Malaysian
Journal of Analytical Sciences Vol 21 No 6 (2017): 1366 - 1372
DOI:
10.17576/mjas-2017-2106-18
SPECIES AND SEROTYPE-SPECIFIC DETERMINATION OF
ENDOTOXINS USING LC-MS
(Penentuan Spesies
dan Serotip-Khusus Endotoksin Menggunakan LC-MS)
Anna Karen Carrasco
Laserna1, Huatao Feng1, Sam Fong Yau Li1,2*
1Department of Chemistry, Faculty of Science,
National University of Singapore, 3 Science
Drive 3, Singapore 11754, Singapore
2NUS Environmental Research Institute,
#02-01 T-Lab Building, 5A Engineering
Drive 1, Singapore 117411, Singapore
*Corresponding author: chmlifys@nus.edu.sg
Received: 7
November 2016; Accepted: 18 September 2017
Abstract
Endotoxins
or lipopolysaccharides (LPS) are characteristic components of the outer
cellular membrane of Gram negative bacteria, which are recognition targets of immune receptors and are responsible for
inciting the bacteria’s toxic effects. The toxicity and physiological effects
of LPS varies diversely within and across different bacterial species. However,
the current standard test for endotoxin analysis, the Limulus Amoebocyte Lysate
(LAL) test, can only provide information on the presence of LPS, regardless of
which bacteria the LPS is coming from. As implied from the name
“lipopolysaccharide”, LPS has a lipid and a carbohydrate portion. The
carbohydrate component is further subdivided to the core oligosaccharide and
the O-antigen polysaccharide chains, which are conserved within species and
unique for each serotype, respectively. In this pilot study, we fingerprinted
the oligosaccharide units generated from mild acid hydrolysis of the LPS from
some serotypes of Escherichia coli
and Salmonella, as well as from Serratia marcesens using liquid
chromatography coupled to a high resolution mass spectrometer (LC-MS) after
labelling them to improve MS ionization. Multivariate analysis showed distinct
clustering of samples belonging to each bacterial species and revealed certain
features that can be potential species and serotype-specific markers of LPS
from the different bacteria under study.
Keywords: endotoxins, lipopolysaccharide, oligosaccharide
fingerprinting, liquid chromatography – mass spectrometry
Abstrak
Endotoksin atau lipopolisakarida (LPS) adalah komponen teras membran selular luaran bakteria Gram negatif yang bertindak untuk mengenalpasti sasaran reseptor imun dan bertanggungjawab menguatkan kesan toksik bakteria tersebut. Terdapat perbezaan dalam ketoksikan dan kesan fisiologi LPS di dalam dan antara spesies bakteria yang berbeza. Walau bagaimanapun, ujian piawai semasa bagi analisis endotoksin, ujian Limulus Amebosit Lisat (LAL), hanya boleh memberikan maklumat mengenai kehadiran LPS tanpa mengira bakteria sumber asal LPS tersebut. Nama lipopolisakarida juga menunjukkan bahawa LPS mempunyai lipid dan sebahagian unsur karbohidrat. Komponen karbohidrat juga terbahagi kepada oligosakarida teras dan rantai polisakarida O-antigen, yang terpelihara di dalam spesies dan unik untuk setiap serotip yang hadir. Dalam kajian rintis ini, kami mengenalpasti unit oligosakarida yang dihasilkan daripada hidrolisis asid lemah LPS dari beberapa serotip dari Escherichia coli, Salmonella dan juga Serratia marcescens menggunakan kromatografi cecair digunakan bersama spektrometri jisim resolusi tinggi (LC-MS) selepas proses pelabelan untuk meningkatkan pengionan MS. Analisis multivariat menunjukkan kelompok tertentu sampel yang datang dari setiap spesies bakteria dan mendedahkan ciri – ciri tertentu yang boleh menjadi spesies yang berpotensi dan penanda serotip spesifik LPS dari bakteria yang berbeza di bawah kajian.
Kata kunci: endotoksin, lipopolisakarida, oligosakarida
cap jari, kromatografi cecair – spektrometri jisim
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