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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 References 36 'BO1
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 <mide. Additional supporting analyses should be employed to verify the identity ot any unknown residue. Anyone noting errors or conflicts is invited to send documentation.
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
Alanine
a-Aminobutyric Acid
Applied Biosystems "Incorporated"
Arginine
Arginine (diallyloxycarbonyl)
Arginine (mesitylene-2-sulfonyl)
Arginine (4-methoxy-2,3,6-trimethylbenzenesulfonyl)
Arginine (4-toluenesulfonyl)
Asparagine
Asparric Acid
Aspartic Acid (cyclohexyl)
Aspartic Acid (O-ailyl)
Aspartic Acid associated peak
Aspartic Acid (O-benzyl)
Aspartic Acid (O-tert-butyl)
Association of Biomolecular Resource Facilities
S-Carboxamidomethyl-cysteine
y-Carboxyg!utamic Acid
S-Carboxymethyl-cy stein e
Citruliine
Cysteine
Cysteine (allyl)
Cysteine (allyloxycarbonyl)
Cysteine (4-methoxybenzyl)
.■"IK
Cysteine (4-methylbcnzyl)
Cysteine (3-nitro-2-pyridylsuIfenyl)
Cysteine (tert-butyl)
Cystine
N-dimethyl, N'-phenylthiourea
O, O-dimethylphosphotyrosine
ACM-Cys
Ala, A
Abu
ABI
Arg.R
Arg(AJoc)2
Arg(Mts)
Arg(Mtr)
Arg(Tos)
Asn, N
Asp, D
Asp(OcHex)
Asp(OAl)
Asp'
Asp(OBzl)
Asp(OtBu)
ABRP
Cys(SCAM)
Gla
Cys(SCM)
Cit
Cys, C
Cys(AI)
Cys(Aloc)
Cys(4-CH3OBzi) or
Cys(SMob)
Cys(4-CH3Bzl) or
Cys(SMeb)
Cys(Npys)
Cys(tBu)
Cys2
DMPTU
Tyr(PO3Me2)
N,N'-diphenyl thiourea
N,N'-diphenylurea
Dithiodireitol
DTT adduct of dehydroalanine
DTT adduct(s) of dehydro-oc-aminoisobutyric acid
Fluorenylmethyloxycarbonyl
Glutamic Acid
Glutamic Acid (O-allyl)
Glucamic Acid associated peak
Glutamic Acid (Obenzyl)
Glutamic Acid (cyciohexyi)
Glutamic Acid (O-9-Fluorenylmethyl)
High Pressure Liquid Chromatography
Histidine
Histidine (allyloxycarbonyl)
Histidine (3-benzyl)
Histidine (3-benyzloxymethyl)
Histidine (tert-butyloxymethyl)
Histidine (2,4-dinitrophenyl)
5-Hydro xyly sine
Hydroxyproline
Isoleucine
Lanthionine
Leucine
Lysine
Lysine (allyloxycarbonyl)
Lysine (chlorobenzyloxycarbonyl)
Lysine (N-e-dinitrophenyl)
Lysine (N-e-9-Fluorenylmethyioxycarbonyl)
Methionine
l-Methylhistidine
3-Methylhistidine
Nitroarginine
Norleucine
Norv aline
Gmithine
DPTU
DPU
DTT
S1
T'
Fmoc
Glu, E
Glu(OAl)
Glu1
GSu(OBzl)
Glu(OcHex)
Glu(OFm)
HPLC
His, H
His(Aloc)
His(3-Bz!)
His(Bom)
His(Bum)
His (Dnp)
Hyl
Hyp
lie, I
Lan
Leu, L
Lys, K
Lys(Aloc)
Lys(ClZ)
Lys (Dnp)
Lys (Fmoc)
Met,M
His(l-Me)
His(3-Me).
