Identification
of
Modified PTH-Amino Acids
in
Protein Sequence Analysis
First Edition
©1993 Association of Biomolecuiar Resource Facilities
Compiled
for
The Association of Biomolecuiar Resource Facilities
by
Mark W. Crankshaw and Gregory A. Grant
Departments of Medicine and Molecular Biology & Pharmacology Washington University School of Medicine St. LouiS) Missouri 63110 CONTENTS
Page
Introduction ^ Suggestions for Using This Guide 5 List of Abbreviations ° Explanation of Numbering Convention 9
PTH-Amino Acids
Reference Standard I - PTH-Amino Acids Applied Biosystems Sequencer Chromatoaram 10 Table 11
Reference Standard II - PTH-Amino Acids Milligen/Biosearch Sequencer ChromatO'iram 13 Table 14
PTC-Amino Acids
Reference Standard m - PTC-Amino Acids Applied Biosytems Sequencer Chromatoaram 15 Table 16
Sequencing Artifacts or Associated Amino Acid Derivatives
Reference Standard IV- Sequencing .Artifacts Applied Biosytems Sequencer Chromatoeram H Table 18
Side Chain Protected Amino Acids Used in Peptide Synthesis
Reference Standard V - Boc Synthesis Applied Biosytems Sequencer ChromatoEram 1" Table 20
Reference Standard VI - Boc Synthesis Porton Sequencer Chromatogram ' Table 22 Reference Standard VII - Fmoc Synthesis Applied Biosytems Sequencer Chromatosram 23 Table 24
Table I - Eludon Times of Selected PTH-Amino Acids on an Applied Biosytems Sequencer 26
Table II - Eludon Times of Selected PTH-Amino Acids on an Applied Biosytems Sequencer 27
Index of Compounds 28
List of Contributors 33
Suggested'BO1 References 36
IV
i..' sit- .if11
r; Introduction
The identification of amino acid residues in modern protein sequence analysis employing automated Edman degradation is dependent on the elution position of the PTH-amino acids on high pressure liquid chromatography systems. This method relies on a comparison of the elution position of the unknown PTH-amino acid with that of reference standards. This is relatively straightforward for the genetically encoded amino acids, but becomes problematic when modified or unusual amino acid residues are present in the sample being sequenced. Since the method does not provide a direct identification of the PTH-amino acid, additional analysis by chemical or phvsicai means is necessary. However, it is often helpful to have some knowledge of where P1H- anuno acids with known modifications elute in these systems. This provides a starting point for the investigator and provides an additional level of knowledge upon which to proceed.
This compilation has been undertaken by the Association of Biomolecular Resource Facilities (ABRF) in an attempt to consolidate information of this type for easy reference It should be noted that an exhaustive review of the literature has not been attempted. Rather, members of the ABRF were asked to submit any information they had in this regard for inclusion in this booklet. This compilation is intended for use by the entire scientific community and is available to.anyone who is interested. Single copies can be obtained for personal use by contacting the ABRb business office by letter or FAX. The address is 9650 RockvUle Pike, Bethesda, MD 20814-^998 and the FAX number is 301-530-7049. Parties interested in large quantities may also inquire through the business office. Also please note that it has not been possible to independently verify the information presented here. In many instances the entries come from personal observations of the contributors and there are no published references to provide rigorous documentation. When literature references have been provided they are included. Therefore, this informaDon is intended to be used only as a It is recognized that this compilation is by no means complete. Anyone who has additional information is encouraged to submit their data for inclusion in subsequent editions. Those wishing to contribute may forward their material either to the ABRF business office or directly to the authors at the Department of Molecular Biology and Pharmacology, Campus Box 8103, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110. Finally we would like