Protein N-glycosylation is initiated within the endoplasmic reticulum through the synthesis of a lipid-linked oligosaccharides (LLO) precursor. This precursor is then transferred en bloc on neo-synthesized proteins through the action of the oligosaccharyltransferase giving birth to glycoproteins. The N-linked glycans bore by the glycoproteins are then processed into oligomannosides prior to the exit of the glycoproteins from the endoplasmic reticulum and its entrance into the Golgi apparatus. In this compartment, the N-linked glycans are further maturated in complex type N-glycans. This process has been well studied in a lot of eukaryotes including higher plants. In contrast, little information regarding the LLO precursor and synthesis of N-linked glycans is available in microalgae. In this report, a user-friendly extraction method combining microsomal enrichment and solvent extractions followed by purification steps is described. This strategy is aiming to extract LLO precursor from microalgae. Then, the oligosaccharide moiety released from the extracted LLO were analyzed by multistage tandem mass spectrometry in two models of microalgae namely the green microalgae, Chlamydomonas reinhardtii and the diatom, Phaeodactylum tricornutum. The validity of the developed method was confirmed by the analysis of the oligosaccharide structures released from the LLO of two xylosyltransferase mutants of C. reinhardtii confirming that this green microalga synthesizes a linear Glc3Man5GlcNAc2 identical to the one of the wild-type cells. In contrast, the analysis of the oligosaccharide released from the LLO of the diatom P. tricornutum demonstrated for the first time a Glc2Man9GlcNAc2 structure. The method described in this article allows the fast, non-radioactive and reliable multistage tandem mass spectrometry characterization of oligosaccharides released from LLO of microalgae including the ones belonging to the Phaeodactylaceae and Chlorophyceae classes, respectively. The method is fully adaptable for extracting and characterizing the LLO oligosaccharide moiety from microalgae belonging to other phyla.

Automotive formulated lubricants are high value products composed of 80% base oil and 20% various additives. During their life service, lubricants are exposed to several factors that will cause degradation over time, such as high temperature, shear, and oxidation. Base oil is a complex combination of hydrocarbons that are relatively sensitive to oxidation. During the initiation phase of oxidation, free radicals are formed, leading to the production of hydroperoxide ROOH and an alkyl radical R^•. These compounds will react with the base oil molecules to form aldehydes, ketones, and carboxylic acids in the termination phase. Owing to the molecular complexity of these mixtures, Fourier transform mass spectrometry seems to be the most appropriate tool to cover their wide range of compounds due to its ultra-high resolving power and mass accuracy. In this study, a native formulated lubricant and its different oxidized states at 140 °C under air flow (3, 5, 7, 8, and 9 days of oxidation) were analyzed by FTICR MS. The combination of atmospheric pressure chemical ionization (APCI) was used to achieve a non-selective ionization of molecules, including base oils, while Electrospray ionization (ESI) was used to selectively ionize acidic molecules. Apparent Kendrick mass defect (aKMD) plots were used to separate homologous series of molecules on different horizontal lines on the basis of the CH₂ repetition unit. Aging of lubricants was mainly characterized by a rapid consumption of certain additive families, such as molybdenum dithiocarbamates (MoDTCs) and zinc dithiophosphate (ZnDTPs), but also by the emergence of many oxidation products. Thus, the presence of aldehydes, ketones, and acids was characterized in the early stage of aging while larger products from polymerization were observed in a more advanced stage of aging. Interaction products between peroxy radicals and hindered phenols/alkyl diphenylamines (ADPAs) antioxidations were elucidated toward the high m/z. The formation of such products can be explained by trapping mechanisms of these additives at high temperature (>120 °C). Other types of interaction products were observed with the formation of antioxidant complexes. Additive degradation products were also characterized. For instance, polyisobutenyl succinimide dispersant oxidation products were clearly evidenced on the aKMD plots due to the gaps of 56 Da between each point. Overall, this study demonstrated the efficiency of the aKMD approach, and the use of ESI/APCI to characterize base oil and additive oxidation products.