Arg(NO2)
Nle
Nva
Orn
7
Ornithine (benzyloxycarbonyl)
Phenylalanine
Phenylalanine (p-amino-benzyloxycarbonyl)
Phenylisothiocyanate
Phenylthiocarbamyl
Phenylthiohydantoin
Phenyltbiohydantoin amino acid
S-pyridylethyi cysteine
Pro line
Serine
Serine (allyloxycarbonyl)
Serine (benzyl)
Solid phase peptide synthesis
Threonine
Threonine (allyloxycarbonyl)
Threonine (benzyl)
Threonine (tert-butyl)
Tryptophan
Tryptophan associated peak
Tryptophan (Nin-formyl)
Tyrosine
Tyrosine (allyl)
Tyrosine (2-bromobenzyloxycarbonyl)
Tyrosine (ten-butyl)
Valine
Om(Z)
Phe, F
Phe(p-amino-Z)
PITC
PTC
PTH
PTH-aa
PECys or
Cys (SPE)
Pro.P
Ser, S
Ser(A!oc)
Ser(Bzl)
SPPS
Thr, T
Thr(Aloc)
Thr(Bzl)
Thr(tBu)
Trp.W
Tip'
Trp(CHO)
Tyr,Y
Tyr(Al)
Tyi(2BrZ)
Tyr(tBu)
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
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
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
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_ Q
O LLJ
— Unknown at PHE/ILE CoorassiB Blue
TRP Associated PaaK_ GLU Associated Peak
— AS? Associated Peak
— H4 Contamination — MethanolPITC
THR Associated Peaks
"5 >SEH Associated Peaks
THIS
""known ASN/SER
— Aniline
q
cd
O
CO CVJ
q
CM CM
q
o
CM
q
CO
q CO
co
q
c\i
q
o
q
co
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
(Z-IO)sA-]-Hld
na
511
-510
-506
-509
-503
-507 ■506'
■505
■504
■503
■502
■501
500
p
d CO
I—> ■4-'
O =3 CVJ C
O
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
SAT U
311
3Hd
nida
1VA
131
OUd
HA1
V1V
SIH
ma
AT9
UHi
H3S
NSV
dSV
604
603
600
602
601
Oi'9
zv% ■ ■
OJ
. CO
OJ
cg
CO
LO
CO
O
eg
. CD
CO
600
CO
CO
CO
in
CD •4—■
c
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
...dJl-Hld
S!H-Hld
B[V-Hld
ni3-Hld nidwa _
A"[9-Hld
u|9-Hld
usv-Hld
-818
818
818
821
820 819
■818J
801
800
O
O
q
o
CO
-4—'
i o ■—
CM ^
p
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 II
3 me his
nlrt
arg
asn
asp
cys
di iodo his
di iodotyr
dimethyl lys
gin
glu
giy his
hydroxy pro
ile
leu
lys
met
mono iodo his
mono iodo Tyr
mono methyl lys
phe
phos-ser
phos-tyr
pro
ser
thr
tip
tyr
val
after his about 1.57/>A (broad shape)
H//X>Y 0.81
ala<X<ser primeZ/approx near methyl his//H
D<X<N(nearer to N)//D(deamidated N)//S'
M
R<X<Y//S'//H//E//G(Q/E ratio approx =, no: as in a Q
call)//T//P//R//X<H(s-p proprioamido Cys-[aerylamide mod.])
//Y//X<P 1.5'
>his 3'
2'>nleu
DMPTU<X<H
E(deamidated Q)//G(ptcE?)//X>A 1'
G(or slighdy after=ptc E)//dptu<X<W//Y or Y<X<P
X<D l'(ptcG)
I//A<X<R(me his)
l">ala ptc hPro l'<his
X>nleu
tyr<X<pro(l/2 way)//M//V<X<dptu//X>W(close to W)
Q<X<T//(S)//X<P(metO?)//dpm<X<W0.5'>dptu
>his 2*
X<W 0.21
X>L
dptu//dptu<X<W//H?
ser/ser'=2.5;//ser/ser"=0.8;//ser
approx. 3.5' into run (w/DTTet al.) need nMol amts.