to thank all those who sent contributions. Their names are included to acknowledge their contribution. Their time and efforts are greatly appreciated and ins hoped that you will find this a useful and worthwhile research tooL.We also thank Edna Siivestn for excellent clerical assistance. v \ n L. *:. Suggestions For Using This Guide This guide has been compiled as an aid to the initial investigation of the identity of an unknown peak in phenykhiohydantoin (PTH) amino acid chromatograms in automated Edman sequencing. The guide is arranged so that you may approach this through one of three routes: 1) Reference Chromatograms. Locate the approximate position of an unknown peak on the appropriate reference standard and refer to the respective listing of modified amino acids which generally elute in that area. Note that there is more than one chromatogram listed, each of which refers to a particular type, source of origin, or instrument. 2) Elution Tables. Tables of elution position have been supplied by some contributors. These overlap to some extent with the reference chromatograms and also contain information not included in the chromatograms. Estimate the unknown's elution time and look through the Tables for a possible match. 3) Index of compounds. If you know or can hypothesize the identity of a modified amino acid that you expect to be present, you can use the master list which refers to the appropriate chromatogram or table. Caution! It is extremely important that you use this information only as a guide to a possible identity for your unknown and that you are aware of the approximate nature of the placement of the contributed modified amino acids on the reference standards and tables. Every effort has been made to present the information as accurately as possible, but no two sequencer-HPLC systems are exactly alike. A major change over die past year affecting Applied Biosystems users is the switch from user adjusted Na-acetate buffers to the new "pre-mix" system. In general the PTH-amino acids elute in relatively the same area, but some significant shifts may occur. Other variables, such as initial conditions, gradient, column temperature, and mobile phase additives, can make it difficult to precisely correlate the elution position from one HPLC system to another. However, we believe this representation is sufficiently accurate that it will be useful. E All amino acids are the PTH derivative unless otherwise stated. PTH-amino acids are referenced with arabic numerals (see explanation of numbering convention on next page), contributors are referenced with upper case letters, and footnotes are designated by lower case roman numerals in italics as superscripts. List of Abbreviations Acetnmidomeihyl-cysteine ACM-Cys Alanine Ala, A a-Aminobutyric Acid Abu Applied Biosystems "Incorporated" ABI Arginine Arg.R Arginine (diallyloxycarbonyl) Arg(AJoc)2 Arginine (mesitylene-2-sulfonyl) Arg(Mts) Arginine (4-methoxy-2,3,6-trimethylbenzenesulfonyl) Arg(Mtr) Arginine (4-toluenesulfonyl) Arg(Tos) Asparagine Asn, N Asparric Acid Asp, D Aspartic Acid (cyclohexyl) Asp(OcHex) Aspartic Acid (O-ailyl) Asp(OAl) Aspartic Acid associated peak Asp' Aspartic Acid (O-benzyl) Asp(OBzl) Aspartic Acid (O-tert-butyl) Asp(OtBu) Association of Biomolecular Resource Facilities ABRP S-Carboxamidomethyl-cysteine Cys(SCAM) y-Carboxyg!utamic Acid Gla S-Carboxymethyl-cy stein e Cys(SCM) Citruliine Cit Cysteine Cys, C Cysteine (allyl) Cys(AI) Cysteine (allyloxycarbonyl) Cys(Aloc) Cysteine (4-methoxybenzyl) Cys(4-CH3OBzi) or .