Download Identification of Modified Amino Acids by Edman Sequencing

Survey
yes no Was this document useful for you?
   Thank you for your participation!

* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project

Document related concepts

Metalloprotein wikipedia, lookup

Protein wikipedia, lookup

Point mutation wikipedia, lookup

Fatty acid synthesis wikipedia, lookup

Fatty acid metabolism wikipedia, lookup

Nucleic acid analogue wikipedia, lookup

Citric acid cycle wikipedia, lookup

Protein structure prediction wikipedia, lookup

Proteolysis wikipedia, lookup

Metabolism wikipedia, lookup

Ribosomally synthesized and post-translationally modified peptides wikipedia, lookup

Genetic code wikipedia, lookup

Peptide synthesis wikipedia, lookup

Biosynthesis wikipedia, lookup

Amino acid synthesis wikipedia, lookup

Biochemistry wikipedia, lookup

Transcript
J.W. Leone – Pfizer Inc., St. Louis, MO, United States
B.J. Madden – Mayo Clinic College of Medicine, Rochester, MN, United States
D. Brune – Arizona State University, Tempe, AZ, United States
J. Pohl – Emory University, Atlanta, GA, United States
R. Kobayashi – UT MD Anderson Cancer Center, Houston, TX, United States
W.S. Lane and J.M. Neveu – Harvard University, Cambridge, MA, United States
N.D. Denslow – University of Florida, Gainesville, FL, United States.
ABRF ESRG 2005 Study: Identification of
Modified Amino Acids by Edman Sequencing
Submitted Amino Acid Calls by Each Participating Facility
Tyr
Arg
Ala
124
Tyr
(Arg)
125
Tyr
126
Tyr
Materials and Methods
127
Tyr
The ESRG 2005 peptide was synthesized on a Milligen-Biosearch 9050+ peptide
synthesizer using Fmoc chemistry. Synthesis was performed on Fmoc-Arg-PEGPS resin (Applied Biosystems) using a 4-fold molar excess (0.8 mmole) of each
Fmoc amino acid and the HATU coupling reagent, except in the cases of Fmoc-3Me-His-OH and Fmoc Lys(Me3)-OH, which were available in smaller amounts.
In these cases, PyAOP (0.7 mmole) was used as the coupling reagent. Three
amino acids, Fmoc-Lys(Me2)-OH, Fmoc Lys(Me3)-OH, and Fmoc-3-Me-His-OH,
were injected manually during the synthesis; all others were dissolved and
injected automatically. Coupling times for the methylated Lys residues were
extended to one hour, and coupling of Gly to N-Me-Ala was 45 minutes; all
others were for 30 minutes. The synthetic peptide was cleaved from the resin
with 92.5% TFA with 2.5% each of triisopropylsilane, ethanedithiol, and water,
precipitated by adding diethyl ether, and dried under vacuum.
128*
Tyr
129
Tyr
130
Tyr
(biotinylLys)
(Me2Lys)
131
Tyr
132*
133
134
Tyr
Tyr
Tyr
135
Tyr
136
Tyr
The monomeric peptide was purified by reversed phase HPLC on a Phenomenex
C12 proteo column (1 cm x 25 cm) using a gradient of 10-20% acetonitrile in
water containing 10 mM TFA over 20 minutes. The peptide was then dissolved in
