Greazy: Open-Source Software for Automated Phospholipid Tandem Mass Spectrometry Identification

Lipid identification from data produced with high-throughput technologies is essential to the elucidation of the roles played by lipids in cellular function and disease. Software tools for identifying lipids from tandem mass (MS/MS) spectra have been developed, but they are often costly or lack the sophistication of their proteomics counterparts. We have developed Greazy, an open source tool for the automated identification of phospholipids from MS/MS spectra, that utilizes methods similar to those developed for proteomics. From user-supplied parameters, Greazy builds a phospholipid search space and associated theoretical MS/MS spectra. Experimental spectra are scored against search space lipids with similar precursor masses using a peak score based on the hypergeometric distribution and an intensity score utilizing the percentage of total ion intensity residing in matching peaks. The LipidLama component filters the results via mixture modeling and density estimation. We assess Greazy’s performance against the NIST 2014 metabolomics library, observing high accuracy in a search of multiple lipid classes. We compare Greazy/LipidLama against the commercial lipid identification software LipidSearch and show that the two platforms differ considerably in the sets of identified spectra while showing good agreement on those spectra identified by both. Lastly, we demonstrate the utility of Greazy/LipidLama with different instruments. We searched data from replicates of alveolar type 2 epithelial cells obtained with an Orbitrap and from human serum replicates generated on a quadrupole-time-of-flight (Q-TOF). These findings substantiate the application of proteomics derived methods to the identification of lipids. The software is available from the ProteoWizard repository: http://tiny.cc/bumbershoot-vc12-bin64

Palmitate and glucose regulate the use of Cer for the biosynthesis of complex sphingolipids in INS-1 cells.

<p>a) Cells were treated for 12 h (left panel) or 24 h (right panel) with 0.4 mM palmitate (P4) or without palmitate in the presence of 5 mM or 30 mM glucose. Cell viability was assessed by the MTT assay. Results are expressed as percentage of cell viability with respect to 5 mM glucose-treated cells (100%). Data are the mean ± S.D. of three independent experiments. *, p<0.05; ** p, <0.01. b) INS-1 cells were treated with 5 mM or with 30 mM glucose ±0.4 mM palmitate and harvested in lysis buffer for immunoblot analysis of GRP78 and GAPDH levels as described in experimental procedures. INS-1 cells were pretreated 30 min ±0.1 µM thapsigargin (Tg). Equal amounts of protein from homogenates were analyzed by immunoblotting with an anti-GRP78 antibody and an anti-GAPDH antibody. c) Cells were treated for 12 h ±0.4 mM palmitate in the presence of 5 mM or 30 mM glucose and then pulsed with 0.3 µCi/ml [C3-³H]sphingosine for 1 h. At the end of pulse, cells were harvested and submitted to lipid extraction and partitioning. The methanolized organic phase and the aqueous phase were analyzed by HPTLC and digital autoradiography of HPTLC (see experimental procedures). G5, 5 mM glucose; G5P4, 5 mM glucose+0.4 mM palmitate; G30, 30 mM glucose; G30P4, 30 mM glucose+0.4 mM palmitate. Data are the mean ± S.D. of at least three independent experiments. *p<0.05 for Ceramide and Sphingomyelin G5P4 or G30 compared with G5 and for GSLs G30P4 compared with G30; **p <0.01 for Cer and SM G30P4 compared with G30.</p

Palmitate and glucose regulate CERT expression and activation in INS-1 cells.

<p>a) INS-1 cells were harvested in lysis buffer as described in material and methods. Equal amounts of protein from homogenates were analyzed by immunoblotting with an anti-CERT antibody, an anti-phosphoserine and an anti-GAPDH antibody; b) the amount of CERT expressed was determined by densitometric quantitation and normalized for GAPDH **p<0.01 for G30+palmitate compared with G30; c) the amount of pCERT expressed was determined by densitometric quantitation and normalized for CERT **p<0.01 for G30+palmitate compared with G30; d) Relative expression of CERT assessed by Real-Time PCR. Results are expressed as fold-change relative to G5 *p<0.05 G30+palmitate cells vs G30. Values are mean ± SD of three independent experiments.</p

Palmitate and glucose affect vesicular-mediated Cer transport.

<p>a) Cells were transfected with a mix of S87 and S522 siRNA for CERT (siCERT) and the corresponding non-targeting corresponding sequences as control (siCT) and harvested in lysis buffer 72 h after transfection. 40 µg of protein from homogenate fractions and 2.4 ng of recombinant CERT (CERT) were analyzed by immunoblotting with polyclonal antibody anti-CERT and monoclonal anti-GAPDH. b) INS-1 cells down-regulated for CERT were treated for 12 h with or without palmitate in the presence of 30 mM glucose. Then the cells were pulsed with 0.3 µCi/ml [C3-³H]sphingosine for 1 h and processed and analyzed as described in the legend of Fig. 1. Data are mean ± S.D. of at least three independent experiments. *p<0.05 for siCERT compared with siCT and in GSL for siCT+palmitate vs siCT and siCERT+palmitate vs siCERT; **p<0.01 for siCT+palmitate compared with siCT and for siCERT+palmitate compared with siCERT.</p

Palmitate and glucose prevent colocalization of CERT and Golgi apparatus in INS-1 cells.