Y<X<P(ptc P)
D(approx 50%of S)//X<Y(r<Y,=S1)//s"=l'>Y//V<X<dptu//
N//Q//G//approx M//ser/ser'=17.9; ser/ser" =5.4
Q//X<P 1.5'(T")//X>dmptu(close to E)//X<F(cIose)// approx M
V<X<dptu l'//F<X,X<K//X<P 0.81
ala<X<tyr(l/2 way)
dptu<X<trp//X<pro(0.2")
fianipfe Translation of Shorthand
Asn- D<X<N(nearer to N)//D(deamidaiedN)//S'<X_<R(?)//M(?) =
•Asn may/will have an unknown(X) peak eluting between Asp and Asn,
and it is nearer to Asn than it is to Asp. fX=the unusual/unknown peak)
•A peak may/will appear at Asp which is deamidated Asn.
•A peak may(?) appear between. Ser1 and Arg (? means this is not verified
by more than one run) •A peak may arise at the Met location. This is not verified by more tiian
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
N-e-Acetyl-Iysine
Alanine
a-Aminoburyric Acid
Aniline
Arginine
Arginine (diallyloxycarbonyl)
Arginine (diallyloxycarbonyl) derivative
Arginine (MesiryIene-2-sulfonyI)
Arginine (4-Methoxy-2,3>6-trimethy I benzene sulfonyl)
Arginine (4-Toluenesulfonyl)
Asparagine
Aspartic Acid
Aspartic Acid (cyclohexyl)
Aspartic Acid (O-allyl)
Aspartic Acid asociated peak
Aspartic Acid (O-benzyl)
Aspartic Acid (O-tert-butyl)
4-O-Benzylhydroxyproline
Biotinylated Iysine
Canavanine
S-Carboxamidomethyl-cysEeine
Carboxamidomethyl-methionine
-y-Carboxyglutamic Acid
S-Carboxymethyl-cysteine
p-Qiloro-phenylalanine
Citrulline
Coomassie Blue
P-Cyclohexylalanine
Cysteine
Cysteine (allyl)
Cysteine (allyioxycarbonyl)
Cysteine (4-methoxybenzyl)
V, VH, Table I, Ref. 17
I, Table I, Ref. 26
All, Tables I & H
I, Table I, Ref. 17
rv
AH, Tables I & II
VH, Ref. 16
VII, Ref. 16
V, Table I
VH, Table I
V. Table I
All, Tables I & H
All, Tables I & H
Table I
VII, Ref. 16
IV
V, VI, Table I, Ref. 23
VII, Ref. 23
Table I
Table I, Ref. 34
I
I
I
I, Ref. 35
I
I
Table I
IV, Ref. 36
Table I
Table U
, Ref. 16
, Ref. 16
V, Table I
28
Cysteine (4-methylbenzyl)
Cysteine (3-nitro-2-pyridylsuIfenyl)
Cystine
Dehydroalanine
Dehydro-a-aminoisobutyric acid
3,4-Dehydroproline
ovy-diaminobutyric acid
0-(2,6-dichlorobenzyl)-tyrosine
3-(2,6-dichlorobenzyl)-tyrosine
N-e-(2,3-dihydroxypropyl)-lysine
Diiodohistidine
Diiodotyrosine
Dimethyilysine
N-dimethyl, N'-phenylthiourea
O, O-dimethylphosphotyrosine
N,N'-diphenylthiourea
N,N'-diphenylurea
Dithioihreitol
DTT adduct of dehyroalanine
DTT adduct(s) of dehydro-a-aminoisobutyric acid
O-Fucosylthreonine
Glutamic Acid
Glutamic Acid (O-allyl)
Glutamid Acid (O-ailyl) derivative
Glutamic Acid associated peak
Glutamic Acid (O-benzyl)
Glutamic Acid (cyclohexyl)
Glutamic Acid (O-9-Fluorenylmethyl)
Glutamine
Glycine
Histidine
Histidine (allyloxycarbonyl)
Histidineazobenzene arsonate
Histidine (3-benzyl)
Histidine (3-benzyloxymethyl)
Histidine (tert-butyloxymethyl)
V, Table I
VII, Ref. 17
I, Ref. 15
Table I
Table I
Table I
I
Table I
Table I
Ref. 25
Table n
