Deconvolution of multichannel LC-MS/MS chromatograms of glucosamine-phosphates: Evidence of a GlmS regulatory...

Resolving isomeric analytes is challenging given their physical similarity - making chromatographic resolution difficult, and their identical masses - making simple mass resolution impossible. MS/MS data provides a means to resolve isomeric analytes if their MS/MS intensity profiles are sufficiently different. Glucosamine-6-phosphate (GlcN-6P) and glucosamine-1-phosphate (GlcN-1P) are early bacterial cell wall intermediates. These and other isomeric hexosamine-phosphates are highly polar and unretained on reverse-phase chromatography media. Three commercially available hexosamine-phosphate standards (GlcN-6P, GlcN-1P, and GalN-1P) were derivatized with octanoic anhydride, and chromatographic conditions were established to resolve these analytes on C18 columns. GlcN-1P and GalN-1P overlapped chromatographically under all tested chromatography conditions. Three MS/MS fragments (79, 97, and 199 m/z) were common to all three commercially available hexosamine-phosphates. Intensity ratios of the three MS/MS fragments from these three hexosamine-phosphate standards were used to deconvolute mixture chromatograms of these standards by non-negative linear regression. This approach allowed the complete resolution of these analytes. The chromatographically overlapping GlcN-1P and GalN-1P, which shared similar but modestly different MS/MS intensity profiles, were fully resolved with this non-negative deconvolution approach. This approach was then applied to MRSA, VSE, and VRE bacterial extracts before and after exposure to vancomycin. This demonstrated a substantial (3-fold) increase in GlcN-6P in vancomycin-treated MRSA samples but not in vancomycin-treated VSE or VRE samples. These observations appear to localize previously observed differences between MRSA and VRE/VSE peptidoglycan biosynthesis regulation to GlmS, which synthesizes GlcN-6P and is the product of a regulatory ribozyme sensitive to the levels of GlcN-6P.