A high-resolution HPLC-QqTOF platform using parallel reaction monitoring for in-depth lipid discovery and rapid profiling.

Here, we developed a robust lipidomics workflow merging both targeted and untargeted approaches on a single liquid chromatography coupled to quadrupole-time of flight (LC-QqTOF) mass spectrometry platform with parallel reaction monitoring (PRM). PRM assays integrate both untargeted profiling from MS1 scans and targeted profiling obtained from MS/MS data. This workflow enabled the discovery of more than 2300 unidentified features and identification of more than 600 lipid species from 23 lipid classes at the level of fatty acid/long chain base/sterol composition in a barley root extracts. We detected the presence of 142 glycosyl inositol phosphorylceramides (GIPC) with HN(Ac)-HA as the core structure of the polar head, 12 cardiolipins and 17 glucuronosyl diacylglycerols (GlcADG) which have been rarely reported previously for cereal crops. Using a scheduled algorithm with up to 100 precursors multiplexed per duty cycle, the PRM assay was able to achieve a rapid profiling of 291 species based on MS/MS data by a single injection. We used this novel approach to demonstrate the applicability and efficiency of the workflow to study salt stress induced changes in the barley root lipidome. Results show that 221 targeted lipids and 888 unknown features were found to have changed significantly in response to salt stress. This combined targeted and untargeted single workflow approach provides novel applications of lipidomics addressing biological questions.

Heterologous co-expression of a yeast diacylglycerol acyltransferase (ScDGA1) and a plant oleosin (AtOLEO3) as an efficient tool for enhancing triacylglycerol accumulation in the marine diatom Phaeodactylum tricornutum.

BACKGROUND: Microalgae are promising alternate and renewable sources for producing valuable products such as biofuel and essential fatty acids. Although this is the case, there are still challenges impeding on the effective commercial production of microalgal products. For instance, their product yield is still too low. Therefore, this study was oriented towards enhancing triacylglycerol (TAG) accumulation in the diatom Phaeodactylum tricornutum (strain Pt4). To achieve this, a type 2 acyl-CoA:diacylglycerol acyltransferase from yeast (ScDGA1) and the lipid droplet (LD) stabilizing oleosin protein 3 from Arabidopsis thaliana (AtOLEO3) were expressed in Pt4. RESULTS: The individual expression of ScDGA1 and AtOLEO3 in Pt4 resulted in a 2.3- and 1.4-fold increase in TAG levels, respectively, in comparison to the wild type. The co-expression of both, ScDGA1 and AtOLEO3, was accompanied by a 3.6-fold increase in TAG content. On the cellular level, the lines co-expressing ScDGA1 and AtOLEO3 showed the presence of the larger and increased numbers of lipid droplets when compared to transformants expressing single genes and an empty vector. Under nitrogen stress, TAG productivity was further increased twofold in comparison to nitrogen-replete conditions. While TAG accumulation was enhanced in the analyzed transformants, the fatty acid composition remained unchanged neither in the total lipid nor in the TAG profile. CONCLUSIONS: The co-expression of two genes was shown to be a more effective strategy for enhancing TAG accumulation in P. tricornutum strain Pt4 than a single gene strategy. For the first time in a diatom, a LD protein from a vascular plant, oleosin, was shown to have an impact on TAG accumulation and on LD organization.

The effect of hypoxia on the lipidome of recombinant Pichia pastoris.

BACKGROUND: Cultivation of recombinant Pichia pastoris (Komagataella sp.) under hypoxic conditions has a strong positive effect on specific productivity when the glycolytic GAP promoter is used for recombinant protein expression, mainly due to upregulation of glycolytic conditions. In addition, transcriptomic analyses of hypoxic P. pastoris pointed out important regulation of lipid metabolism and unfolded protein response (UPR). Notably, UPR that plays a role in the regulation of lipid metabolism, amino acid metabolism and protein secretion, was found to be upregulated under hypoxia. RESULTS: To improve our understanding of the interplay between lipid metabolism, UPR and protein secretion, the lipidome of a P. pastoris strain producing an antibody fragment was studied under hypoxic conditions. Furthermore, lipid composition analyses were combined with previously available transcriptomic datasets to further understand the impact of hypoxia on lipid metabolism. Chemostat cultures operated under glucose-limiting conditions under normoxic and hypoxic conditions were analyzed in terms of intra/extracellular product distribution and lipid composition. Integrated analysis of lipidome and transcriptome datasets allowed us to demonstrate an important remodeling of the lipid metabolism under limited oxygen availability. Additionally, cells with reduced amounts of ergosterol through fluconazole treatment were also included in the study to observe the impact on protein secretion and its lipid composition. CONCLUSIONS: Our results show that cells adjust their membrane composition in response to oxygen limitation mainly by changing their sterol and sphingolipid composition. Although fluconazole treatment results a different lipidome profile than hypoxia, both conditions result in higher recombinant protein secretion levels.

Choline transporter-like1 (CHER1) is crucial for plasmodesmata maturation in Arabidopsis thaliana.