25 mM Na Phosphate, pH 7.6, at a final concentration of 1.8 mM (determined
form its 280 nm absorbance) followed immediately by adding diamide to a final
concentration of 0.9 mM to cause dimerization (Kosower et al., 1987). After
allowing the reaction to proceed for several minutes, the diamide concentration
was increased to 1.3 mM, in order to drive the reaction to completion.
137
Tyr
18 ABI 49X-HT (1-10 years old: avg age of 7 yrs)
7 ABI 49X-cLC (5-10 years old: avg age of 6.7 yrs)
1 ABI 477A (10 yrs old)
22 used all instrument manufacturer reagents
1 used some mfg. R1, R2c, R4, R5, & Premix
1 said almost mfg. reagents
1 said partially
1 said S4 home made
21 GFF, 4 PVDF,
1 said both GFF and PVDF (probably answered in general)
Chemistry Cycle
All users seemed to use the cycles that matched the support (ie. GFF or PVDF)
except one PVDF with GFF cycle
DTT in S2
6 yes and 20 no
Other Additives
1TCEP to R4A, 1 DTT in R4A & 1 DTT in S3
Sample Solvent
13 used 0.1% TFA/30% acetonitrile
4 used 0.1% TFA/20% acetonitrile
2 used 0.1% TFA/ water
1 used 1% TFA/ 60% acetonitrile
1 used 0.01%TFA/ 50% acetonitrile
1 used 0.0005% TFA/ 0.05%HAc/ 30% acetonitrile
1 used 30% acetonitrile/ water
3 users were unclear
Ranged from 2 to 100%
Me3Lys
Arg
X
Gly
Gly
Gly
N-MeAla
X
Me3Lys
Tyr
Tyr
Tyr
Lys
Lys
Lys
His
His
His
X
X
(Cystine)
Ala
Ala
Ala
(Cystine)
X
X
Tyr
Tyr
Tyr
(N-MeAla)
X
Tyr
Gly
Gly
Gly
(Me3Lys)
X
X
Tyr
Tyr
Tyr
Ala
Ala
Ala
(isoAsp)
X
Ala
X
Lys
His
Cys-SPam
Ala
pSer
Tyr
Gly
Me2Arg
Tyr
Ala
Ala
Ala
(3MeHis)
Lys
His
X
Ala
(Cystine)
Tyr
Gly
Me3Lys
Tyr
Ala
X
(X)
Ala
3MeHis
Lys
His
(X)
Ala
Gly
(X)
Me2Lys
Ala
X
Arg
Ala
Me3Lys Me2Lys Cystine
X
Tyr
X
Gly
Cys
N-MeAla 3MeHis isoAsp
Me3Lys (Me2Lys) His HomoCit Ala
NHydroxyMet HydroxySuccinyl- Lan CAM-Met
pSer
Me2Lys
Lys
sulfone
Lys
Lys
Arg
X
Tyr
HydroxyPro
Gly
Abu
Tyr
Cys
Tyr
(NmethylAla)
Gly
(Me2Lys)
Tyr
Ala
(MeLys)
Ala
3MeHis
Lys
His (CAM-Cys) Ala
(Cystine)
Tyr
(Me2Lys)
Gly
(Me3Lys)
Tyr
Ala
(isoAsp)
Ala
(3MeHis)
Lys
His (HomoCit) Ala
(Cys)
Tyr
(N-MeAla)
Gly
(Me3Lys)
Tyr
Ala
(isoAsp)
(Me2Lys)
Ala
3MeHis
Lys
His
HomoCit
Ala
Cystine
Tyr
Me3Lys
Gly
N-MeAla
Tyr
Ala
(iso-Asp)
Me2Lys
3MeHis
(Arg)
Ala
Ala
Ala
3MeHis
N-MeAla
(3MeHis)
Lys
Lys
Lys
His
His
His
HomoCit
isoAsp
X
Ala
Ala
Ala
Cystine
(Ser)
(MeLys)
Tyr
Tyr
Tyr
Me3Lys
HomoCit
X
Gly
Gly
Gly
N-MeAla
Cystine
X
Tyr
Tyr
Tyr
Ala
Ala
Ala
isoAsp
Ala
X
(N-MeAla) Ala
3MeHis
Lys (HomoCit)(isoAsp)
Ala
Cys
Tyr
X
Gly
(Tyr)
Ala
Glu
Ala
3MeHis
Lys
His
Ala
Cystine
Tyr
Me3Lys
Gly
N-MeAla
Tyr
Ala
(iso-Asp)
(3MeHis)
Lys
His (HomoCit) Ala
(Cystine)
Tyr
(Me2Lys)
Gly
(Me3Lys)
Tyr
Ala
(iso-Asp)
(N-MeAla) Ala
-0.30
-0.27
-0.22
-0.20
-0.18
-0.17
-0.13
-0.04
0.00
0.12
0.16
0.33
0.38
0.40
0.55
0.63
0.66
0.68
0.70
494-HT
494-HT Av
Std Dev of
Full RT
RTnA's
0.03
0.02
0.01
0.01
0.01
0.01
0.01
0.02
0.00
0.02
0.01
0.00
0.01
0.00
0.01
0.02
0.02
0.03
0.03
4.19
4.61
5.24
5.52
5.74
6.00
6.48
7.69
8.22
9.78
10.36
