5 Dimensional Structural

Characterization of Synthetic Peptides

Dr Osama Chahrour, Dr John Malone, Diego Cobice, Dr Dan Fletcher, Dr Alan Thompson,
and Cliodhna McGurgan
Almac Sciences, 20 Seagoe Industrial Estate, Craigavon, Northern Ireland, BT63 5QD
Background
There is currently growing interest in the therapeutic benefits of synthetic peptides. These molecules, like small molecule pharmaceuticals, require comprehensive characterization to unequivocally
confirm the structure as per the USP guidelines. Full identity elucidation requires integration of orthogonal techniques to overcome the limitations associated with each technique. Presented is an
example of the characterization protocol developed at Almac for synthetic peptides. This process confirms peptide sequence by Nuclear Magnetic Resonance and Tandem Mass Spectrometry,
peptide molecular formula by accurate mass determination, chiral purity by pre-column derivatization of the peptide hydrolysates followed by liquid chromatography with mass spectrometry detection
as well as ratio determination of each amino acid in the sequence. This characterization protocol goes beyond the requirements of the USP and gives confidence in the identification of synthetic
peptides. The protocol has already been used for peptides containing up to 34 amino acids . A case study is presented herein.

Peptide Sequencing by NMR

~25 mg of a 20-mer peptide was dissolved in DMSO-d6. Spectra were obtained using a
Bruker Avance NMR spectrometer operating at 500 MHz running under TopSpin 2.1. The
sample was run at 28 and 50C in the presence and absence of TFA. 1H and 13C spectra
were assigned using DEPT-135, HSQC, HMBC, ROESY and TOCSY experiments.
Individual peaks were picked out in the HSQC (finger-printing) and their multiplicity
determined with the aid of the DEPT-135 spectrum. The correct number and multiplicity of
carbons and protons was also confirmed.
COSY-DQF, Transverse-ROESY and HSQC-TOCSY data was to assign the protons to
individual amino acid side chains and confirm they agreed with the expected residues. The
ROESY and HMBC data was used to determine the relative positions and sequence of
amino acids within the molecule

Precursor ion

m/z (prec.)

Number of charges Z

(M+H)+

1X36.9849

Product
Ion

m/z

m/z (prod.)

Diff (ppm)

Sequence

b6

3X7.1777

3X7.183

-13.3

R1GGR2GA

b10

78X.3853

78X.3791

7.8

R1GGR2GAGR3LQ

y5

5X2.3074

5X2.3089

-2.9

GSLR6K

1
1

y6

6X1.3497

6X1.3515

-2.8

R5GSLR6K

y7

7X4.431

7X4.4356

-5.9

LR5GSLR6K

y8

8X5.4695

8X5.4727

-3.8

ALR5GSLR6K

y10

105X.6084

105X.6095

-1.1

R4LALR5GSLR6K

y11

118X.6594

118X.6681

-7.4

QR4LALR5GSLR6K

b10-H2O

7X4.3609

7X4.3686

-10

R1GGR2GAGR3LQ

y11-NH3

1X66.648

1X66.6416

5.5

QR3LALR4GSLR5K

ppm

3.7

3.8

3.9

4.0

4.1

Figure 4: Example of peptide precursor ion and corresponding product ions.

4.2

Chirality Determination by HPLC-MS

4.3

4.4

8.30

8.25

8.20

8.15

8.10

8.05

8.00

7.95

7.90

7.85

7.80

7.70

7.75

7.60

7.65

7.50

7.55

4.5
ppm

7.45

Figure 1: Intra-residue NH-Ha COSY correlations (green/blue).

ROESY
Inter-residue correlations (red) across the amide bonds from the a-H of residue n to the NH
of the residue n+1 were used to confirm the correct peptide sequence .

The chiral purity of the amino acids in the peptide were determined by acid hydrolysis
followed by amino acid separation. Racemisation was circumvented by hydrolysing
peptides in DCI/ [D4] acetic acid (1:1). Amino acids were then derivatised with Marfey's
reagent and analysed by LC-MS. This method allows the original chiral purity of each
amino acid in a peptide to be determined using common achiral reversed-phase HPLC
columns. Use of ESI prevented fragmentation during ionisation and allowed the
protonated molecular ion to be monitored for each amino acid
.