■"IK Cys(SMob) Cysteine (4-methylbcnzyl) Cys(4-CH3Bzl) or Cys(SMeb) Cysteine (3-nitro-2-pyridylsuIfenyl) Cys(Npys) Cysteine (tert-butyl) Cys(tBu) Cystine Cys2 N-dimethyl, N'-phenylthiourea DMPTU O, O-dimethylphosphotyrosine Tyr(PO3Me2) N,N'-diphenyl thiourea DPTU N,N'-diphenylurea DPU Dithiodireitol DTT DTT adduct of dehydroalanine S1 DTT adduct(s) of dehydro-oc-aminoisobutyric acid T' Fluorenylmethyloxycarbonyl Fmoc Glutamic Acid Glu, E Glutamic Acid (O-allyl) Glu(OAl) Glucamic Acid associated peak Glu1 Glutamic Acid (Obenzyl) GSu(OBzl) Glutamic Acid (cyciohexyi) Glu(OcHex) Glutamic Acid (O-9-Fluorenylmethyl) Glu(OFm) High Pressure Liquid Chromatography HPLC Histidine His, H Histidine (allyloxycarbonyl) His(Aloc) Histidine (3-benzyl) His(3-Bz!) Histidine (3-benyzloxymethyl) His(Bom) Histidine (tert-butyloxymethyl) His(Bum) Histidine (2,4-dinitrophenyl) His (Dnp) 5-Hydro xyly sine Hyl Hydroxyproline Hyp Isoleucine lie, I Lanthionine Lan Leucine Leu, L Lysine Lys, K Lysine (allyloxycarbonyl) Lys(Aloc) Lysine (chlorobenzyloxycarbonyl) Lys(ClZ) Lysine (N-e-dinitrophenyl) Lys (Dnp) Lysine (N-e-9-Fluorenylmethyioxycarbonyl) Lys (Fmoc) Methionine Met,M l-Methylhistidine His(l-Me) 3-Methylhistidine His(3-Me). Nitroarginine Arg(NO2) Norleucine Nle Norv aline Nva Gmithine Orn 7 Ornithine (benzyloxycarbonyl) Om(Z) Phenylalanine Phe, F Phenylalanine (p-amino-benzyloxycarbonyl) Phe(p-amino-Z) Phenylisothiocyanate PITC Phenylthiocarbamyl PTC Phenylthiohydantoin PTH Phenyltbiohydantoin amino acid PTH-aa S-pyridylethyi cysteine PECys or Cys (SPE) Pro line Pro.P Serine Ser, S Serine (allyloxycarbonyl) Ser(A!oc) Serine (benzyl) Ser(Bzl) Solid phase peptide synthesis SPPS Threonine Thr, T Threonine (allyloxycarbonyl) Thr(Aloc) Threonine (benzyl) Thr(Bzl) Threonine (tert-butyl) Thr(tBu) Tryptophan Trp.W Tryptophan associated peak Tip' Tryptophan (Nin-formyl) Trp(CHO) Tyrosine Tyr,Y Tyrosine (allyl) Tyr(Al) Tyrosine (2-bromobenzyloxycarbonyl) Tyi(2BrZ) Tyrosine (ten-butyl) Tyr(tBu) Valine Val,V Explanation of Numbering Convention Category Modified PTH-aa on Applied Biosystems Instruments Modified PTH-aa on Milligen/Biosearch Instruments Phenylthiocarbamyl amino acids Applied Biosystems Instruments Side chain protected amino acids used in Boc SPPS on Applied Biosystems Instruments Side chain protected amino acids used in Boc SPPS on Porton Instruments 600-699 21 Side chain protected amino acids used in FMOC SPPS on Applied Biosystems Instruments 800-899 23 Reference Standard I Modified PTH Amino Acids on Applied Biosystems 03 CD 10 Modified Amino Acids for Reference Standard I Applied Biosystems Instruments PTH# Name Contributor 1 y-Carboxyglutamic Acid (Gla) I, RW* 2 O-Fucosyl threonine K"'" 3 S-Carboxymethyl cysteine J,M,R 4 Homoserine M,R 5 S-Carboxamidomethyl cysteine D,J 6 Carboxamidomethyl methionine T 7 Methionine suifone C,M,R,T 8 N-e-Succinyl Iysine B,D 9 5-Hydroxy Iysine derivative R iv 10 Hydroxyproline (Hyp) B,D,F,G,I,J,R,T 11 N-e-Acetyl Iysine M.P, Q, T 12 Methyl histidine O,R 13 O-Methyl threonine M 14 Cysrine O 15 O-Methyl glutamic acid B, U " 16 N-e-Methyl Iysine B, C, J, M, P, Q, R 17 N-e-dimethyi Iysine C, J, P, Q v 18 N-e-trimethyl Iysine C, E, J, P, Q v 19 Canavanine C 20 a-Amino butyric acid (Abu) G 21 Methyl arginine R 22 S-Methyl cysteine M 23 DL-Homocystine M 24 5-Hydroxylysine (Hyl) D.K.R 25 Iodotyrosine C,T 26 ct,Y-diaminobutyric acid I 27 Omithine (Orn)' R,T 28 O-Methyl tyrosine t' 29 Lanthionine (Lan) R vi 30 Norleucine (Nle) C,M 31 p-Chlorophenylalanine T 32 diiodotyrosine C i In systems where Gla is efficiently extracted from the cartridge it runs as a broad peak immediately in front of Asp. A certain percentage (- 5-10%) can decarboxylate to Glu, which is the only PTH-aa seen if Gla is not extracted from the cartridge. 11 ii Reference 35 shows Gla as di-O-mcthyl-GIa using a methylation procedure and a modified gradient. In addition, O-methyi Asp and O-methyi GIu are also described. itt Successive Edman cycles results in deglycosylation. iv Of three 5-Hydroxy-Lys contributors, this is the only one to indicate this peak. v The methyl lysines (mono-, di-, and tri-) have proven to be particularly problematic in establishing their elurion position. Different contributors have shown diem eluting in basically two places. The majority show diem eluting in a wide area between alanine and DPTU and also after leucine. In some instances, a contributor will indicate both positions, and in others, only one of the two positions. In an effort to resolve this discrepancy, we obtained samples of the mono-, di- and tri- methyl lysines and ran them on a standard Applied Biosystems 477A sequencer