67th ASMS Conference on Mass

Applications of a novel hydrolysis system for deconvolution of cyclotides
on a bead and super rapid amino acid analyses

Spectrometry and allied topics
June 2 - 6, 2019, Atlanta, Georgia

Yuki Tominaga, Takeshi Kasama, Haruyuki Fujino, Atsushi Kitagawa and Kiyoshi Nokihara

HiPep Laboratories, Kyoto, JAPANSummary
Recently demands on discovery and QC in drug development are increasing, hence facileand rapid hydrolyses of peptides and proteins are required. We have constructed a novelhydrolysis system which adapted to wide range of temperature without leakage of HCl
fume. Two applications of AHST-16 are shown.
(1) A library consisting of cyclic octa-peptides as capturing molecules consisting of 24
natural and non-proteinogenic amino acids on the manner of “one peptide on one bead(OPOB)” focusing on drug discovery. The system are applied for rapid deconvolution ofOPOB using MS/MS, of which methods has been improved based on the previouslyreported. (2) As an another application, super rapid amino acid analyses was performed,
while 16 samples could be analyzed with rapid hydrolyses followed by the rapid
determination by LC/MS using unique separation columns in a single day. Thus AHST-16 isuseful for deconvolution immobilized peptides and for high throughput amino acid analyses
in QC of peptides and proteins.

Problems in deconvolution
1.LC-MS of partial hydrolysate of cyclotides
on beads gave difficulties to identify thecorresponding peaks
2.Termini were unclear since target was
cyclicImproved deconvolution method for cyclic peptides immobilized on
Gel-type beads constructed for drug discovery tool
OPOB* has been prepared for drug discovery [*one single peptide immobilized on onebead, LIBRARY of cyclic octa-peptides (involving two D-Cys) immobilized on a gel-typeresin (TentaGel®) which allows bioassays in an aqueous media
High quality cyclic peptide-beads as capturing molecules constructed with 24 amino acids
and cyclized with two D-Cys = Diversity 246 = ca. 200 millions; No inter-moleculardisulfides; ca 100 mm particle; Peptide loading ca 80 pmole/bead; 2.3 million beads/g
Selected single bead are deconvoluted by partial hydrolysis with MS/MSThe present report is the improved method from previously reported: Nokihara et.
al., Amino Acids, 48, 2492-2499, 2016. DOI 10.1007/s00726-016-2269-1
OPOB is a useful tool for drug discovery
Discovery for interacted captured
molecules

Construction of OPOB
Split & combine method, Deprotection and coupling
are carried out using PetiSyzer® with LibraTubes®;
On-resin cyclization in 10% DMSO (pH6.0) at room
temperature for 4 days.
Detail: Nokihara et. al., Amino Acids, 48, 2492, 2016

OPOB: available from HiPep Labs
(http://hipep.jp/eng/?p=751)
as research products
and/or Nakarai Tesque US.

1. A bead was reduced and labeled in 0.83
mM tris(2-carboxyethyl) phosphine),
0.14 M DAABD-Cl
2. Washed with MeOH, H2O
3. Resulted bead was partially hydrolyzed
in a vial H255 with 5.7 N HCl (20 µL)
under sealing at 60 ℃ for 30 min, 1 h,
2 h using AHST-16
Flow rate : 0.3 mL/min
Gradient : B = 20-80 in 30 min
A: 0.1% TFA, B: 0.1% TFA
in 90% ACN
Column: HiPep-Cadenza C18
3.0x150 mm
UV 325 nm

Fluorescent intensity(UV375 nm) of thelabeled bead wasgradually decreasing
depends on duration ofhydrolysis = theimmobilized peptide wasgradually liberated(detached from a bead).

Beads
recognizedcell surface
proteins
Screening

Deconvolution

Partial hydrolysis with 5.7N HCl at 60 °C
for 3 h by the use of the AHST-16 ➡
LCMS／MALDI-TOF-MS/MS

Throughput depends onthe column length
50 mm

ca 10 min

100 mmca 15 min

150 mmca 20 minVial broken, HCl fume destroys oven…Not completelyhydrolyzed……

A typical separation profiles of standard aminoacids without derivatization:
Column: Intrada Amino Acid 3 id x 100 mm
Flow rate 0.6 mL/min,35 ℃; detection byLCMS (ESI positivemode)
Eluent:A = ACN, THF,
25 mM
NH4COOH,HCOOH(9:75:16:0.3v/v);
B = ACN, 100mM NH4COOH(20:80 v/v);
Gradient: B% =0 (0-3 min),
0-17 (3-9 min),
100 (9-16 min)Detection by a
Mass
Spectrometer,
Not UV
Lower cost MS,
such as single Qpole MS is enough