I, Table H
I, Table II, Ref. 26
All, Table I, Ref. 36
Table I
All, Table I, Ref. 36
Table I, Ref. 36
All, Ref. 36
All, Table I
IV, Table I
I, Ref. 19
All, Table I, Table H
VII, Ref. 16
VE, Ref. 16
IV
V, Table I, Ref. 23
Table I
Table I
All, Table I, Table H
All, Table I, Table II
All, Table I, Table n
VH, Ref. 16
Ref. 33
Table I
Table I
, Ref. 17
29
Histidine (2,4-dinitrophenyl)
Homoarginine
DL-Homocystine
Homophenylalanine
Homoserine
5-Hydroxylysine
5-Hydroxylysine derivative
Hydroxyproline
N-y-hydroxyethyl-glutamine
lodotyrosine
Isoleudne
Lanthionine
Leucine
Lysine
Lysine (allyloxycarbonyl)
Lysineazobenzene arsonate
Lysine (N-e- Chlorobenzyloxycarbonyl)
Lysine (N-e-dinitrophenyl)
Lysine (N-E-9-Fluorenylmeihyloxy car bony 1)
Methanoi/PITC conversion artifact
Methionine
Methionine sulfone
N-a-methylalanine
Methylarginine
S-methylcysteine
O-methyl glutamic acid
N-y-methyl glutamine
Methylhistidine
1-Methy Ihistidin e
3-Methylhistidine
N-E-methyllysine
N-a-methylphenylalanine
O-Methylthreonine
O-Methy 1 tyrosine
Naphthylalanine
Nitroarginine
Table I
Table I
I
Table I
I, Table I
I, Ref. 30
I, Ref. 30
I, Ref. 30
Table I
I, Table n
All, Table I, Table II
I, Ref. 30
All. Table I, Table II
All, Table I, Table II
VII, Ref. 16
Ref. 33
V, Table I
Table I
Table I
IV, Ref. 36
All, Table I, Table D
I, Ref. 30
Table I
I, Ref. 30
I
I, Ref. 35
Table I
I, Ref. 30
Table I
Table I
I, Table E, Ref. 14, 26, 30
Table I
I
I
VI, Table I
Table I
30
p-Nitrophenylalanine
3-Nitrotyrosine
Norleucine
Norv aline
Omi thine
Omithine (benzyloxycarbonyl)
Phenylaianine
Phenylalanine (p-amino-benzyloxycarbonyl)
Phenylaianine (p-amino-benzyloxycarbonyl) prime
Phenylthiocarbamyl aianine
Phenylthiocarbamyl glycine
Phenylthiocarbamyl isoleucine
Phenylthiocaibamyl Ieucine
Phenylthiocarbamyl lysine
Phenylthiocarbamyl methionine
Phenylthiocarbamyl vaiine
O-phosphoserine
O-phosphotyrosine
P -(3 -pyridyl) aianine
S-Pyridylethyl cysteine
Proline
R4 contamination peak
Resumption of interrupted sequence artifacts
Serine
Serine (allyloxycarbonyl)
Serine (allyloxycarbonyl) derivative
Serine associated peaks (Ser1)
Ser (benzyl)
N-e-succinyl lysine
Threonine
Threonine (allyloxycarbonyl)
Threonine associated peaks (Thr1)
Threonine (benzyl)
Threonine (benzyl) prime
Threonine (ten-butyl)
N-e-trimethyl lysine
Table I
Ref. 24
I, Table I
Table I
I
Table I
All, Table I, Table II
Table I
Table I
m, Ref. 30
in
m, Ref. 30
in, Ref. 30
m. Table I, Ref. 30
m, Ref. 30
HI, Ref. 30
H, Table H
Table I, Table n
Table I
i, rrr, rv
AU, Table I, Table II
IV
IV
AU, Table I, Table II
VII, Ref. 16
VH, Ref. 16
rv, Table I
V,VI
I, Ref. 14
All, Table I, Table II
VH, Ref. 16
TV, Table I
V
V
vn
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
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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