12.63
13.34
13.65
15.61
16.64
17.08
17.36
17.61
494-cLC
494-cLC
Average
494-cLC Av
Std Dev of
RTnA's
Full RT
RTnA's
n=10
-0.30
-0.27
-0.23
-0.21
-0.19
-0.17
-0.14
-0.05
0.00
0.11
0.16
0.33
0.38
0.41
0.57
0.62
0.66
0.68
0.70
0.01
0.01
0.01
0.01
0.01
0.00
0.00
0.01
0.00
0.01
0.01
0.00
0.01
0.00
0.01
0.01
0.01
0.01
0.01
6.14
6.59
7.34
7.68
7.92
8.24
8.77
10.13
10.89
12.57
13.39
16.06
16.86
17.23
19.84
20.66
21.15
21.48
21.79
477
RTnA's
n=1
-0.24
-0.21
-0.17
-0.16
-0.14
-0.12
-0.10
0.00
0.00
0.13
0.16
0.37
0.42
0.44
0.61
0.66
0.70
0.73
0.76
477 Full
RT
7.91
8.50
9.35
9.75
10.10
10.50
11.00
13.00
13.10
15.80
16.64
21.10
22.20
22.60
26.15
27.30
28.23
28.73
29.38
Porton
RTnA's
n=1
-0.31
-0.28
-0.23
-0.21
-0.20
-0.16
-0.15
-0.05
0.00
0.12
0.15
0.36
0.39
0.40
0.57
0.62
0.71
0.67
0.69
% loaded
% loaded
90
80
70
60
50
20
40
30
10
20
10
01
Mean 21.35%
3
1
3
5
5
7
7
9
9
11
11
Val*
Arg*
Porton
Full RT
6.26
6.75
7.46
7.79
8.02
8.60
8.88
10.49
11.23
13.23
13.61
16.93
17.49
17.72
20.44
21.23
22.66
22.00
22.31
Time Lines for Elution of Standard and Modified
Amino Acids on the Procise HT and Procise cLC
30
25
To compensate for variations in the actual
elution times of amino acid standards, the
retention time (RT) for each standard was
normalized to Ala using the following
procedure: The retention time of Ala was
subtracted from the retention time of each
amino acid and this was divided by the total
time interval between Asp and Leu (the first
and last standards to elute). RTnA is the
decimal fraction of this interval between
the time when each amino acid eluted and
the time when Ala eluted, with negative
values indicating amino acids eluting
before Ala. Std Dev RTnA is the
calculated standard deviation for each
RTnA value. Av Full RT were determined
by multiplying the RT difference between
Asp and Leu by RTnA and adding the
product to the RT for Ala. In cases where
data were available from only one
instrument of a particular type, only RTnA
values and Full RT are reported.Results in
this table include data from instruments
operated by members of the Edman
Sequencing Research Group.
ABI Procise HT
20
X = "X" + "-"
15
3-Me
His
Homo
Citr
Tri-Me Di-Me
N-me
Lys Lys Cys Ala
W = PW + TW
C = PC + TC
D N
SQTG
E
H
A
R
Y
P
MV
W
F
I KL
10
5
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
minutes
0
ABI Procise cLC
Homo
Citr
D N
3-Me
His
Di-Me Tri-Me
Lys
Lys Cys
N-me
Ala
SQT G
E
H
A
R
Y
P
M V
W
F
I K L
Average PTH Yields
4.00
200
Normalized Retention Times for Amino Acids in Peptide
% of Sample Loaded
30
Me3Lys)
Arg*
Accuracy of Identification
100
40
(Me2 or
Val*
HydroxypThr
Lys
(3MeHis)(HomoCit) His
HomoCit
Ala
Me3Lys)
Ala
Me2Lys
(iso-Asp)
(Me2 or
70
0
% Loaded
Tyr
Tyr
Tyr
90
50
isoAsp
X
X
Cys
X
(Arg) (2)
100
60
Ala
Ala
Ala
His HomoCit Ala
His (Cys-S-Pam) Ala
His
X
Ala
6.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
22.00
24.00
26.00
minutes