Accurate Mass Determination by LC-ESI-TOF

The accurate mass of the peptide was determined using an Agilent LC-QTOF to within
2ppm of theoretical. The Find By formula Algorithm of the MassHunter software was used
to map the MS(1) data. It identified all major cluster ions and the algorithm confirmed the
empirical formula
.
Figure 5: Labelled Hydrolysis.

Figure 6: Marfey Reaction.

M alone ,
Joh n
Opera tor:
Ti me:
1 0:23: 47
Print ing
May
27 ,
201 4
Tue,

X IC

of

+Q 1

MI

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File :
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io n s ):

3 8 4 .2

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S a m p le

(K

A cq.
A cq.
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2 6 M A Y20 1 4A 0 0 04 )

of

T ime:
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3 65_K

1 2:19
M onday ,
May
26,
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I nst
I .D.
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2 6 M ...

M a x.

2 .3 e 6

cp s.

2 8 .6 5

2 .3 e 6
2 .2 e 6

2 9 .1 6

2 .0 e 6

L-Isoleucine
L-Leucine

1 .8 e 6
1 .6 e 6

ID Source

Formula

Diff (ppm)

TRUE

FBF

C79H133N23O27

-1.60

Intensity, cps

Best

1 .4 e 6
1 .2 e 6
1 .0 e 6
8 .0 e 5

D-Leucine

D-Isoleucine

6 .0 e 5
4 .0 e 5
3 1 .7 2
3 1 .4 0

2 .0 e 5
0 .0
2 7 .0
X IC

+Q 1

2 7 .5
MI

(1 9

2 8 .0
io n s ):

2 8 .5

4 1 8 .2

amu

2 9 .0

fro m

S a m p le

2 9 .5
4

(K

3 0 .0
3 0 .5
T im e , m in
2 6 M A Y20 1 4A 0 0 04 ) o f

3 1 .0
K

3 1 .5

3 2 .0

3 2 .5

2 6 M ...

M a x.

3 3 .0
3 .3 e 6

cp s.

2 8 .8 7

3 .3 e 6
3 .2 e 6

Figure 2: Isotopic distribution matching between actual spectrum (black line) and FBF
calculated theoretical distribution (red boxes) and table of mass confirmation.

3 .0 e 6
2 .8 e 6

L-Phenylalanine

2 .6 e 6
2 .4 e 6
2 .2 e 6

Intensity, cps

Peptide Sequencing by LC-ESI-QTOF

of

2 .0 e 6
1 .8 e 6
1 .6 e 6
1 .4 e 6

D- Phenylalanine

1 .2 e 6
1 .0 e 6
8 .0 e 5
6 .0 e 5

The peptide precursor ions identified during the accurate mass determination were
fragmented in the tandem mass spectrometer and the product (fragment) ions were
subsequently mass analysed. The implemented low energy collision induced dissociation
(CID) primarily produced y and b type ions. The data were processed using the Molecular
Feature Extractor algorithm of MassHunter and matched with theoretical peptide fragment
ions. The majority of y and b ions observed were within 10 ppm difference.

3 0 .8 7

4 .0 e 5
2 .0 e 5
0 .0
2 7 .0

2 7 .5

2 8 .0

2 8 .5

2 9 .0

Page

of

2 9 .5

3 0 .0
T im e , m in

3 0 .5

3 1 .0

3 1 .5

3 2 .0

Scien ces
* Almac
0 641E0 002C

3 2 .5

P rojec t

3 3 .0

No.

Figure 7: Example representative L and D amino acid standard chromatograms.

Amino Acid Analysis

Amino acid analysis was carried out by acid, oxidative and alkaline hydrolysis. The
individual amino acids were then analysed with HPLC-UV/FLU and/or Biochrom amino
acid analysers using classical ion-exchange liquid chromatography with post-column
Ninhydrin derivatisation and photometric detection. Li-Citrate as well as Na-Citrate
buffer system elution was used to cover all the amino acids. The presence and correct
number of amino acids in the peptide sequence were confirmed.

Conclusion
X

A summary of the results is given in the table below:

X

R1
X

R2

R3

R4

R5

R6

Sequence Confirmed by NMR

Sequence confirmed by CID Hybrid MS
Chirality Confirmed- by LC MS
-

Figure 3: Isotopic distribution matching between actual spectrum (black line) and FBF
calculated theoretical distribution (red boxes).

The protocol for peptide characterization has been fully implemented by Almac has been
used routinely to unequivocally confirm the structure of an number of peptides for our
clients
.