simply by loading an aliquot into the reaction vessel and running a sequencer cycle. Both mono- and dimethyl lysine show both early and late peaks, while trimediyl lysine shows only the early peak. The eiution order of die early peaks is mono- before di- before tri-, with a fairly limited range between Ala and Met The late eludng peaks tend to co-elute just after leu (and nleu). So, what is the explanarion ? Without chemical proof we can only speculate, but we offer die following possiblity. Mono- and dimethyl lysine are alkyl amines that may be capable of becoming protonated and assuming a positive charge. Trimediyl lysine is a quaternary amine that is always positively charged. As such, the charge should cause diem to run relatively early in the chromatogram and, like His and Arg, their elution position will probably be very sensitive to ionic strength. Hence, varying ionic strength from different systems may explain the wide variance reported in the elurion positions of die early peaks. The late peaks may be due to a portion of die mono- and dimethyl lysine side chains reacting with PITC in a manner similar to that of lysine, since they retain a free pair of electrons on the nitrogen that can participate in nucleophilic attack on the PFTC. In our hands, this appears to be a major reaction for monomethyl lysine and a minor reaction for dimethyl lysine, but others have reported variable ratios. This variability may be cycle dependent. Trimediyl lysine does not possess an unbonded pair of electrons and thus would not be expected to react with PITC at this position. Hence, we do not see a late eluting peak. Again, it must be stressed that this is only a hypothesis and particular care must be taken in interpreting your results, However, the general behavior of the methylated lysines, whatever the reason, is well documented and should aid in their identiiication. Vi Also reports minor peaks berween Ser and Gin and at dehydroalanine. 12 Reference Standard II Modified PTH Amino Acids on Miiligen/Biosearch q o q cd q 13 Modified Amino Acids for Reference Standard II Milligen/Biosearch Instruments PTH# Name Contributor 200 Hydroxy-Pro (Hyp) L 201 O-Phospho-Ser L1' Both Ser and O-Phospho-Ser are convened to dehvdroalanine. 14 Reference Standard III PTC Amino Acids on Applied Biosystems 15 PTC Amino Acids from Reference Standard III Applied Biosystems Instruments PTH# Name Contributor D R R F, R R F, R R 16 Reference Standard IV Sequencing Artifacts q cd — Unknown at PHE/ILE CoorassiB Blue O CO CVJ TRP Associated PaaK_ GLU Associated Peak Q_ Q — AS? Associated Peak q CM CM — H4 Contamination q O — MethanolPITC LLJ o CM THR Associated Peaks q CO "5 >SEH Associated Peaks q co CO q c\i THIS q o ""known ASN/SER q co — Aniline q CD 17 Sequencing Artifacts or Associated Derivatives of Normal Amino Acids on Applied Biosystems Reference Standard IV PTH# Name Contributor i Probably derived from PITC as a consequence of high residual acid during sample loading. H Broad peak between Asn and Ser occasionally seen in very high sensitivity runs. '" Only on instruments using methanolic conversion. Results from reaction between methanol and PITC carried into the flask with S3. i'v Unknown contaminant. Peak results from heating and drying in the conversion flask. v Unidentified derivatives associated with Asp or Glu in addition to PTH-Asp and PTH- Glu respectively. vi Unidentified derivative of Trp often seen as the major peak. v" Sharp peak often seen between Phe and lie. vizi Consists of peaks at 1) Ser and Ser", 2) between Ser' doublet and Pro, and 3) between Trp and Phe. ix Peroxides in R4 may degrade Lys. 18 Reference Standard V Sida-chain Protected Amino Acids Used in Boc SPPS on Applied Biosystema O d 511 -510 -506 (Z-IO)sA-]-Hld -509 -503 -507 ■506' p ■505 d ■504 CO ■503 ■502 ■501 I—> ■4-' O =3 CVJ C 500 O na d 19 Side Chain Protected Amino Acids used in Boc SPPS Applied Biosystems Instruments Reference Standard V1 PTH# Name Contributor 500 Acetamidomethyl cysteine (ACM-Cys) J 501 Arg-p-toluenesulfonyl (Tos) N 502 Trp-Nin-formyl (CHO) N 503 Ser-benzyl (Bzl) N 504 Arg-mesirylene-2-sulfonyl (MTS) 505 Asp-O-benzyl (OBzl) N 506 Thr-benzy] (Bzl) N 507 Glu-O-benzyl (OBzl) N 508 Cys-4-methoxybenzyl (4-CH3OBzl) N 509 Lys-chlorobenzyloxycarbonyl (C1Z) 510 Cys-4-methyIbenzyl (4-CH3Bzl) 511 Tyr-2-bromobenzyloxycarbonyl (2BrZ) N i Standard chromatogram provided by Michael Kochersperger of AppUed Biosystems. See reference 23 . it Lys-{2C1Z) is more commonly used given its greater stability in long syntheses and it co-eiutes with Lys-(CIZ). 