Plasmodesmata (PD) are microscopic pores connecting plant cells and enable cell-to-cell transport. Currently, little information is known about the molecular mechanisms regulating PD formation and development. To uncover components of PD development we made use of the 17 kDa movement protein (MP17) encoded by the Potato leafroll virus (PLRV). The protein is required for cell-to-cell movement of the virus and localises to complex PD. Forward genetic screening for Arabidopsis mutants with altered PD binding of MP17 revealed several mutant lines, while molecular genetics, biochemical and microscopic studies allowed further characterisation. Map-based cloning of one mutant revealed a point mutation in the choline transporter-like 1 (CHER1) protein, changing glycine247 into glutamate. Mutation in CHER1 resulted in a starch excess phenotype and stunted growth. Ultrastructure analysis of shoot apical meristems, developing and fully developed leaves showed reduced PD numbers and the absence of complex PD in fully developed leaves. This indicates that cher1 mutants are impaired in PD formation and development. Global lipid profiling revealed only slight modifications in the overall lipid composition, however, altered composition of PD-associated lipids cannot be ruled out. Thus, cher1 is devoid of complex PD in developed leaves and provides insights into the formation of complex PD at the molecular level.

A Caenorhabditis elegans model for ether lipid biosynthesis and function.

Ether lipids are widespread in nature, and they are structurally and functionally important components of membranes. The roundworm, Caenorhabditis elegans, synthesizes numerous lipid species containing alkyl and alkenyl ether bonds. We isolated C. elegans strains carrying loss-of-function mutations in three genes encoding the proteins required for the initial three steps in the ether lipid biosynthetic pathway, FARD-1/FAR1, ACL-7/GNPAT, and ADS-1/AGPS. Analysis of the mutant strains show that they lack ether lipids, but possess the ability to alter their lipid composition in response to lack of ether lipids. We found that increases in de novo fatty acid synthesis and reduction of stearoyl- and palmitoyl-CoA desaturase activity, processes that are at least partially regulated transcriptionally, mediate the altered lipid composition in ether lipid-deficient mutants. Phenotypic analysis demonstrated the importance of ether lipids for optimal fertility, lifespan, survival at cold temperatures, and resistance to oxidative stress.Caenorhabditis.

An enhanced plant lipidomics method based on multiplexed liquid chromatography-mass spectrometry reveals additional insights into cold- and drought-induced membrane remodeling.

Within the lipidome of plants a few bulk molecular species hamper the detection of the rest, which are present at relatively low levels. In addition, low-abundance species are often masked by numerous isobaric interferences, such as those caused by isoelemental species and isotopologues. This scenario not only means that minor species are underrepresented, but also leads to potential misidentifications and limits the structural information gathered by lipidomics approaches. In order to overcome these limitations we have developed a multiplexed liquid chromatography-mass spectrometry lipidomics platform able to achieve an enhanced coverage of plant lipidomes. The platform is based on a single extraction step followed by a series of ultra-performance liquid chromatography separations. Post-column flow is then directed to both a triple quadrupole analyzer for targeted profiling and a time-of-flight analyzer for accurate mass analysis. As a proof of concept, plants were subjected to cold or drought, which are known to trigger widespread remodeling events in plant cell membranes. Analysis of the leaf lipidome yielded 393 molecular species within 23 different lipid classes. This enhanced coverage allowed us to identify lipid molecular species and even classes that are altered upon stress, allowing hypotheses on role of glycosylinositolphosphoceramides (GIPC), steryl glycosides (SG) and acylated steryl glycosides (ASG) in drought stress to be addressed and confirming the findings from numerous previous studies with a single, wide-ranging lipidomics approach. This extended our knowledge on membrane remodeling during the drought response, integrating sphingolipids and sterol lipids into the current glycerolipid-based model.

Isolation and characterization of the plasma membrane from the yeast Pichia pastoris.

Despite similarities of cellular membranes in all eukaryotes, every compartment displays characteristic and often unique features which are important for the functions of the specific organelles. In the present study, we biochemically characterized the plasma membrane of the methylotrophic yeast Pichia pastoris with emphasis on the lipids which form the matrix of this compartment. Prerequisite for this effort was the design of a standardized and reliable isolation protocol of the plasma membrane at high purity. Analysis of isolated plasma membrane samples from P. pastoris revealed an increase of phosphatidylserine and a decrease of phosphatidylcholine compared to bulk membranes. The amount of saturated fatty acids in the plasma membrane was higher than in total cell extracts. Ergosterol, the final product of the yeast sterol biosynthetic pathway, was found to be enriched in plasma membrane fractions, although markedly lower than in Saccharomyces cerevisiae. A further characteristic feature of the plasma membrane from P. pastoris was the enrichment of inositol phosphorylceramides over neutral sphingolipids, which accumulated in internal membranes. The detailed analysis of the P. pastoris plasma membrane is discussed in the light of cell biological features of this microorganism especially as a microbial cell factory for heterologous protein production.

The lipidome and proteome of microsomes from the methylotrophic yeast Pichia pastoris.

The methylotrophic yeast Pichia pastoris is a popular yeast expression system for the production of heterologous proteins in biotechnology. Interestingly, cell organelles which play an important role in this process have so far been insufficiently investigated. For this reason, we started a systematic approach to isolate and characterize organelles from P. pastoris. In this study, we present a procedure to isolate microsomal membranes at high purity. These samples represent endoplasmic reticulum (ER) fractions which were subjected to molecular analysis of lipids and proteins. Organelle lipidomics included a detailed analysis of glycerophospholipids, fatty acids, sterols and sphingolipids. The microsomal proteome analyzed by mass spectrometry identified typical proteins of the ER known from other cell types, especially Saccharomyces cerevisiae, but also a number of unassigned gene products. The lipidome and proteome analysis of P. pastoris microsomes are prerequisite for a better understanding of functions of this organelle and for modifying this compartment for biotechnological applications.