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1.  A novel strategy for the comprehensive analysis of the biomolecular composition of isolated plasma membranes 
A methodology for rapid, high-purity isolation of plasma membranes using superparamagnetic nanoparticles is described. The method is illustrated with high-resolution proteomic, glycomic and lipidomic analyses of presenilin-deficient cells.
We present a novel strategy based on cationic phospholipids-coated superparamagnetic nanoparticles (SPMNPs) to isolate plasma membranes with very high purity and at a preparative scale.The SPMNP-based isolation method is compatible with subsequent analysis of the biomolecular composition of plasma membranes, including proteomics, lipidomics and N-glycomics analysis.A comparative ‘omics' analysis of plasma membranes from wild-type, presenilin-deficient and human presenilin-1-rescued fibroblasts revealed convergent changes in proteins and lipids, suggesting an underlying endosomal transport defect.Our methodology allows for the systematic set-up of comprehensive plasma membrane inventories: alterations in the composition of the cell surface may potentially identify novel biomarkers or drug targets.
One of the major goals of this paper was to establish a robust method for plasma membrane (PM) isolation in order to perform a full analysis of their biomolecular composition. Using thermal decomposition, we manufactured superparamagnetic nanoparticles (SPMNPs) that we rendered water soluble and monodisperse by subsequent coupling of NH2 phospholipids. When incubated with cell monolayers, these cationic phospholipids-SPMNPs remain predominately localized at the cell surface. We applied these unexpected feature of phospholipids-SPMNPs to establish a novel protocol to isolate high yields of highly pure PMs. Due to the superb quality and quantity of isolated PM fractions, we could perform a comprehensive and comparative biomolecular profiling on this subcellular compartment that included proteomics, N-glycoproteomics, lipidomics and N-glycan profiling. This method was subsequently applied to compare the biomolecular composition of PMs isolated from wild-type and presenilin-deficient and human presenilin-1-rescued mouse embryonic fibroblasts (MEFs). For the first time, we succeeded in identifying convergent changes in the biomolecular composition of the cell surface caused by presenilin gene deficiency. Furthermore, the observed proteomic/cholesterol changes were restored in presenilin-deficient MEFs rescued with the human presenilin-1 ortholog. These (subtle) changes in protein and lipid composition suggest an underlying endosomal transport defect in presenilin-deficient cell lines that is the subject of ongoing research.
Moreover, and extending the versatility of our method, we could show that our PM isolations are compatible with fluorophore-assisted carbohydrate electrophoresis (FACE), allowing for N-glycan profiling of the cell surface. Hitherto, the N-glycan composition was measured on total cell extracts where the sensitivity to detect mature N-glycan chains is significantly hampered by the higher abundances of intracellular immature N-glycan intermediates. Finally, using the γ-secretase protein complex as a model, we confirm that we can study the activity and composition of active protein complexes in their native membrane environment. Our strategy incorporates for the first time most available omics analyses and this on a single isolated membrane compartment. As such, it allows for the monitoring and identification of systematic changes at the cell surface, for instance during differentiation and polarization or as a consequence of environmental insults, ER-stress, apoptotic stimuli and altered lipogenesis. The identification of alterations in the PM protein and lipid composition, as they occur as a consequence of disease, is therefore of paramount importance in several fields of experimental medicine, including immunology, cancer and stem cell research. Our methodology provides also a foundation to systematically start collecting PM ‘fingerprints' of an increasing number of cells. The resulting integrated and comprehensive databases may become the starting point of a ‘subcellular systems biology' approach of the cell's limiting membrane. Since PM proteins provide many pathological relevant biomarkers representing two-thirds of the currently used drug targets, this novel technology has great potential for biomedical and pharmaceutical applications.
We manufactured a novel type of lipid-coated superparamagnetic nanoparticles that allow for a rapid isolation of plasma membranes (PMs), enabling high-resolution proteomic, glycomic and lipidomic analyses of the cell surface. We used this technology to characterize the effects of presenilin knockout on the PM composition of mouse embryonic fibroblasts. We found that many proteins are selectively downregulated at the cell surface of presenilin knockout cells concomitant with lowered surface levels of cholesterol and certain sphingomyelin species, indicating defects in specific endosomal transport routes to and/or from the cell surface. Snapshots of N-glycoproteomics and cell surface glycan profiling further underscored the power and versatility of this novel methodology. Since PM proteins provide many pathologically relevant biomarkers representing two-thirds of the currently used drug targets, this novel technology has great potential for biomedical and pharmaceutical applications.
doi:10.1038/msb.2011.74
PMCID: PMC3261717  PMID: 22027552
eukaryotic cell systems; glycomics; lipidomics; presenilin, proteomics
2.  Disruption of the SapM locus in Mycobacterium bovis BCG improves its protective efficacy as a vaccine against M. tuberculosis 
EMBO Molecular Medicine  2011;3(4):222-234.
Mycobacterium bovis bacille Calmette-Guerin (BCG) provides only limited protection against pulmonary tuberculosis. We tested the hypothesis that BCG might have retained immunomodulatory properties from its pathogenic parent that limit its protective immunogenicity. Mutation of the molecules involved in immunomodulation might then improve its vaccine potential. We studied the vaccine potential of BCG mutants deficient in the secreted acid phosphatase, SapM, or in the capping of the immunomodulatory ManLAM cell wall component with α-1,2-oligomannoside. Both systemic and intratracheal challenge of mice with Mycobacterium tuberculosis following vaccination showed that the SapM mutant, compared to the parental BCG vaccine, provided better protection: it led to longer-term survival. Persistence of the SapM-mutated BCG in vivo resembled that of the parental BCG indicating that this mutation will likely not compromise the safety of the BCG vaccine. The SapM mutant BCG vaccine was more effective than the parental vaccine in inducing recruitment and activation of CD11c+MHC-IIintCD40int dendritic cells (DCs) to the draining lymph nodes. Thus, SapM acts by inhibiting recruitment of DCs and their activation at the site of vaccination.
doi:10.1002/emmm.201000125
PMCID: PMC3377067  PMID: 21328541
Mycobacterium; SapM; tuberculosis; vaccine; BCG

Results 1-2 (2)