet to demonstrate sufficient electrochemical activity so that sensitive electrochemical detection methods can be employed. In other words, the PTH derivatives have not shown uniform capacity for electrochemical detection. It is thus a primary object of the present invention to provide a novel and improved system, i.e. a novel method of precolumn derivatization, which collectively overcomes the aforesaid and other problems and limitations of the prior art. Another primary object includes the preparation of an isothiocyanate compound which is more effective in coupling with amino acids to provide quantitative derivatization without the need for subsequent treatment with the isothiocyanate compound to remove unreacted free amino groups. Yet a further object is to provide a novel and improved method for preparing amino acid derivatives that are electrochemically active in order to qualitatively and/or quantitatively determine the presence of selected substances in the sample. A more specific object is to provide a novel and improved electrochemical detection method for analyzing the eluant from a liquid chromatographic system separation. Yet other objects of the invention will in part appear obvious and will in part appear hereinafter. BRIEF DESCRIPTION OF THE INVENTION 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:

S O S ll ii n -N-OS;-N-00;-C-8-R; and -C-NH-C-R

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. TABLE 2: mPTH-Amino Acid Limits of Detection

AMINO ACID RETENTION RANGE (MIN) LOD(fmol)

2.31-2.37 25

2.69-2.76 50

2.97-3.09 50

3.66-3.81 50

4.73-4.99 50

6.67-7.10 50

7.00-7.58 50 valine/methionine 11.41-12.10 50 proline 12.01-1313 25 lysine 17.58-18.27 250 isoleucine 22.35-23.80 100 phenylalanine 23.38-25.08 100 leucine 25.80-26.99 250

In the inventive process, a method for determining the identity of an N-terminal amino acid of the reagent polypeptide complex is described which 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. a wash step; (d) converting the N- terminal amino acid complex from step (c) to a stable compound; and (e) identifying the stable compound formed in step (d) using electrochemical detection techniques. In a further embodiment, the inventive process further comprises sequencing the polypeptide by repeating the steps (a) - (e) on the cleaved polypeptide from step (c).

BRIEF DESCRIPTION OF THE DRAWINGS 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.

DETAILED DESCRIPTION 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. Accordingly, 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:

S O S n n n -N-c-S;-M-00;-C-S-fζ and -C-NH-C-R

The D moieties comprise groups that are attached to the aromatic ring. In a preferred embodiment the D moieties are selected from the group consisting of -OH, -OCH3, -Cl and -Br. In an even more preferred embodiment, 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:

wherein 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. In a preferred embodiment, the moieties X, Y and Z are selected from those groups that have an electron-donating effect on the aromatic ring. In an even more preferred embodiment, the moieties X, Y and Z are selected from the group consisting of -OH, -OCH3 , -Cl and -Br. Furthermore, when one or two of the X, Y or Z moieties are hydrogen, the remaining moieties can also be selected from the above group. In a preferred embodiment, 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. Such 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 top trace shows the amino acids detected by UV absorbance at 269 nm. The bottom trace shows the same amino acids detected electrochemically. Most of the PTH amino acids without the metahydroxyl group are, evidentially, not electrochemically active. In the inventive process, 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. a wash step; (d) converting the N- terminal amino acid complex from step (c) to a stable compound; and (e) identifying the stable compound formed in step (d) using electrochemical detection techniques. In a further embodiment, 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. By way of additional description, 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. For the method of chromatographic analysis the sample is introduced into a reverse phase column. Column temperature is maintained at 50°C. The mobile phase comprises: A- 15.0mM NH4~acetate (pH=4.5) :CH3CN (90%:10%); B- 15.0mM NH₄-acetate (pH=4.5) :CH₃CN (10%:90%). 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. Complete elution of 21 amino acids occurs within 10 minutes. A Model 5100A Coulochem Multielectrode Electrochemical Detector is employed for electrochemical detection. Other preferred mobile solutions include the following: A- 0.03M NH₄-acetate, pH=4.2; B- 0.0225M NH -acetate in 90% CH3CN and the gradient proceeds in the following manner:

%B-%B Time in Minutes

0-0 4 0-2 1 2-2 20 2-4 2 4-4 6.5 4-14 3.5 14-16 13 16-22 4 22-25 20 25-100 2 100-100 1 100-0 2

In another preferred embodiment a base deactivated column was employed with the following separation method: Mobile Phase: 0.03M NaOAc (sodium acetate) pH 4.91 with H3P0₄in 24% acetonitrile Column: Supelco pKblOO, 25 cm, 4.6 mm ID Temperature: ambient Flow: 1.0 ml/minute Other preferred mobile solutions comprise: (1) 0.03 M sodium acetate, 0.02% SDS, 5% methanol, pH=3.66; (2) 0.03M sodium acetate, 0.02% SDS, 40% methanol, pH=3.66. By way of example, the method comprises the following steps when employed for the manual sequencing of β-lactogobulin:

