PeptideSequencingby                                                                         Secondaryarticle
            EdmanDegradation                                                                               Article Contents
                                                                                                . Introduction
            JohnBryanSmith,CelltechChiroscienceplc,Slough,UK                                    . TheBasisoftheMethod
                                                                                                . TheRoleofPeptideSequencingwithEdmanChemistry
            Edmanchemistryisalong-establishedapproachtodeterminationoftheprimarystructure,
            or sequence, of polypeptides.
            Introduction                                                  sion(seethenextsection),andcouldundertakesolid-phase
            Bythemiddle of the twentieth century it was known that        sequencing as well. Later developments were automated
            proteinswerecomposedofaminoacids,butnothowthese               (‘on-line’) phenylthiohydantoin-amino acid identification
            were joined together. Were they arranged in blocks of         by high-performance liquid chromatography, and later
            similar residues? or randomly mixed together? or in           still, morethanonereactioncartridgesothatonesequence
            repeated patterns? At about that time, methods were           run could automatically be followed by another. Today,
            devised to provide an answer to these questions:namely,       manual sequencing is rarely done. Automation has
            that each individual protein has its constituent amino acid   broughtsavingsinresourcesandtimeand,withminimiza-
            residues arranged linearly in a unique order, or sequence.    tion of interference by oxygen and water and improved
            ThestrategyofSangerandcolleaguesforthesequencingof            reagents and reaction conditions, a marked increase has
            insulin was to characterize series of small overlapping       beenachievedintheefficiencyofthewholeprocess.Inthis
            peptides produced by cleavage of the parent molecule.         waytheamountofsamplerequiredtoobtainasignificant
            Determination of the overall amino acid content and the       sequenceisnowordersofmagnitudelessthanitwas,atlow
            identity of the amino- (N-)terminal residue for each          or subpicomolar levels.
            peptide allowed deduction of the sequence of the whole
            molecule(Sanger,1959).Analternativeapproachwasthat
            describedbyPehrEdman(1950).Thisalloweddetermina-              TheBasisoftheMethod
            tion of extended sequences of peptides or whole proteins,
            and has been used widely up to the present day. The
            method employs a series of chemical reactions to remove       Peptide sequencing by Edman chemistry may be divided
            and identify the amino acid residue that is at the N-         into steps as follows, and as illustrated in Figure 1.
            terminus of the polypeptide chain, i.e. the residue with a
            free a-amino group. At the same time, the next residue in     Coupling
            the sequence is made available and subjected to the same
            round of chemical reactions. Reiteration of this process      Phenyl isothiocyanate (PITC) reacts with an a-amino
            reveals the sequence of the polypeptide.                      group(orinthecaseofprolylresiduewithaniminogroup)
              Originally described as a manual method, it was later       at the N-terminal end of the polypeptide chain, to form a
            partly automated (Edman and Begg, 1967) in an instru-         phenylthiocarbamyl derivative of the terminal residue.
            menttermeda‘sequenator’,thedesignofwhichwasusedin             Basic conditions are required for this reaction. Edman
            the commercially available ‘spinning cup’ sequenator          originally used pyridine to generate a pH of 8.6.
            marketed by Beckman in the late 1960s. In 1971 Laursen        Alternatives used since include ‘Quadrol’, trimethylamine
            describedadifferentdesignofautomatedpeptide‘sequen-            and N-methylpiperidine. Clearly, a free a-amino group is
            cer’ that used the same chemistry but with the sample         required for this reaction to occur. If the amino group has
            covalently attached to a solid support that minimized the     become modified and will no longer react with PITC, the
            loss that can occur during steps involving extraction of      polypeptide is said to be ‘blocked’. Many proteins are
            reaction products from solution (Laursen, 1971). This         blocked by acetyl, formyl or pyroglutamyl groups, for
            ‘solid-phase’ sequencing approach was useful for sequen-      instance.Variousremedialtechniqueshavebeendescribed
            cing of short peptides that were especially easily lost in    to deblock such molecules (Hirano et al., 1977). Blockage
            extraction steps. A significant advance came with the ‘gas-    may also occur artificially during the handling of the
            phase’ peptide sequenator (Hewick et al., 1981), so-called    sampleinthelaboratory.Tracecontaminantsincommer-
            because some reagents were delivered as vapour. First         cial preparations of non-ionic detergents are known to
            commercialized as the Applied Biosystems model 470A,          cause blockage, for instance. Again, a peptide may not
            the design included automation of the process of conver-      haveanN-terminusatall:cyclosporin is just one example
                                                                          ofanaturalpeptidethatiscyclic.Incaseswherethereisno
                                 ENCYCLOPEDIAOFLIFESCIENCES/&2001MacmillanPublishersLtd,NaturePublishingGroup/www.els.net           1
                   PeptideSequencingbyEdmanDegradation
                                                  1                 2                              Currently, trifluoroacetic acid (TFA) is used for this
                                                 R     O           R
                                         H NCHC NH CH peptide                                      cleavage reaction. Conditions are as anhydrous as is
                                          2                                                        practically possible in order to minimize acid hydrolysis at
                                                             +                                     pointswithinthepolypeptidechain.Wherethishappens,a
                                                     Ph N C S                                      new N-terminus is generated and becomes subject to
                                                                                                   sequencing.Thisthencontributestobackground‘noise’in
                                                                                                   thesubsequentanalysis.Minimizationofthisacidcleavage
                                                                                                   leads to longer and clearer sequence runs.
