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The study presents the characterization of a distinct arabinofuranosyltransferase in Mycobacterium smegmatis, which is then used to explain the biosynthesis of cell wall arabinan. These transferases are found to be very helpful for the assembly of very complex structures.

A study presents opportunities for the better understanding of the structural level by mapping and sequencing of the digestion products, which were dominated by distinctive [Ara.sub.18] and [Ara.sub.19] structural units, together with [Ara.sub.7] and lesser amount of [Ara.sub.11] and [Ara.sub.12]. The establishment of linkage-specific MS/MS fragmentation characteristics led to the identification of a galactosamine substituent of the C2 position of a portion of the internal 3,5-branched Ara residue of the arabinogalactan (AG) of Mycobacterium tuberculosis.

Data dependent acquisition (DDA) of higher collision energy dissociation (HCD)-MS2 followed by electron transfer dissociation (ETD)-MS2 upon detection of glycan-specific oxonium is one of the better approaches in current LC-MS2 analysis of intact glycopeptides. Although impressive numbers of glycopeptide identification by a direct database search have been reported, false positives remained high and difficult to determine. Even in cases when the peptide backbones were correctly identified, the exact glycan moieties were often erroneously assigned. Any attempt to fit the best glycosyl composition match by mass only is problematic particularly when the correct monoisotopic precursor cannot be determined unambiguously. Taking advantage of a new trihybrid Orbitrap configuration, we experimented with adding in a parallel ion trap collision induced dissociation (CID)-MS2 data acquisition to the original HCD-product dependent (pd)-ETD function. We demonstrated the feasibility and advantage of identifying the peptide core ion directly from edited HCD-MS2 data as an easy way to reduce false positives without compromising much sensitivity in intact glycopeptide positive spectrum matches. Importantly, the additional CID-MS2 data allows one to validate the glycan assignment and provides insight into possible glycan modifications. Moreover, it is a viable alternative to deduce the glycopeptide backbone particularly in cases when the peptide backbone cannot be identified by ETD/HCD. The novel HCD-pd-CID/ETD workflow combines the best possible decision tree dependent MS2 data acquisition modes currently available for glycoproteomics within a rapid Top Speed DDA duty cycle. Additional informatics can conceivably be developed to mine and integrate the rich information contained within for simultaneous N- and O-glycopeptide analysis. [ABSTRACT FROM AUTHOR]

The mycobacterial D-arabinofuran is a common constituent of both cell wall mycolyl-arabinogalactan (AG) and the associated lipoarabinomannan (LAM), and is thus accorded critical structural and immunological roles. Despite a well-recognized importance, progress in understanding its full structural characteristics beyond the nonreducing terminal motifs has hitherto been limited by available analytical tools. An endogenous arabinanase activity recently isolated from Mycobacterium smegmatis was previously shown to be capable of releasing large oligoarabinosyl units from AG. Advanced tandem mass spectrometry utilizing both low and high energy collision induced dissociation now afforded a facile way to map and directly sequence the digestion products which were dominated by distinctive Ara18 and Aral9 structural units, together with Ara7 and lesser amount of Ara11 and Ara12. Significantly, evidence was obtained for the first time which validated the linkages and branching pattern of the previously inferred Ara22 structural motif of AG, on which the preferred cleavage sites of the novel arabinanase could be localized. The established linkage-specific MS/MS fragmentation characteristics further led to identification of a galactosamine substituent on the C2 position of a portion of the internal 3,5-branched Ara residue of the AG of Mycobacterium tuberculosis, but not that of the nonpathogenic, fast growing M. smegmatis. [ABSTRACT FROM AUTHOR]