Reagents: mPTIC (solid) weigh lOmg into screw cap tube and dissolve with 1 ml ethyl acetate (ABI). Store -20°C. TFA anhydrous (Porton Instruments) TFA 25% aqueous, butyl chloride, heptane (ABI) milliQ water pyridine (Pierce Sequanal grade)

Coupling

dissolve β-lactoglobulin (25nmol, ABI) in 500 Ml 50% aq. pyridine pipet 50μl into a tapered glass tube (500ul volume) add 25μl 1% solution of mPITC place in PICOTAG (Water, Inc. or Millipore) vial, flush with nitrogen incubate at 50°C for lhr in water bath

Wash

add 200μl heptane:ethyl acetate (2:1 vol:vol), vortex, and centrifuge for 10 s remove upper organic layer with a fine-tipped pipet and discard protein is in the lower aqueous phase or at the interface with the organic layer repeat extraction of aqueous phase, remove as much organic layer as possible dry for 20 min in vacuo, generally, or Waters Picotag workstation specifically

Cleavage add 25 μl anhydrous TFA flush with nitrogen incubate at 50°C for 15 min in water bath dry in vacuo for 30 min

Extraction

add 25 μl H2O and 200 μl butyl chloride, vortex, and centrifuge remove upper butyl chloride layer and dry on speedvac for 15 min dry lower aqueous layer on Picotag workstation for 30 min, ready for coupling

Conversion

add 25 μl 25% TFA to dried butyl chloride extract, incubate in temp block for 15 min at 50°C dry on speed vac for 15 min resuspend in 50μl 10% acetonitrile inject 10μl onto HPLC While only certain embodiments of the invention have been described in specific detail, it will be apparent to those skilled in the art that other specific embodiments may be practiced, and many other changes may all be made within the spirit of the invention, and it is intended that all such embodiments and changes be considered within the scope of the invention which resides wholly within the claims hereinafter appended.

Claims

CLAIMS 1. A method for determination of the identity of an N-terminal amino acid of a reagent polypeptide complex comprising the steps of:

(a) providing a reagent polypeptide complex of 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 comprises 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;

(b) cleaving the terminal amino acid from the polypeptide complex to form a mixture of the cleaved N- terminal amino acid complex B-C-D and the cleaved polypeptide which then contains an N-terminal amino acid unit B¹ ;

(c) separating the N-terminal amino acid complex B-C-D from the cleaved polypeptide;

(d) converting the N-terminal amino acid complex from step (c) to a stable compound; and

(e) identifying the stable compound formed in step (d) using electrochemical detection techniques.

2. The method of claim 1 wherein the N-terminal amino acid complex is separated from the cleaved polypeptide by extraction with an organic solvent.

3. The method of claim 1 wherein the identification of the stable compound formed in step (d) comprises passing the stable compound reaction mixture through a chromatographic column wherein the effluent contains the stable compound which is detected electrochemically and identified according to its position on the chromatogram.

4. The method of claim 1 wherein 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.

5. The method of claim 1 wherein the anilinothiazolinone type N-terminal amino acid complex is separated from the cleaved polypeptide and converted to a stable phenylthiohydantoin type amino acid compound and identified by passing the meta-hydroxy phenylthiohydantoin amino acid compound through a chromatographic column to form an effluent wherein the meta-hydroxy phenylthiohydantoin amino acid compound is detected electrochemically and identified according to its position on the chromatogram.

6. A method of sequencing a polypeptide by repeated electrochemical determination of the identity of an N- ter inal amino acid of a polypeptide complex comprising the steps of: (a) providing a reagent polypeptide complex of 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 comprises 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;

(b) cleaving the reagent polypeptide complex to form a mixture of cleaved polypeptide (A) which contains an N-terminal amino acid unit (B* ) and an N-terminal amino acid complex (B-C-D) ;

(c) separating the cleaved polypeptide from the N-terminal amino acid complex;

(e) identifying the stable compound formed in step (d) .

7. The method of claim 6 wherein the N-terminal amino acid complex is separated from the cleaved polypeptide by extraction with an organic solvent.

8. The method of claim 6 including the steps of eluting the stable compound reaction mixture from step (d) through a chromatographic column to form an effluent; and electrochemically detecting and identifying the compound according to its position on the chromatogram.

9. The method of claim 6 wherein 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.

10. The method of claim 9 wherein the meta-hydroxy anilinothiazolinone type N-terminal amino acid complex is separated from the cleaved polypeptide and converted to a stable phenylthiohydantoin type amino acid compound and identified by passing the meta-hydroxy phenylthiohydantoin amino acid compound through a chromatographic column wherein the phenylthiohydantoin type amino acid compound is detected electrochemically and identified according to its position on the chromatogram.