                                                          1                 2
                                                         R     O           R
                                 Ph   NH C NH CH C NH CH peptide                                   Conversion
                                              S
                                                                    +                              The ATZ residue is separated from the peptide by
                                                                   H  (trifluoroacetic acid)       extraction in organic solvent (ethyl acetate or chlorobu-
                                                                        2                          tane), andisthenconvertedtoamorestableformtoallow
                                                                      R                            better analysis. Conversion to the more stable phenylthio-
                                       1                                     peptide
                                     R     CH C O   +   H N           CH                           hydantoin(PTH)formisdoneinaqueousacid(25%TFA,
                                                                2
                                         HN       S                                                v/v in water). Some modified amino acid residues (such as
                  anilinothiazolinone          C               shortened peptide                   glycosylatedasparaginyl)maybepoorlysolubleinorganic
                         (ATZ)                 NH                                                  solvent andsogiveablankattheircorrespondingpointin
                       amino acid                                                                  thesequence.Insolid-phasesequencing,wherethepeptide
                                               Ph                                                  is covalentlylinkedtoasolidsupport,alternativemeansof
                                                                +                                  extraction of the ATZ residue may be tried, in the
                                                       H O + H  (trifluoroacetic acid )
                                                        2                                          knowledge that the remaining peptide will not also be
                                                                                                   extracted and lost (giving large drops in yield).
                                       1
                                      R     CH C O
                                          HN      N Ph                                             Analysis of PTH residues
                    phenylthiohydantoin        C
                           (PTH)                S                                                  The PTH residue generated by each cycle of Edman
                         amino acid                                                                chemistry is typically identified by chromatography,
               Figure 1    EdmanchemistryforN-terminalsequencingofpolypeptides.                    originally thin-layer chromatography and latterly re-
                                                                                                   versed-phase high-performance liquid chromatography.
               free N-terminus, the polypeptide must be cleaved by                                 The PTH amino acid residue derived from each cycle in
               chemical or enzymatic means to yield fragments that do                              turn is identified and quantified by comparison with
               havefree N-termini. On a practical note, it is necessary to                         standards, and the sequence is described by the order of
               avoidcontaminationofthesamplewithamine-containing                                   residues from the N- to the C-terminus. If radioactive
               nonpeptidic species, such as trizma base, since these, too,                         amino acid residues are present they may be detected by
               mayreactwithPITCandgenerateproductsthatinterfere                                    their activity at this stage.
               with subsequent analysis. Reaction of PITC also occurs
               withtracesofwaterandothermoleculesthataredifficultto                                  Theefficiencyofsequencing
               exclude completely from the reaction, so byproducts such
               asdiphenylthioureaarecommonlyfound.Minimizationof                                   The different amino acid residues, being structurally
               side reactions such as these helps to improve the efficiency                          different, react at each stage with different degrees of
               ofthechemistryandsubsequentanalysis,andtheabilityto                                 efficiency. The overall efficiency (‘repetitive yield’) is less
               dothis is one of the advantages of automated sequencing                             than100%(usuallyoftheorderof95%),sooverthecourse
               methodsoveroldermanualmethods.                                                      ofanumberofcyclestheyieldofsequencedeclines,andthe
                                                                                                   degreeofstagger,or‘lag’,graduallyincreases.Atthesame
               Cleavage                                                                            time,theamountofbackgroundnoiseincreases.Whenthe
                                                                                                   sequence signal reaches the level of the background, the
               In the presence of strong acid, cleavage occurs at the first                         sequence becomes uninterpretable. The number of cycles
               peptide bond, giving the peptide (minus the first residue)                           at which this occurs may be small or large (50 or more),
               and the liberated first residue as the anilinothiazolinone                           depending to a great extent on the size and amino acid
               (ATZ)form.Onceotherreactantsandproductshavebeen                                     content of the polypeptide itself, since this dictates the
               washed away, the shortened polypeptide can be taken                                 degreeofrandomacidhydrolysisandothersidereactions.