20 REF STD VI Sids-chain Protected Amino Acids Used in Boc SPPS on Porton Instruments OJ 604 . CO OJ cg CO LO 603 600 CO SAT U 311 3Hd 602 O eg nida . CD CO in 1VA CD 131 601 •4—■ OUd c CO HA1 CO V1V SIH ma AT9 UHi CO H3S NSV Oi'9 dSV zv% ■ ■ 600 Side Chain Protected Amino Acids used in Boc SPPS Porton Instruments Reference Standard VI* PTH# Name Contributor Chromatogram provided by Audree Fowler. Reference Standard VII Side-chain Protected Amino Acids Used in FMOC SPPS on Applied Biosystems -818 O O ...dJl-Hld 818 818 821 820 819 q o ■818J CO -4—' i o ■— CM ^ S!H-Hld 801 B[V-Hld 800 p nidwa _ ni3-Hld A"[9-Hld u|9-Hld usv-Hld p o 23 Side chain Protected Amino Acids used in FMOC Synthesis Applied Biosystems Instruments Reference Standard VII *>" PTH# Name Contributor i Sauidard chromatogrnm provided by Michael Kochersperger of Applied Biosystems. See reference 23. li Locations are approximate. Originally done with different gradient conditions and are now represented on a typical resin bound sequencing standard (ABI "rez" cycle) lit Side chain deprotecrion occurs during conversion. iv Byproduct resulting from conversion. v Side chain deprotection accumulates during repeated Edman cycles. Tables The following tables of eiution positions were submitted already assembled and are reproduced here essentially as received. 25 TABLE I s Om, omithinc; Trp', unidentified derivative of PTH-Trp; 26 TABLE II 3 me his after his about 1.57/>A (broad shape) nlrt H//X>Y 0.81 arg ala fianipfe Translation of Shorthand Asn- D one run. Gin- E(deamidatedQ)I!G(ptcE?)llX>A V = •A peak may/will appear at Glu. This is deamidated Gin. •A peak may/will appear at Gly, possibly PTC Glu. •An unknown(X) peak may/will arise after Ala by about 1 minute. 27 Index of Compounds (Roman numeral refers to chromatogram number) Acetamidomethyl-cysteine V, VH, Table I, Ref. 17 N-e-Acetyl-Iysine I, Table I, Ref. 26 Alanine All, Tables I & H a-Aminoburyric Acid I, Table I, Ref. 17 Aniline rv Arginine AH, Tables I & II Arginine (diallyloxycarbonyl) VH, Ref. 16 Arginine (diallyloxycarbonyl) derivative VII, Ref. 16 Arginine (MesiryIene-2-sulfonyI) V, Table I Arginine (4-Methoxy-2,3>6-trimethy I benzene sulfonyl) VH, Table I Arginine (4-Toluenesulfonyl) V. Table I Asparagine All, Tables I & H Aspartic Acid All, Tables I & H Aspartic Acid (cyclohexyl) Table I Aspartic Acid (O-allyl) VII, Ref. 16 Aspartic Acid asociated peak IV Aspartic Acid (O-benzyl) V, VI, Table I, Ref. 23 Aspartic Acid (O-tert-butyl) VII, Ref. 23 4-O-Benzylhydroxyproline Table I Biotinylated Iysine Table I, Ref. 34 Canavanine I S-Carboxamidomethyl-cysEeine I Carboxamidomethyl-methionine I -y-Carboxyglutamic Acid I, Ref. 35 S-Carboxymethyl-cysteine I p-Qiloro-phenylalanine I Citrulline Table I Coomassie Blue IV, Ref. 36 P-Cyclohexylalanine Table I Cysteine Table U Cysteine (allyl) , Ref. 16 Cysteine (allyioxycarbonyl) , Ref. 16 Cysteine (4-methoxybenzyl) V, Table I 28 Cysteine (4-methylbenzyl) V, Table I Cysteine (3-nitro-2-pyridylsuIfenyl) VII, Ref. 17 Cystine I, Ref. 15 Dehydroalanine Table I Dehydro-a-aminoisobutyric acid Table I 3,4-Dehydroproline Table I ovy-diaminobutyric acid I 0-(2,6-dichlorobenzyl)-tyrosine Table I 3-(2,6-dichlorobenzyl)-tyrosine Table I N-e-(2,3-dihydroxypropyl)-lysine Ref. 25 Diiodohistidine Table n Diiodotyrosine I, Table H Dimethyilysine I, Table II, Ref. 26 N-dimethyl, N'-phenylthiourea All, Table I, Ref. 36 O, O-dimethylphosphotyrosine Table I N,N'-diphenylthiourea All, Table I, Ref. 36 N,N'-diphenylurea Table I, Ref. 36 Dithioihreitol All, Ref. 36 DTT adduct of dehyroalanine All, Table I DTT adduct(s) of