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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