Structural characterization of Mycobacterium tuberculosis lipoarabinomannans by the combination of capillary electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) associated with capillary electrophoresis (CE) has been used for structural characterization of mannooligosaccharide caps from Mycobacterium tuberculosis H37rv mannosylated lipoarabinomannans (ManLAMs). The mannooligosaccharide caps were released by mild acid hydrolysis, labeled with 1-aminopyrene-3,6,8-trisulfonate (APTS) prior to being separated by CE, collected, and analyzed by MALDI-TOF-MS and post-source decay experiments. This approach was optimized using standard APTS-labeled oligosaccharides. With the selected (9:1) mixture of 2,5-dihydroxybenzoic acid (DHB) and 5-methoxysalicylic acid (MSA) as matrix and the on-probe sample cleanup procedure with cation-exchange resin, standard APTS-maltotriose was successfully detected down to 50 fmol using linear-mode negative MALDI-TOF-MS. Moreover, using extraction delay time, only 100 and 500 fmol of this standard were required, respectively, to obtain accurate reflectron mass measurements and sequence determination through post-source decay experiments. Applied to only 5 microg (294 pmol) of M. tuberculosis ManLAMs, this analytical approach allowed successful mass characterization of the mannooligosaccharide cap structures from the deprotonated molecular ions [M - H]- and the y-type ion fragments obtained in post-source decay experiments. This powerful analytical approach opens new insights into both the characterization of oligosaccharides and the capping motifs displayed by ManLAMs purified from mycobacteria isolated from tubercular patients without in vitro culturing.
Ludwiczak
P
P
Insitut de Pharmacologie et de Biologie Structurale, UMR 5089, CNRS, Toulouse, France.
Brando
T
T
Monsarrat
B
B
Puzo
G
G
D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D019075:Electrophoresis, Capillary / Q000379:methods*; D008070:Lipopolysaccharides / Q000737:chemistry*; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D008070:Lipopolysaccharides; C050016:lipoarabinomannan