dehydro-a-aminoisobutyric acid IV, Table I O-Fucosylthreonine I, Ref. 19 Glutamic Acid All, Table I, Table H Glutamic Acid (O-allyl) VII, Ref. 16 Glutamid Acid (O-ailyl) derivative VE, Ref. 16 Glutamic Acid associated peak IV Glutamic Acid (O-benzyl) V, Table I, Ref. 23 Glutamic Acid (cyclohexyl) Table I Glutamic Acid (O-9-Fluorenylmethyl) Table I Glutamine All, Table I, Table H Glycine All, Table I, Table II Histidine All, Table I, Table n Histidine (allyloxycarbonyl) VH, Ref. 16 Histidineazobenzene arsonate Ref. 33 Histidine (3-benzyl) Table I Histidine (3-benzyloxymethyl) Table I Histidine (tert-butyloxymethyl) , Ref. 17 29 Histidine (2,4-dinitrophenyl) Table I Homoarginine Table I DL-Homocystine I Homophenylalanine Table I Homoserine I, Table I 5-Hydroxylysine I, Ref. 30 5-Hydroxylysine derivative I, Ref. 30 Hydroxyproline I, Ref. 30 N-y-hydroxyethyl-glutamine Table I lodotyrosine I, Table n Isoleudne All, Table I, Table II Lanthionine I, Ref. 30 Leucine All. Table I, Table II Lysine All, Table I, Table II Lysine (allyloxycarbonyl) VII, Ref. 16 Lysineazobenzene arsonate Ref. 33 Lysine (N-e- Chlorobenzyloxycarbonyl) V, Table I Lysine (N-e-dinitrophenyl) Table I Lysine (N- E-9-Fluorenylmeihyloxy car bony 1) Table I Methanoi/PITC conversion artifact IV, Ref. 36 Methionine All, Table I, Table D Methionine sulfone I, Ref. 30 N-a-methylalanine Table I Methylarginine I, Ref. 30 S-methylcysteine I O-methyl glutamic acid I, Ref. 35 N-y-methyl glutamine Table I Methylhistidine I, Ref. 30 1-Methy Ihistidin e Table I 3-Methylhistidine Table I N-E-methyllysine I, Table E, Ref. 14, 26, 30 N-a-methylphenylalanine Table I O-Methylthreonine I O-Methy 1 tyrosine I Naphthylalanine VI, Table I Nitroarginine Table I 30 p-Nitrophenylalanine Table I 3-Nitrotyrosine Ref. 24 Norleucine I, Table I Norv aline Table I Omi thine I Omithine (benzyloxycarbonyl) Table I Phenylaianine All, Table I, Table II Phenylalanine (p-amino-benzyloxycarbonyl) Table I Phenylaianine (p-amino-benzyloxycarbonyl) prime Table I Phenylthiocarbamyl aianine m, Ref. 30 Phenylthiocarbamyl glycine in Phenylthiocarbamyl isoleucine m, Ref. 30 Phenylthiocaibamyl Ieucine in, Ref. 30 Phenylthiocarbamyl lysine m. Table I, Ref. 30 Phenylthiocarbamyl methionine m, Ref. 30 Phenylthiocarbamyl vaiine HI, Ref. 30 O-phosphoserine H, Table H O-phosphotyrosine Table I, Table n P -(3 -pyridyl) aianine Table I S-Pyridylethyl cysteine i, rrr, rv Proline AU, Table I, Table II R4 contamination peak IV Resumption of interrupted sequence artifacts IV Serine AU, Table I, Table II Serine (allyloxycarbonyl) VII, Ref. 16 Serine (allyloxycarbonyl) derivative VH, Ref. 16 Serine associated peaks (Ser1) rv, Table I Ser (benzyl) V,VI N-e-succinyl lysine I, Ref. 14 Threonine All, Table I, Table II Threonine (allyloxycarbonyl) VH, Ref. 16 Threonine associated peaks (Thr1) TV, Table I Threonine (benzyl) V Threonine (benzyl) prime V Threonine (ten-butyl) vn N-e-trimethyl lysine I, Ref. 26 Tris artifact IV, Ref. 36 Tryptophan All, Table I, Table II Tryptophan associated peak IV, VII, Table I Tryptophan (N^-formyl) V, Table I Tyrosine All, Table I, Table II Tyrosine (allyl) VII, Ref. 16 Tyrosine azobenzene arsonate Ref. 33 Tyrosine (2-bromobenzyloxycarbonyl) V, VI, Table I Tyrosine (tert-butyi) VII Unknown at Asparagine/Serine IV Unknown at Phenylalanine/Isoleucine IV Valine AH, Table I, Table II Note: " All" indicates that the compound is represented in each of the reference standards. 32 LIST OF CONTRIBUTORS Instrument A. Andersen, Thomas T. Porton Dept Biochem & Mol Biol A-10 Albany Med Col Protein Chemistry Core Facility Albany NY 12208 B. Barra, Donatella Applied Biosystems Dept di Scienze Biochimiche Univ La Sapienza Piazzale Aido Moro 5 00185 Rome, Italy C. Beach, Carol M. Applied Biosystems Dept of Biochemistry Univ of Kentucky Chandler Medical Center Lexington KY 40536-0084 D. Cook, Richard F. Applied Biosystems MTT E17-310 Cambridge MA 02139 E. Crimmins, Dan L. Applied Biosysiems Howard Hughes Medical Inst. Washington Univ Sch of Med 660 South Euclid - PO Box 8022 St. Louis MO 63110 F. Dorwin, Sarah A. Applied Biosystems