bovis BCG, lipoprotein modifications of LprF, LpqH, LpqL and LppX from Δlnt check details mutant were analyzed at the molecular level. In Δlnt, signals with molecular masses indicating Lgt- and LspA- modified and glycosylated peptides were found. The differences in molecular mass of 550.87 Da for LprF, LpqH and LppX and 576.91 Da for LprF and LpqH between the experimentally found peptide and the unmodified

N-terminal peptide (Table 1) indicate (Lgt and LspA, but not Lnt modified peptides carrying) a diacylglycerol modification carrying ester-linked C16 and C16 or ester-linked C16 and C18 fatty acid, respectively. The differences in molecular mass of 592.96 Da for LprF, LpqH, LpqL and LppX refer to a diacylglycerol modification with ester-linked C16 and C19 fatty acid. The differences in molecular mass of 755.20 Da for STAT inhibitor LprF and LppX refer to a diacylglycerol modification with ester-linked C16 and C19 fatty acid plus RG7112 chemical structure glycosylation with one hexose

(592.96 Da + 162.24 Da). The difference in molecular mass of 917.90 Da for LppX refers to a diacylglycerol modification with ester-linked C16 and C19 fatty acid plus modification with two hexoses (592.96 Da + 162.24 Da + 162.24 Da). In contrast to the MS from parental strain, no molecular masses which we calculated for modifications with three fatty acids were found in the Δlnt mutant strain. In particular, the differences in molecular mass of 238.4 Da (831.36 Da – 592.96 Da) or 280.49 Da (1035.69 Da – 162.24 Da – 592.96 Da) between the C16/C19/C16 or C16/C19/C19 triacylated

modification found in the parental strain and the corresponding estimated C16/C19 modification in the Δlnt mutant indicate a lack of N-acylation with a C16 or C19 fatty acid in the Δlnt mutant. In MS/MS analysis, this indication of missing N-acylation in the mutant was confirmed by identification of the estimated modifications and information about its linkage (Table 2). Modifications with C16/C19 diacylglyceryl residue were confirmed by eliminations of fragments with the molecular mass of 626.53 Da, corresponding Nutlin-3 to the elimination of a diacylthioglyceryl carrying C16 and C19 fatty acid. The O-linked C16 or C19 fatty acids were confirmed by neutral losses of 256.24 Da or 298.29 Da, corresponding to the elimination of palmitic acid or tuberculostearic acid, respectively. Further, the neutral loss of 370.29 Da corresponds to the elimination of C19 fatty acid α-thioglyceryl ester. A glycosylation at other amino acids than the conserved cysteine was confirmed by the release of a fragment of 162.24 Da for a hexose. These findings indicate that N-acylation is not a prerequisite for glycosylation. As mentioned before, only diacylglyceryl residues composed of a C16 and a C19 fatty acid were identified in mycobacterial lipid anchors so far [12, 13]. However, the eliminations of fragments with the molecular mass of 584.44 Da or 256.