               throughanotherroundofcouplingandcleavagetorelease                                      Thetimetakentosequenceasampleisconsiderablyless
               the second residue, and so on in a cyclical fashion.                                than it used to be – the time for one cycle of Edman
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                                                                                        PeptideSequencingbyEdmanDegradation
            chemistry has been reduced to 20 minutes. Current             particular states of development or disease. For this
            sequencers may have more than one reaction cartridge,         purpose, a short sequence just a few residues long is all
            sothemachinescanbeusedforafull24hourseachdayand               that is necessary for screening against databases of known
            generate much information in a short time.                    gene sequences, when used in conjunction with other
              The amount of sample required currently to provide a        properties such as molecular weight, pI or masses of
            sequence of a few to 20 or so residues is of the order of 1   peptides. Peptide sequencing also has an important role in
            picomole or less. Automated sequencers can accept             quality assurance of ‘biopharmaceuticals’, recombinant
            samples that are covalently bound to solid supports, such     therapeutic proteins that are the result of the molecular
            as synthesized peptides remaining on the supports on          biology revolution.
            which they were made, or samples that are noncovalently          Edmanchemistryisnowastandardmethodforpeptide
            boundtoglassfibreorpolyvinylidene difluoride (PVDF).            sequencing.Inrecentyearsithasbeencomplementedbya
            Thelatter maybeusedtotrapproteinstransferredfroma             varietyofmassspectrometricmethodsthathavebeen,and
            polyacrylamide gel, a rapid method for resolution of          continuetobe,refinedsuchthattheycandeterminemasses
            complex mixtures, and this approach to sample prepara-        of proteins and of peptides derived from them, and can
            tion is very common.                                          determinepeptidesequencefromthepatternsoffragmen-
              The simplest case is one where there is just one            tation from peptide into individual amino acid residues.
            polypeptide in the sample, but it is common to have           While the methods may complement each other today, it
            differently processed forms of the same protein or other       could be that in the next decade or so the mass spectro-
            proteins present as contaminants. These mixtures can be       metrymethodwillreplacetheEdmanchemistryapproach.
            resolved into individual sequences if they are in signifi-     Nevertheless,itremainsacredittoEdman’schemistrythat
            cantlydifferentquantities,ifoneofthesequencesisknown           it hasprovedsorobustandgivensuchvaluableserviceover
            andmaybesubtractedfromtheother(s),orif,inthecaseof            decades of rapid technological change in the field of
            a variously processed protein, the frameshift or delayed      biochemistry.
            repeat in the sequence can be spotted. Again, mixtures of
            sequencescanbeidentifiedbyscreeningagainsttherapidly           References
            growing database sequences.
                                                                          EdmanP(1950)Methodfordeterminationoftheaminoacidsequencein
                                                                            peptides. Acta Chimica Scandinavica 4:283–293.
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            decades. Originally the aim was to determine the sequence     Hirano H, Komatsu S and Tsunasawa S (1977) On membrane
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            wasrequiredfordesignoftheoligonucleotideprobesused            Sanger F (1959) Chemistry of insulin. Science 129:1340–1344.
            in the process of gene cloning and for confirmation that
            isolated clones were indeed the relevant ones. As sequen-     FurtherReading
            cing of whole genomes progresses, we approach the time
            whenallgene(andthereforeall protein) sequences will be        ReimDFandSpeicherDW(1995)N-terminalanalysisofproteinsand
            known.Currently,however,knowledgeofthesequenceof                peptides. In:Coligan JE, Dunn BM, Ploegh HL, Speicher DW and
            a gene does not give us complete information on matters         WingfieldPT(eds)CurrentProtocolsinProteinScience,vol.2,section
            that are significant in the function of a protein, such as       11.10. Chichester:Wiley.
            inter- and intramolecular disulfide bonding patterns or        Smith BJ (ed.) (1997) Protein Sequencing Protocols. Methods in
            modification and processing events. Determination of             Molecular Biology, vol. 64. Totowa, NJ:Humana Press.
                                                                          SmithBJandChapmanJR(1998)Proteinsequencing.In:RapleyRand
            protein sequence has a role in providing this, and in           Walker JM (eds) Molecular Biomethods Handbook, pp. 503–525.
            identification of proteins that are found to correlate with      Totowa,NJ:HumanaPress.
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