cLC
HT
% Loaded by Facility
80
Gla
Tyr
13
15
13
17
15 17 19 21
participant
#
19
23
21
23
150
Relative Peak Area
Sample Support
Ala
Cystine
di
m
D
N
S
Q
T
G
E
H
A
R
Y
P
M
V
W
F
I
K
L
Sequencers Information
23 liquid, 2 gas phase and 1 both
Tyr
Ala
Arg*
Ty
r
494-HT
Average
RTnA's
n=23
Kosower, N.S. and E. M. Kosower (1987) Formation of disulfides with diamide. Meth.
Enzymol. 143, 264-270.
TFA Cleavage
Abu,
Canavn.,
MeArg
OMeLys,
OMeGlu
Val*
Normalized retention times of standards relative to Ala by instrument type
Reference:
Reagents
isoAsp
(1) Comment: Coelutes with Ser
(2) Comment: Cystine
(3) Comment: Order of residues 3,4,8, and 15: 8<15<3<4. Would be very easy to call all Ala.
* These facilities correstly assigned residues 17 and 18 as Val and Arg, respectively, by MS/MS.
The dimeric peptide was then purified by reversed phase HPLC as described for
the monomer, using a gradient of 12 to 22% acetonitrile in water containing 10
mM TFA over 20 minutes. The dimeric nature of the product was indicated by (1)
elution at a different (higher) percentage of acetonitrile during reversed phase
HPLC, (2) the presence of a peak with twice the molecular mass of the monomer
in MALDI-TOF mass spectrometry, and (3) and earlier elution time than the
monomer in size exclusion chromatography on a Phenomenex SEC 2000 column
(0.78 x 60 cm).
Manufacturer and Model
Ala
As
p
123
(3MeHis)
X
X
Tyr
a
(N-MeAla) Ala
X
Ala
X
Ala
N-MeAla
iso
Tyr
Tyr
Tyr
Gly
X
Al
120
121
122
Me3Lys
Ala
Ty
r
Ala
Ala
Ala
His
CAMMet
Gly
Tyr
X
a
Me2Lys
Arg
N-MeAla
Cystine
(Me3Lys)
18
Arg
eA
l
Tyr
Tyr
Tyr
Ala
Gly
17
Val
Nm
117
118
119
HomoCit
16
isoAsp
X
isoAsp
Glu
G
ly
Ala
His
15
Ala
Ala
X (3)
Ala
eL
ys
Tyr
3MeHis Lys
NAcetylLys
Lys
3MeHis Lys
(Ala)
Lys(1)
HomoCit Lys
Cystine (N-MeAla)
14
Tyr
Tyr
Tyr
Me3Lys N-MeAla
m
116
OMeThr
X
13
N-MeAla
Arg
X
Ty
r
Ala
Ala
Me3Lys
X
X
3MeCys
12
Gly
Gly
Gly
Gly
tri
Me2Lys
X
11
a
Cy
st
in
e
Tyr
His
10
Tyr
Tyr
Tyr
Tyr
Al
115*
9
Cystine
X
Cystine
Tyr
H
Ho is
m
oC
it
Ala
8
Ala
Ala
X
Ala
Ly
s
(Me-His)
7
HomoCit
X
X
HomoCit
is
Tyr
6
His
His
X
His
eH
114
4
5
3MeHis Lys
Ala
Lys
Ala
Lys
3MeHis Me2Lys
X
Me2Lys
a
Me2Lys
Arg
Arg
iso-Asp
3
Ala
Ala
X
Ala
3M
The Edman Sequencing Research Group (ESRG) of the Association of
Biomolecular Resource Facilities (ABRF) has directed numerous studies focused
on various aspects of Edman degradation of proteins and peptides. These studies
provide a means for participating laboratories to compare their analyses against a
benchmark of those from other laboratories that provide this valuable service. The
ABRF ESRG 2005 sample is a continuation of a similar study conducted with the
ESRG 2004 sample in which laboratories were asked to identify the sequence of a
synthetic peptide containing both standard amino acids and posttranslationally
modified or uncommon amino acids that are occasionally encountered in
