Structural characterization of phosphatidyl-myo-inositol mannosides from Mycobacterium bovis bacillus calmette gúerin by multiple-stage quadrupole ion-trap mass spectrometry with electrospray ionization. II. Monoacyl- and diacyl-PIMs

Abstract

The multiple-stage ion-trap mass spectrometric approaches towards to the structural characterization of the monoacyl-PIM (triacylated PIM) and the diacyl-PIM (tetracylated PIM), namely, the PIM (diacylated PIM) consisting of one or two additional fatty acid substituents attached to the glycoside, respectively, were described. While the assignment and confirmation of the fatty acid substituents on the glycerol backbone can be easily achieved by the methods described in the previous article, the identity of the glycoside moiety and its acylation state can be determined by the observation of a prominent acylglycoside ion arising from cleavage of the diacylglycerol moiety ([M -Hdiacylglycerol] − ) in the MS 2 -spectra of monoacyl-PIM and diacyl-PIM. The distinction of the fatty acid substituents on the 2-O-mannoside (i.e., R 3 CO 2 H) from that on the inositol (i.e., R 4 CO 2 H) is based on the findings that the MS 3 -spectrum of [M -H -diacylglycerol] − contains a prominent ion arising from further loss of the fatty acid at the 2-O-mannoside (i.e., the [M -H -diacylglycerol -R 3 CO 2 H] − ion), while the ion arising from loss of the fatty acid substituent at the inositol (i.e., the [M -H -diacylglycerol -R 4 CO 2 H] − ion) is of low abundance. The fatty acyl moiety on the inositol can also be identified by the product-ion spectrum from MS 4 of the [M -H -diacylglycerol -R 3 CO 2 H] − ion, which gives rise to a prominent ion corresponding to loss of R 4 CO 2 H. An [M -Hacylmannose] − ion was also observed in the MS 2 -spectra and, thus, the identity of the fatty acid substituent attached to 2-O-mannoside can be confirmed. The combined information obtained from the multiple-stage product-ion spectra from MS 2 , MS 3 , and MS 4 permit the assignment of the complex structures of monoacyl-PIMs and diacyl-PIMs in a mixture isolated from M. bovis Bacillus Calmette Guérin.Although ion-trap tandem mass spectrometry with ESI for structural characterization of complex structures of acyl-PIM 2 was recently reported [1], structural detail revealed by multiple-stage ion-trap tandem mass spectrometry has not been described previously.In the first part of this report, we described the IT multiple-stage mass spectrometric approaches for characterization of PI and PIM molecules from a lipid extract from M. bovis BCG [2]. In this subsequent report, we will describe the structural determination of the monoacyl-and diacyl PIMs in the extract, using the same mass spectrometric approaches. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript the fatty acid substituents attached to the glycosides of monoacyl-PIM and of diacyl-PIM is based on the understanding that the fatty acyl moieties are connected to the O-6 position of the 2-O-linked Manp and to the O-3 position of the myo-inositol, respectively, based on the previous studies using NMR [3][4][5][6][7][8]. Materials and MethodsThe isolation of PIMs from M. bovis Bacillus Calmette Guérin (BCG) and the IT multiplestage mass spectrometry for structural chara...

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

“…These results indicate that the ion at m/z 741 probably represents a deprotonated palmitoyl-Man-Ino-Man anion arising from further loss of a bicyclic glycerophosphate ester moiety (136 Da) (discussed later in 2-lyso-PIMs), suggesting that the ion at m/z 1175 may also consist of a minor 16:0-(19:0/0)-PIM 2 isomer, as previously described by Gilleron et al [25].°The°presence°of°the°16:0-(19:0/0)-PIM 2 isomer is also consistent with the observation of a minor peak at m/zé 803°in°Figure°4a,°signifying°the°attachment°of°a palmitoyl°residue°to°mannose°1° [28].°However,°Khoo°et°al. reported that monoacyl-2-lyso-PIM 2 s, rather than PIM 2 s (diacylated PIM 2 ) are the predominant species found in the°lipid°extract°from°Mycobacteriumétuberculosisé [23].…”

“…forms a phosphoester intermediate, followed by elimination of the glycerol moiety in various forms (Scheme 1). Although these ions are of low abundance, the analogous ions from the similar fragmentation processes are prominent in the MS 3 spectra of the [M -H -R 1(or 2) CO 2 H] Ϫ ions arising from monoacyl-or diacylPIMs and are indicative for the fatty acyl substituents that reside at glycerol backbone (that is, R 1 CO-and R 2 CO-), allowing us to distinguish them from those residing°at°the°glycoside° [28].…”

“…In this first part of the report, we shall describe the mechanism(s) underlying the fragmentation processes of PI and PIMs upon collisional activation decomposition (CAD) in an ion-trap, as well as the structural characterization of PI and of the subclass of PIMs (diacylated PIMs). The mass spectrometric methods for structural characterization of monoacyl-PIMs (triacylated PIMs) and diacyl-PIMs (tetraacylated PIMs) will be presented separately in a companion paper of this report [28].°This°unique°multiple-stage°tandem°mass spectrometric method permits the complex structures of PIMs, including various isomers, to be revealed in detail.…”

“…The IT MS 2 These features in the MS 3 spectra are useful for the confirmation of positions of the fatty acyl substituents, in particular, when the fatty acyl residues attached to the inositol and to the mannosyl residues are to be distinguished. The multiple-stage IT mass spectrometric approach toward to structural characterization of complex PIM molecules, including monoacyl-PIMs (triacylated PIMs) and diacyl-PIMs (tetraacylated PIMs), is described°in°a°companion°paper°that°follows° [28] …”

Electron transfer dissociation of doubly sodiated glycerophosphocholine lipids

“…Among the various complex lipids, glycerophospholipids (GPLs) perform two important biological functions: one is making up most of the membranes of mammalian cells, and the other is acting as secondary messengers in metabolism [11,12]. The structural determination (identities and positions of the fatty acid substituents) of GPLs for all five subclasses has been made via CID of negative ions formed by either ESI, fast atom bombardment (FAB), or MALDI [5,13,14]. Among the five main subclasses of GPLs, which are differentiated by the head-group (e.g., ethanolamine, choline, serine, glycerol, or inositol), glycerophosphocholine (GPC) species generally give stronger signals in positive ion mode than in negative mode due to the fixed charge on the quaternary nitrogen.…”