Quantitative analyses by a LC-MSAAFI
LVE
DNHTG
QCRSPKM

regression equationy = 1E+09x+7E+08
y= 9E+07x+2E+08
y= 1E+08x+1E+08
y= 1E+08x+7E+07
y= 1E+08x+4E+07
y= 5E+07x+1E+07
y= 6E+07x+3E+07
y= 2E+08x+5E+07
y= 6E+07x-3E+07
y= 1E+07x+3E+06
y= 2E+08x-2E+08
y= 7E+07x-4E+07
y= 4E+08x-3E+08
y= 5E+07x-3E+07
y= 4E+08x-3E+06-8
y= 2E+08x-1E+08
y= 4E+08x-4E+08

R2
0.96130.75820.99880.95540.98330.97700.94870.99790.9746
0.98170.95090.99970.9097
10.94450.94110.9257Quantitative analyses by a LC-MS

Hydrolysate: Analyzed
by LC-MS [LC: Agilent
1100+ MS: HCTultra (ITMS, Bruker)]
LC-MS of hydrolysate of the above DAABD-peptide:
was labeled with DAABD (5.7 N HCl for 2 hrs)

Conclusion of Part 1

By reducing and derivatizing for Cys-cyclotides with DAABD, the sensitivity on LC-MS
was enhanced. Fluorescent labeling facilitated identification of the fragments
generated by the partial hydrolysis, and sequence analyses were efficiently improved.

Xn = 19 natural and5 non-proteinogenic
amino acids (Cha,Hyp, Nle, Nva, Phg)

composition can be analyzed by Mass
Chromatograms ➡ Racemization
caused [2]The present column cannot
discriminate D & L-isomers.

4-[2-(Dimethylamino)ethylamino-sulfonyl]-7chloro-2,1,3-benzoxadiazole

Fluorescent intensity changes afterhydrolysis of the labeled bead
during hydrolysis (UV 375 nm)

Part 1High throughput Amino Acid Analysis by the Novel System without any derivatization➡ up to 16 Samples in a single day
Hydrolysis with HCl (5.7 N) & TFA (2:1
Smart analyses Labor savingv/v) at 165 ∘C for 25 min [1], AA-d= DAsp; c= DCys

Methods

Advantages(1) Easy vial sealing no-use by “open flame” ie. without burners; (2) For various chemical
processes under sealed conditions. ; (3) Easy operation without skills; (4) Contamination
free, semi-microscale, one touch sealing (just ca 1 min) after reaction excess reagents canbe rapidly removed by nitrogen stream for ca 30 sec using the same apparatus.

www.hipep.jp/eng

Part 2

Conventional and previous manneracid hydrolysis:
Improved
sensingmechanism
allowsrapidlyreaching tothe setting
temperatureof eachpositionPepTenChip@hipep.jp

Auto sampler during lunch time

AHST-16 Hydrolysis system, reactions under sealing, higher efficiency & labor saving in
AHST-16：

PEG-grafted polystyrene
Beads: 90 μm,Tenta
0.27 mmol/gGel®
246 = ca 200 million
S-NH2

Cysteine residues at both termini of
cyclotide immobilized on bead werederivatized with DAABD*-Cl(fluorescent labelling agent) by the
one-pot reaction (10 min)

MP577

① Cyclic peptide beads were reductively derivatized with DAABD-Cl. The progress ofthe hydrolysis has been shown as fluorescence intensities of the bead wasdecreased. Thus the peptide on a bead was fragmented and released by thehydrolysis.
② The peptide has been partially decomposed by hydrolysis at lower temperaturefor shorter time.
③ In the case of a peptide consisting of Cys at both termini, fluorescent labeling ofCys residues resulted higher efficient deconvolution.
④ Hydrolysate of the OPOB-bead for 2hours several fragment peaks were observed
at LC-MS analysis. The mass indicated in the spectrum gave agreement with thetheoretical value of the corresponding fragment generated by hydrolysis.

Conditions
AreaColumn: Intrada AminoAcid (150 x 3 id mm)
A2721573
Flow Rate: 0.6 mL/min
B5004978 Gradient :B%=20 (0-5 min), 35 (5-11 min),
C 697261246 100 (11-20 min)
A: 0.3% HCOOH in ACN, B: 100 mM
Ammonium formate/ACN = 80/20, at 40℃;
HCTplus, Bruker

Conclusion of Part 2

Current difficulties in High Throughput analyses by MS ➡ Determination: Gln vs Lys; Leu vsIle; N- & C-termini : free or modified?; Gln & Lys give similar m/z, Leu Ile give the same m/z;High resolution MS (= costly instrument) + contaminants causes noise & disturbing interpretation① High throughput amino acid analysis was performed by AHST-16, with the column Intrada
Amino Acid, and LC-MS.
② Acid hydrolysis: HCl:TFA = 2:1 v/v (166℃ for 25 min) ➡ Throughput at least 16 samples
in a day③ The present AA-analysis gives useful information without costly instruments, such as
Amino acid analyzers.
The KEYs for MS-de novo sequencing①N-terminus: acetylation gave peak shift +42 Da②C-terminus: methyl esterification gave peak shift +42 Da