11. A method of sequencing a polypeptide by repeated electrochemical determination of the identity of an N- terminal amino acid of a polypeptide complex comprising the steps of: (a) providing a reagent polypeptide complex of 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 comprises those moieties attached to the aromatic ring that contribute to or do not interfere with 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;

(b) cleaving the reagent polypeptide complex to form a mixture of cleaved polypeptide (A) which contains an N-terminal amino acid unit (B¹) and an N-terminal amino acid complex (B-C-D);

(c) separating the cleaved polypeptide from the N-terminal amino acid complex by extracting with an organic solvent which removes said N-terminal amino acid complex;

(d) converting the N-terminal amino acid complex to a stable compound;

(e) identifying the stable compound formed in step (d) ; and (f) repeating steps (a)-(e) after first providing a reagent polypeptide complex of claim 1 formed from the cleaved polypeptide separated in step (c) .

12. The method of claim 11, including the step of eluting the stable compound reaction mixture formed in step (d) through a chromatographic column to form an effluent wherein the stable compound is detected electrochemically and identified according to its position on the chromatogram.

13. The method of claim 12 wherein the stable compound reaction mixture is eluted using a mobile phase which comprises: (a) 15.0mM ammonium acetate (pH=4.5): acetonitrile (90%:10%); (2) 15.0mM ammonium acetate (pH=4.55); and acetonitrile (10%:90%).

14. The method of claim 12 wherein the stable compound reaction mixture is eluted using a mobile phase which comprises: (1) 0.03M ammonium acetate, pH=4.2; (2) 0.0225 M ammonium acetate in 90% acetonitrile.

15. The method of claim 12, including the step of eluting the stable compound reaction mixture formed in step (d) through a reverse phase column using a mobile phase which comprises: (1) 0.03 M sodium acetate, pH 4.9 with H3P0₄ in 24% acetonitrile.

16. The method of claim 12 including the step of eluting the stable compound reaction mixture formed in step (d) through a reverse phase column using a mobile phase which comprises: (1) 0.03 M sodium acetate, 0.02% SDS, 5% methanol, pH=3.66; (2) 0.03M sodium acetate, 0.02% SDS, 40% methanol, pH=3.66.

17. The method of claim 12, including the step of regulating the mobile phase gradient and effecting a resolution of the stable N-terminal amino acid as it traverses a flow path through the column.

18. A method for determination of the identity of an N-terminal amino acid comprising the steps of:

(a) providing a reagent peptide complex comprising the formula B-C-D of an N-terminal amino acid B wherein C is an aromatic moiety which contains a functional group which can react with and bind to the N- terminal amino acid and D is a moiety attached to the aromatic ring that contributes to the electrochemical activity of the peptide complex and is selected from those functional groups that do not interfere with the reactivity of the functional groups of the C moiety in binding to the N-terminal amino acid; and

(b) converting the reagent peptide complex to a stable compound; and (c) identifying the stable compound formed in step (b) using electrochemical detection techniques.

19. The method of claim 18 including the steps of eluting the reaction mixture containing the stable compound formed in step (b) through a chromatographic column to form an effluent, and electrochemically detecting and identifying the stable compound according to its position on the chromatogram.

20. A reagent polypeptide complex comprising the formula A-B-C-D for subsequent electrochemical detection of the identity of a unit derivatized N-terminal amino acid from said polypeptide complex 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 or do not interfere with 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.

21. The reagent polypeptide complex of claim 20 wherein 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:

S O S

II II II

-M-OS;-N-00;-C-S-R; and -C-NH-C-R

22. The reagent polypeptide complex of claim 20 wherein the D moieties comprise groups that are electron- donating when attached to the aromatic ring.

23. The reagent polypeptide complex of claim 20 wherein the D moieties are selected from the group consisting of -OH, -OCH3, -Cl and -Br.

24. The reagent polypeptide complex of claim 20 wherein 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.

25. The reagent polypeptide complex of claim 20 wherein the.unit derivatized N-terminal amino acid comprises an anilinothiazolinone type derivative.

26. The reagent polypeptide complex of claim 20 wherein the unit derivatized N-terminal amino acid comprises a phenylthiocarbonyl type derivative.

27. The reagent polypeptide complex of claim 20 wherein the unit derivatized N-terminal amino acid comprises a phenylthiohydantoin type derivative.

28. An electrochemically active amino acid derivative of the following structure:

wherein 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.

29. The electrochemically active amino acid derivative of claim 28 wherein the moieties X, Y and Z are selected from those groups that have an electron-donating effect on the aromatic ring.

30. The electrochemically active derivative of claim 28, wherein the moieties X, Y and Z are selected from the group consisting of -OH, -OCH3, -Cl and -Br.

31. The electrochemically active derivative of claim 28 wherein one of the X, Y or Z moieties is hydrogen, the other two moieties are selected from the group consisting of -OH, -OCH3, -Cl and -Br.

32. The electrochemically active derivative of claim 28,. wherein two of the X, Y or Z moieties are hydrogen and the remaining moiety is selected from the group consisting of -OH, -OCH3, -Cl and -Br.