Resulting

peptides were prepared and analyzed by MALDI-TOF/TOF mass spectrometry [35]. For the identification Osimertinib concentration of the modification we determined the structure and calculated the expected monoisotopic molecular masses of the unmodified N-terminal tryptic or AspN-digested peptides of LprF, LpqH, LpqL and LppX (without signal peptide). Phospholipids found in mycobacteria mainly consist of palmitic (C16:0), click here palmitoleic (C16:1), oleic (C18:1) and tuberculostearic acid (10-methyloctadecanoic acid) (C19:0) [39]. In E. coli, fatty acids of membrane phospholipids, i.e. myristic (C14:0), palmitic, palmitoleic, oleic (C18:1 ω9) or vaccenic (18:1 ω7) acid are used for the modification of lipoproteins [40–44]. Therefore we calculated the theoretical mass of the N-terminal peptides of the four lipoproteins with all possible combinations of the above mentioned fatty acids observed in mycobacterial phospholipids to identify putative modifications. Glycosylations are also commonly found in lipoproteins [45, 46]. Some of the analyzed N-terminal peptides carry putative O-glycosylation sites, therefore we also calculated the

masses with hexose modifications. [M+H]+ signals at m/z values which we calculated for MEK inhibitor the unmodified N-terminal peptides were not found. Instead, we found MS signals at m/z values which indicate that the N-terminal peptides are modified in a lipoprotein-specific manner with different combinations of saturated and unsaturated C16, C18 and C19 fatty acids. The calculated m/z values are summarized and compared with the experimentally determined m/z values in Table 1. Table 1 Comparison of m/z values of N-terminal AspN-digested/tryptic peptides of LprF, LpqH, LpqL and LppX found in BCG parental and Δ lnt mutant strain   Peptide Calculated m/z Parental strain m/z Δlnt m/z LprF CGK…ILQ 2496.24 – -   CGK…ILQ 3047.11 – 3046.70    + Diacylglycerol (C16/C16) (+550.87)   (+550.46)   CGK…ILQ 3073.15 – 3072.71    + Diacylglycerol (C16/C18) (+576.91)

  (+576.47)   CGK…ILQ 3089.20 – 3088.74    + Diacylglycerol (C16/C19) (+592.96) see more   (+592.50)   CGK…ILQ 3251.44 – 3251.65    + Diacylglycerol (C16/C19) (+755.20)   (+755.41)    + Hexose         CGK…ILQ 3327.60 3326.83 –    + Diacylglycerol (C16/C19) (+831.36) (+830.59)      + N-acyl (C16)         CGK…ILQ 3531.93 3530.56 –    + Diacylglycerol (C16/C19) (+1035.69) (+1034.32)      + N-acyl (C19)          + Hexose       LpqH CSSNK 538.23 – -   CSSNK 1089.10 – 1088.60    + Diacylglycerol (C16/C16) (+550.87)   (+550.37)   CSSNK 1115.14 – 1114.68    + Diacylglycerol (C16/C18) (+576.91)   (+576.45)   CSSNK 1131.19 1130.79 1130.71    + Diacylglycerol (C16/C19) (+592.96) (+592.56) (+592.48)   CSSNK 1369.59 1369.04 –    + Diacylglycerol (C16/C19) (+831.36) (+830.81)      + N-acyl (C16)       LpqL CIR 391.21 – - CIR 984.17 984.50 983.