Liquid chromatography/tandem mass spectrometry of dolichols and polyprenols, lipid sugar carriers across evolution

Abstract

Across evolution, dolichols and polyprenols serve as sugar carriers in biosynthetic processes that include protein glycosylation and lipopolysaccharide biogenesis. Liquid chromatography coupled with electrospray ionization mass spectrometry offers a powerful tool for studying dolichols and polyprenols in their alcohol or glycan-modified forms in members of all three domains of life. In the following, recent examples of the how different versions of this analytical approach, namely reverse phase liquid chromatography-multiple reaction monitoring, normal phase liquid chromatography/tandem mass spectrometry and normal phase liquid chromatography-precursor ion scan detection have respectively served to address novel aspects of dolichol or polyprenol biology in Eukarya, Archaea and Bacteria. This article is part of a Special Issue entitled Lipodomics and Imaging Mass Spectrometry.

N-Linked Glycosylation in Archaea: a Structural, Functional, and Genetic Analysis

“…The adjacent gene, saci0092 (gds), codes for a geranylgeranyl pyrophosphate synthase. In S. shibatae, this bifunctional enzyme synthesizes both farnesyl pyrophosphate and geranylgeranyl pyrophosphate (145,146), the latter reportedly serving as a precursor in DolP biosynthesis (147). Such clustering of isoprenoid biosynthesis genes directly upstream of aglH could reflect a need for coordinated regulation and expression of genes involved in the biosynthesis of dolichol or DolP.…”

Mutants Resistant to LpxC Inhibitors by Rebalancing Cellular Homeostasis*

“…Liquid Chromatography-Mass Spectrometry (LC-MS)-The method of normal phase LC-MS was described previously (27). Reverse phase LC-MS was performed using a Shimadzu LC system (consisting of a solvent degasser, two LC-10A pumps, and an SCL-10A system controller) coupled to a QSTAR XL quadrupole time-of-flight tandem mass spectrometer.…”

Identification of Genes Involved in the Acetamidino Group Modification of the Flagellin N-Linked Glycan of Methanococcus maripaludis

“…The flagellin digests were analyzed by nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) using a QTOF Ultima hybrid-quadrupole time-of-flight mass spectrometer coupled to a NanoAquity UPLC system (Waters, Milford, MA). The digests were injected onto a 5-mm by 300-m-internal-diameter Acclaim PepMap100 18 -precolumn (Dionex/Thermo Scientific, Sunnyvale, CA) and separated on a 100-m by 100-mm-internal-diameter by 1.7-m BEH130 C 18 column (Waters) under the following gradient conditions: 5% to 45% acetonitrile (ACN) in 0.2% formic acid over 35 min and 45% to 95% ACN over 5 min, at 450 nl/min. MS/MS spectra were acquired on doubly, triply, and quadruply charged ions and searched against the NCBInr database by using the Mascot search engine (Matrix Science Ltd., London, United Kingdom).…”

“…However, archaea use dolichol mono-or pyrophosphate as the lipid carrier (like eukaryotes), while bacteria use undecaprenol pyrophosphate (18,19,31). A series of glycosyltransferases assemble the glycan from nucleotide-activated sugars, and once complete, the glycan is flipped across the membrane by an as yet unidentified flippase in archaea.…”