Development of peptide chip for rough diagnosis

Deguchi NaoE-mail address : ac1210nd@apps.kct.ac.jpDepartment of Material Sciences & Chemical Engineering, Kitakyushu National College of Technology

introduction

・ Severe illness that are difficult to cure cause significant impact on our daily lives.

・ However, with early detection, it is quite possible to slow the progression, or even cure the disease with appropriate treatment.

As a method to easily diagnose these diseases, I have been developing a peptide chip that could potentially be a break-thorough for simple diagnostic methodologies.

It is understood that the majority of the causes of untreatable diseases originate in some kinds of abnormality in the intracellular signal transduction system. If there are methodologies to which we can monitor these intercellular signal transductions, diagnosis of these untreatable diseases will be possible.

it is practically impossible to completely understand the intracellular signal transductions.

however

Protein phosphorylation signalingwhich is one of the most important and versatile

intracellular signaling methods

－ a reaction of the g-phosphate group of ATP transferring to a hydroxy group at the side chain of serine, threonine and tyrosine residues, which are included in substrate peptides or proteins in cells.

protein phosphorylation

200 kinds of protein kinase, which is an enzyme catalyzing phosphorylation reaction, in human body.

In order to monitor all protein kinase activities at once, I have tried to develop a analytical technique utilizing peptide array and MALDI-TOF-MS.

principle of MALDI-TOF-MS

+ -

P

P

substrate peptide

substrate

photocleavage compound

P

P

detector

mass of phosphorylated peptide is increased by 80 rate of phosphorylation of MS spectra

purpose

In order to conduct mass spectrometry using a peptide array, Our team needs substrate peptides with the photo-cleavable part by ionization laser

synthesis

　 2-bromo-2-(2-nitrophenyl)acetic acid (BNPA)

method

1

OH

O

Cl

O

Cl

O

Br

N

Br

OO

O

OHBr

NO2NO2

NO2NO2SOCl2

CCl4 CCl4

H2O

●BNPA

2-(2-nitrophenyl)acetic acid (5.0 g)+

thionyl chloride (8 ml)

In carbon tetrachloride (5 ml)

N-bromosuccinimide (6.0 g)+

CCl4 (25 ml) +

catalytic amount of benzoyl peroxide

65℃1.5 ｈ

ice(25 g)extract 3 x 25 ml CH2Cl2

recrystallization　　　 from CH2Cl2

75℃4.5 ｈ 1.0 ｈ

+

+

result

Figure.1H-NMR

mass of observed fragment ion

proposed structure

242

244

214

216

Table. mass spectrometry

・ shape: Solid needle・ color: Clear・ mp: 59.6 ~ 63.0 ℃

NO2

BrO H

O

O

BrO H

NO2

NH2

substrate

HN

NHCOCH3

substrate peptideOO

S

NO2

NH2

substrate

Immobilized concept

conclusionI've tried the synthesis of the photo-cleavable compound.The synthesized one was identified with BNPA by 1H-NMR and mass spectrometry.