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

Abstract

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 -R 2 CO 2 H] Ϫ and [M -H -R 2 CH¢CO] Ϫ ions), are respectively more abundant than the ions arising from the analogous losses at sn-1 ions, x ϭ 1, 2), respectively, probably arising from various losses of the glycerol. The profile of the ion-pair in the MS 3 spectrum of the [M -H -R 2 CO 2 H] Ϫ ion is readily distinguishable from that in the MS 3 spectrum of the [M -H -R 1 CO 2 H] Ϫ 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] Ϫ 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 MS 2 and MS 3 product-ion spectra permit the complex structures of PIMs that consist of various isomers to be unveiled in detail. (J Am Soc Mass Spectrom 2007, 18, 466 -478)

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.…”

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

“…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.…”

Algorithm for processing raw mass spectrometric data to identify and quantitate complex lipid molecular species in mixtures by data-dependent scanning and fragment ion database searching

“…First, peak list files of both MS and MS/MS spectra are loaded into memory and undergo data pretreatment that includes smoothing (5-point Triangular Smooth)/noise filtering. MS/MS data are searched against a fragment ion database from a library of reference spectra for complex lipids that we have acquired [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28] or constructed from established fragmentation rules [29] to identify lipid molecular structures, and deduced chemical formulae are used to calculate theoretical isotope distributions subsequently used in the deisotoping algorithm. The m/z values of those ions in the MS spectra that do not correspond to a recorded MS/MS spectrum are searched against a lipid chemical formula database, and identified formulae are used to calculate theoretical isotope distributions for deisotoping, as described further below.…”

“…Rules to explain fragmentation patterns of a wide variety of glycerolipids comprising hundreds of molecular species from different head-group classes have been established [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29], and we have constructed a lipid fragment ion database that includes species with fatty acid side-chain lengths of 10 -30 carbon atoms and 0 -6 double bonds. Searching the MS 2 spectra of different lipid species against this database permits deduction of their structures and chemical formulae.…”

“…Unfractionated lipid extracts are directly infused and analyzed by ESI/MS with data-dependent switching to MS/MS mode. MS/MS data are searched against a library of reference spectra for complex lipids constructed from studies of standard glycerophospholipids of all major head-group classes [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28], which we used to establish fragmentation rules [29] that facilitate identification of glycerophospholipids in biological mixtures [30,31]. Structure assignment permits determination of an elemental formula, and a theoretical isotope profile is then calculated and used in a deisotoping algorithm.…”