Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry of lipopolysaccharide species separated by slab-polyacrylamide gel electrophoresis: high-resolution separation and molecular weight determination of lipooligosaccharides from Vib
Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry of lipopolysaccharide species separated by slab-polyacrylamide gel electrophoresis: high-resolution separation and molecular weight determination of lipooligosaccharides from Vib
复制标题
通过平板聚丙烯酰胺凝胶电泳分离的脂多糖种类的基质辅助激光解吸/电离时间飞行质谱法：来自 Vib 的脂寡糖的高分辨率分离和分子量测定
DOI：
10.1002/elps.200405980
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发表时间：
2004
期刊：
影响因子：
2.9
通讯作者：
Hardy,Eugenio
中科院分区：
文献类型：
--
作者：
Pupo,Elder;Phillips,NancyJ;Gibson,BradfordW;Apicella,MichaelA;Hardy,Eugenio
We recently demonstrated that the combined use of lipopolysaccharide (LPS) reverse staining and high‐efficiency passive elution techniques can be successfully used as a suitable interface between LPS slab‐gel separation and electrospray ionization‐mass spectrometry (ESI‐MS) of LPS‐derived oligosaccharides. Here, we extend our micropurification strategy for the analysis ofO‐deacylated LPS forms fromVibrio fischeriHMK after recovery from single reverse‐stained LPS bands using matrix‐assisted laser desorption/ionization‐time of flight‐mass spectrometry (MALDI‐TOF‐MS). The quantities (30–40 μg) obtained from the two gel‐resolved LPS bands were sufficient to allow MALDI‐TOF‐MS detection ofO‐deacylated LPS glycoforms atm/z3767.1, 3890.1 for the high‐molecular‐weight or atm/z2522.5, 2645.4, 2725.7, and 2848.7 for the low‐molecular‐weight LPS band. These LPS band heterogeneities resulted not only from variations in the oligosaccharide region of the LPS but also from two phosphorylation states of the lipid A (diphosphoryl and diphosphoryl plus a single phosphoethanolamine substitution). On the other hand, MALDI‐TOF mass spectra of the separated LPS bands displayed reduced heterogeneity and increased signal‐to‐noise ratios as compared to spectra of the unpurified LPS. Furthermore, micropurification of LPS bands prior MALDI‐TOF‐MS led to a higher sensitivity of detection of less abundant low‐molecular‐weight LPS glycoforms. Taken together, this and our previous study on gel‐micropurified LPS using ESI definitively show how one can unambiguously determine the different molecular species contained within each gel‐separated LPS band, their relative abundance and oligosaccharide sequences.