D-93D/AP-9A-Corporate Mol Biol Abbott Laboratories One Abbott Park Road Abbott Park IL 60064-3500 G. Fields, Gregg B. Applied Biosystems Dept Lab Medicine & Pathology Univ of Minnesota 420 Delaware St. SE - Box 107 Minneapolis MN 55455-0392 33 Instrument H. Fowler, Audree V. Porton Dept of Biological Chemistry UCLA Sch of Med Los Angeles CA 90024-1737 I. Gaathon, Ariel Applied Biosystems Bletterman Macromol Res Lab Interdepartment Equipment Div P.O. Box 1172 Jerusalem 91010 Israel J. Grant, Gregory A. Applied Biosystems Crankshaw, Mark W. DepL Moiec Biology & Pharmacol Washington Univ Sch of Med Campus Box 8103 St Louis, MO 63110 K. Harris, Reed J. Applied Biosystems #62 Genentech Inc 460 Point San Bruno Blvd So San Francisco CA 94080 L. Hoffman, Donald R. Milligen/Biosearch Dept of Pathology & Lab Med East Carolina Univ Sch of Med 7S-10 Brody Sciences BIdg Greenville NC 27858-3254 M. Hoogerheide, John G. Applied Biosystems 1140-230-3 The Upjohn Co 7000 Portage Road Kalamazoo MI 49001-0199 N. Kochersperger, Michael Applied Biosystems Applied Biosystems 850 Lincoln Centre Drive Foster City CA 94404 O. Lane, William S. Applied Biosystems Microchemisiry Facility Harvard Univ 16 Divinity Avenue Cambridge MA 02138 34 Instrument P. Man del, Lydia C. Applied Biosystems Molec Biology Core Facility Univ Missouri Sch Bas Life Sci 5100 RockhiU Road - 109-BSB Kansas City MO 64110-2499 Q. Niece, Ronald L. Applied Biosystems Biotechnology Center Univ ofWisconsin 1710 University Avenue Madison WI 53705 R. Paroutaud, P.S. Applied Biosystems Applied Biosystems S.A.R.L. 13 Rue de la Perdrix, BP 50086 Z.A.C. Paris Nord II 95948 Roissy Charies de Gaulle Cedex, France S. Pohl, Jan Applied Biosystems Microchemical Facility - Rm. 5220 Emory Univ 1327 Clifton Road NB Atlanta GA 30322 T. Siegel, Ned R. Applied Biosystems Smith, Christine Biological Sciences - AA21 Monsanto Co 700 Chesterfield Pkwy North Chesterfield MO 63198 ■ > U. Williamson, Matthew K. Applied Biosystems DepLofBiology-0322 ... ■ University of California-San Diego 9500 Gilman Drive LaJollaCA 92093-0322 35 SUGGESTED REFERENCES (* indicates a reference included by a contributor) Books 1. Sequencing of Proteins and Peprides. Allen. G., Elscvier, 1981. 2. Techniques in Protein Chemistry HI. Angelciti, R.H., Ed., Academic Press, 1992. 3. Techniques in Protein Chemistry TV. Angeletti, R.H., Ed., Academic Press, 1993- 4. Practical Protein Chemistry - A Handbook. Darbre, A., Ed., John Wiley and Sons, 1986. 5. Methods in Protein Sequence Analysis. Elzinga , M., Ed., Humana Press, 1982. 6. Protein Sequencing -- A Practical Approach, Findlay, J.B.C. and Geisow, MX,- IRL Press, 1989. 7 Handbook of HPLC For the Separation of Amino Acids. Peprides and - Proteins. Hancock, W.S., Ed., CRC Press, Vol. I &. II, 1984. 8. Techniques in Protein Chemistry I. Hugli, T.E., Ed., Academic Press, 1989. 9. High-Perfonnance Liquid Chromatographv of Peprides and Proteins; Separation. Analysis and Conformation. Mant, C.T. and Hodges, R.S., CRCPress, 1991. 10. -■ Methods of Protein MjcTocharacterizarion - A Practical Handbook. Shively, J.E., Ed., Humana Press, 1986. 11. Post-Translation Modifications of Proteins. Tuboi, S., Taniguchi, CRC Press, 1993. ''..ii... . -1 . ' "3 12. Current Researrh in Protein Chemistry: Techniques. Structure, and :- Function. Villafranca, J.J., Ed., Academic Press, 1990. ( 13 Techniques in Protein Chemistry TT. Villafranca, J.J., Ed., Academic Press, 1989. Articles 14.* The protein sequence of glutamate dehydrogenase from Sulfolobus solfataricus, a thennoacidophilic archacbacterium, Barra, D., Eur. J. Biochem., 203, 81-87, 1 1992. 36 15.* Complete assignment of neurophysin disulfides indicates pairing in two separate domains, Burman, S., Wellner, D., Chait, B., Chaudhary, T., Breslow, E., Proc.Nad.Acad.Sci. USA, 86, 429-433, 1989. 16.* The Development of High-Performance Liquid Chromatographic Analysis of Ally! and Allyloxycarbonyl Side-Chain-Protected Phenylthiohydantoin Amino Acids, Fields, C.G., Loffet, A., Kates, S.A., and Fields, G.B., Anal. Biochem., 203, 245-251, 1992. 17.