Structural Characterization of Phosphatidyl-myo-inositol Mannosides from Mycobacterium bovis Bacillus Calmette Guérin by Multiple-Stage Quadrupole Ion-Trap Mass Spectrometry with Electrospray Ionization. I. PIMs and Lyso-PIMs

We described a multiple-stage ion-trap mass spectrometric approach to characterize the structures of phosphatidylinositol and phosphatidyl-myoinositol mannosides (PIMs) in a complex mixture isolated from Mycobacterium bovis Bacillus Calmette Guérin. The positions of the fatty acyl substituents of PIMs at the glycerol backbone can be easily assigned, based on the findings that the ions arising from losses of the fatty acid substituent at sn-2 as molecules of acid and of ketene, respectively (that is, the [M – H – R2CO2H]− and [M – H – R2CHCO]− ions), are respectively more abundant than the ions arising from the analogous losses at sn-1 (that is, the [M – H – R1CO2H]− and [M – H – R1CHCO]− ions) in the MS2 product-ion spectra of the [M – H]− ions desorbed by electrospray ionization (ESI). Further dissociation of the [M – H – R2CO2H]− and [M – H – R1CO2H]− ions gives rise to a pair of unique ions corresponding to losses of 74 and 56 Da (that is, [M – H – RxCO2H – 56]− and [M – H – RxCO2H – 74]− ions, x = 1, 2), respectively, probably arising from various losses of the glycerol. The profile of the ion-pair in the MS3 spectrum of the [M – H – R2CO2H]− ion is readily distinguishable from that in the MS3 spectrum of the [M – H – R1CO2H]− ion and thus the assignment of the fatty acid substituents at the glycerol backbone can be confirmed. The product-ion spectra of the [M – H]− ions from 2-lyso-PIM and from 1-lyso-PIM are discernible and both spectra contain a unique ion that arises from primary loss of the fatty acid substituent at the glycerol backbone, followed by loss of a bicyclic glycerophosphate ester moiety of 136 Da. The combined structural information from the MS2 and MS3 product-ion spectra permit the complex structures of PIMs that consist of various isomers to be unveiled in detail.