submitted samples. Laboratories requesting a sample were provided with 1
nanomole of an 18 amino acid synthetic peptide and asked to provide amino acid
assignments at each cycle along with the retention time and peak area. Details
about instruments and parameters used in the analysis were also collected.
Participants were also provided with several modified amino acid elution
references posted on the ESRG website, and had the option of viewing a list of
the modified amino acids present in this peptide. Together with the ESRG 2004
results, this study will provide a valuable reference for Edman sequencing
laboratories of the retention times of uncommonly encountered amino acids
which will be accessible at the ABRF ESRG website.
2
Al
1
expected Tyr
111
Tyr
112
Tyr
113
Tyr
eL
ys
Abstract
25
25
participant #
Effect of Premix Concentration on Elution Profiles
Elution Profiles of 3meHis, Me2Lys & Me3Lys on cLC
100
(21ml/L Premix vs 14.5ml/L Premix)
3meHis
3meHis
vs
21ml/L
lag Ala
lag Ala + 3meHis
Ala
(Std & Cycle 4)
14.5ml/L
50
Description of the Sample
The sample was a synthetic, cysteine-9 disulfide-linked 18-mer peptide
with the following sequence:
H
N
H
H3 C
N
CH3
H 3C
N
H2N
O
H3 C
NH
N
N
CH3
CH3
S
S
H
H
N
H
OH
O
R2 -N,N-Dimethyl Lysine
N
H
H
OH
CH3
O
H
R4 3- Methyl Histidine
N
H
OH
O
R7 -N-Carbamyl Lysine
H
OH
N
H
O
R9 Cystine
H
OH
N
O
H
H
N
OH
OH
N
H
O
R13 N-Methyl Alanine
R11 -N,N,N- Trimethyl Lysine
Ala
Tyr
NmeAla
Gly
trimeLys
Tyr
Cystine 1
Ala
HomoCit
His
Lys
3MeHis
Ala
Me2Lys
Me2Lys - 2
vs
Me3Lys - 11
Me3Lys
(Cycle 2 & 11)
Me2Lys - 2
lag Tyr
14.5ml/L
21ml/L
lag Tyr
Me3Lys - 11
1. Relative retention times of the modified amino acids between similar
instruments were very consistent.
CH3
OH
Tyr
Conclusions
Structures of the modified amino acids are shown below:
O
dimeLys
1
2
3
4
5 6
7 8 9 10
11 12
13 14 15
16 17 18
Tyr-[Me2-Lys]-Ala-[3-Me-His]-Lys-His-[homoCit]-Ala-Cys-Tyr-[Me3-Lys]-Gly-[N-Me-Ala]-Tyr-Ala-[isoAsp]-Val –Arg

S-S

Tyr-[Me2-Lys]-Ala-[3-Me-His]-Lys-His-[homoCit]-Ala-Cys-Tyr-[Me3-Lys]-Gly-[N-Me-Ala]-Tyr-Ala-[isoAsp]-Val –Arg
0
O
HO
O
R16 iso-Aspartic Acid
2. Sequencing and elution properties of the modified amino acids on the ABI
Procise HT and cLC were well characterized. In addition we have profiles
for these amino acids on the single participating ABI 477 and Porton
sequencer.
3. Assignment of the positively charged modified amino acids proved to be
challenging due to their poor behavior on silica based reverse phase supports
Acknowledgements
Thanks to all the participating laboratories who agreed to run this sample
and who were willing to share the data with our committee. Without
their contributions this study would not have been possible.
Thanks to Bachem and AnaSpec for generously contributing modified
amino acids to this study.
Thanks to Melinda Miller for removing identifiers from the contributing
laboratories