* Edman Degradation Sequence Analysis of Resin-Bound Peprides Synthe-sized by 9-FIuorenyImethoxycarbonyl Chemistry, Fields, C.G., VanDirisse, V. and Fields, G.B., Pep. Res., 6, 39-46, 1993. 18. Solvent system for the rapid identification of phenylthiohydantoin derivatives of amino acids by high-performance liquid chromatography, Fonck, C, Frutigef, S., and Hughes, G.J., J. Chromatogr., 370-2, 339-343, 1986. 19.* O-Linked Fucose Is Present in the First Epidermal Growth Factor Domain -of Factor XII but Not Protein C, Harris, R.J., J. Biol. Chem., 267-8, 5102-5107, 1992. .-1 jv_. 20. Microsequence Analysis of Peptides and Proteins: n. Separation of:Arnino_Acid Phenylthiohydantoin Derivatives by High-Perfonnance Liquid Chromatography on Octadecylsilane Supports, Hawke, D., Yuan, P-M., and Shiveiy, J.E., Anal. Biochem. 120, 302-311, 1982. __. 21. Isocratic separation of phenylthiohydantoin-amino acids by reversed-phase high- performance liquid chromatography, Hayakawa, K. and Oizumi, Ji, J. Chromatogr., 487-1, 161-166, 1989. 22. Instability of phenylthiohydantoin amino acids, Jansecu_E.H. &. Both-Miedema, R., J. Chromatogr., 435-2, 363-367, 1988. 23.* Sequencing of peptides on solid phase supports, Kocherspe-gerJ&i Blacher, R., Kelly, P., Pierce, L., and Hawke, D.H., American-:rBiotechnoTogy Laboratory, 1989. _ - s£ a. ' "c 24. Preparation and characterization of 5-{4-hydroxy-3-nitrobenzyl)-3-p'henyl- 2- thiohydantoin, the phenylthiohydantoin derivative of 3-nitrotyrosine, Lilova, A. Kleinschmidt, T., Nedkov, P., and Braunitzer, G.^BiolrXhenx Hoppe Seyler, 367-10,1055-9,1986. .. .x. ■. A 'jfi 25. Preparation and characterization of N epsilon-(2,3-dihy.drjQ3!ypropyl)-Lrlysine_and its phenylthiohydantoin derivative, Lilova, A., Biol. Chem. Hoppe Seyler, 368-11, 1489-1493, 1987. 26.* Sequence Analysis of Acetylation and Methylation in Two Histone H3 Variants of Alfalfa, Mandel L.C., J. Biol. Chem., 265-28, 17157-17161, 1990. 27. Separation of amino acid phenylthiohydantoin"'derivjati^es by high-pressure .liquid chromatography, Meuth, J.L. and Fox, J.L., AnaI.Jbiochem., 154, 478-84,',1986. 37 28 High-sensitivity phenylthiohydantoin amino acid analysis on-line to a gas phase protein sequencer, Murphy, R., J. Chromatogr., 408, 388-392, 1987. 29. Retention behaviour of phenylthiohydantoin amino acids in micro high-performance liquid chromaiography with ociadecyl bonded glasses and silicas, Okamoto, M., J. Chromatogr., 396, 345-349, 1987. 30.* Unpublished Studies on Unusual and Post Translational Modified^mino Acids — available upon request; and poster reprint from Protein Society Meeting, San Diego, CA, July 1993 - will be available in future, by Paroutaud, P.S. Contact Ruth Steinbrich, Applied Biosystems USA, 1-8OO-874-9868. 31. An optimized procedure for the separation of amino acid phenylthiohydantoins by reversed-phase HPLG, Persson, B. and Eaker, D., J. Biochem. Biophys. Methods, 21-4, 341-350, 1990. 32. Analysis of phenylthiohydantoin amino acid mixtures for sequencing by thermospray liquid chromatography/mass spectrometry, Pramanik, B.C., Hinton, S.M., Mtlliagton, D.S., Dourdeville, T.A., and Slaughter, CA.., Anal. Biochem., 175-1, 305-318, 1988. 33. Protein modification by diazotized arsanilic acid: synthesis and characeterization of the phenylthiohydantoin derivative of azobenzene arsonate-coupledtyrosine, histidine, and lysine residues and their sequential alotment in labeled peptides, Schwallcr, B. & Sigrist, H., Anal. Biochem., 177-1, 193-187, 1989. 34. Biotinylatedpeptides/proteins. I. Identification of biotinylated lysyl phenylthiohydantoins, Smith, J.S., Anal. Biochem., 197-1, 254-257, 1991. 35.* Direct Identification of 7-Carboxyglutamic Add in the Sequencing of Vitamin K Dependent Proteins, Williamson, M.K., Anal. Biochem., 199,93- 97, 1991. Bulletins 36. Artifact Peaks In HPLC Analysis of PTH Amino Acids, User Bulletin #5, Applied Biosystems, 1984. 37. Sequence Analysis of Synthetic, Side-Chain Protected, Resin-Bound Peptides, User Bulletin #13, Applied Biosystems, 1985. 38. PTH Amino Acid Analysis, Hunkapiller MW, Applied Biosystems, 1985. 38