<p>INS-1 cells grown on a glass coverslip were transfected with the plasmid CERT-GFP as described in experimental procedures. 24 h later, the cells were treated with or without palmitate in the presence of 5 mM or 30 mM glucose for 12 h. Cells were then fixed and immunostained with WGA texas red-conjugated, a specific marker for the Golgi apparatus. a) Representative confocal microscopy images are shown; all images were processed and printed identically. b) The co-localization between CERT and WGA has been quantified through the Image J software and reported as Pearson colocalization coefficient. *p<0.05 G30+palmitate cells vs G30. c) The percentage of cells with co-localization of CERT and WGA was determined. The data are means ± the SD. **p<0.01 for G30+palmitate compared with G30.</p

Palmitate and glucose inhibit vesicular-mediated Cer traffic through downregulation of PI3K/Akt pathway.

<p><b>a</b>) INS-1 cells were treated with 5 mM or with 30 mM glucose ±0.4 mM palmitate and harvested for immunoblot analysis of phospho-Akt and GAPDH levels as described in experimental procedures. INS-1 cells were pretreated 30 min with or without 20 µM LY294002. The cells were then <b>b</b>) treated for 12 h with 5 mM glucose in the presence or absence of LY294002 or of Wm; <b>c</b>) treated for 12 h with 30 mM glucose in the presence or absence of LY294002 or Wm; <b>d</b>) treated for 12 h with 5 mM glucose plus 0.4 mM palmitate in the presence or absence of LY294002 or Wm; <b>e</b>) treated for 12 h with 30 mM glucose plus 0.4 mM palmitate in the presence or absence of LY294002 or Wm. Then cells were pulsed 1 h with [³H]Sph in the absence (<i>opened and dotted bars</i>) or presence of 20 µM LY294002 (<i>striped bars</i>) or 10 nM Wm (<i>square bars</i>). At the end of pulse, cells were harvested and submitted to lipid extraction and analyzed as described in the legend of Fig. 1. All values are the mean ± S.D. of at least three individual experiments. *p<0.05 for Cer and GSLs G5+LY294002 and G5+Wm compared with G5 and for GSLs G30+LY294002 and G30+Wm compared with G30; **p<0.01 for SM G5+LY294002 and G5+Wm compared with G5 and G30+LY294002 and G30+Wm compared with G30.</p

Schematic representation of the model showing the involvement of ceramide traffic in ER stress induced by glucolipotoxicity.

<p>Glucolipotoxicity impairs CERT- and vesicular-mediated Cer traffic. Glucolipotoxicity decrease the amount of active CERT significantly decreasing a) the total amount of the protein and b) the phosphorylation of CERT SR motif that is no longer able to localize at the Golgi apparatus. Moreover glucolipotoxicity inhibits PI3K/Akt pathway that could in turn impairs vesicular trafficking of Cer from the ER to the Golgi apparatus. Both transport systems contribute to the accumulation of Cer at the ER, thereby inducing ER stress. Furthermore ceramide synthase 4 (CerS4) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110875#pone.0110875-Veret1" target="_blank">[12]</a> and serine palmitoyltransferase (SPT) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110875#pone.0110875-Shimabukuro1" target="_blank">[16]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110875#pone.0110875-Kelpe1" target="_blank">[17]</a>, both residing in the endoplasmic reticulum (ER), have been shown to be involved in regulating Cer levels in β-cells in response to lipotoxicity and/or glucolipotoxicity.</p

Dynamic remodeling of lipids coincides with dengue virus replication in the midgut of <i>Aedes aegypti</i> mosquitoes

<div><p>We describe the first comprehensive analysis of the midgut metabolome of <i>Aedes aegypti</i>, the primary mosquito vector for arboviruses such as dengue, Zika, chikungunya and yellow fever viruses. Transmission of these viruses depends on their ability to infect, replicate and disseminate from several tissues in the mosquito vector. The metabolic environments within these tissues play crucial roles in these processes. Since these viruses are enveloped, viral replication, assembly and release occur on cellular membranes primed through the manipulation of host metabolism. Interference with this virus infection-induced metabolic environment is detrimental to viral replication in human and mosquito cell culture models. Here we present the first insight into the metabolic environment induced during arbovirus replication in <i>Aedes aegypti</i>. Using high-resolution mass spectrometry, we have analyzed the temporal metabolic perturbations that occur following dengue virus infection of the midgut tissue. This is the primary site of infection and replication, preceding systemic viral dissemination and transmission. We identified metabolites that exhibited a dynamic-profile across early-, mid- and late-infection time points. We observed a marked increase in the lipid content. An increase in glycerophospholipids, sphingolipids and fatty acyls was coincident with the kinetics of viral replication. Elevation of glycerolipid levels suggested a diversion of resources during infection from energy storage to synthetic pathways. Elevated levels of acyl-carnitines were observed, signaling disruptions in mitochondrial function and possible diversion of energy production. A central hub in the sphingolipid pathway that influenced dihydroceramide to ceramide ratios was identified as critical for the virus life cycle. This study also resulted in the first reconstruction of the sphingolipid pathway in <i>Aedes aegypti</i>. Given conservation in the replication mechanisms of several flaviviruses transmitted by this vector, our results highlight biochemical choke points that could be targeted to disrupt transmission of multiple pathogens by these mosquitoes.</p></div

Chain-length specificity of ceramide, sphingomyelin and glucosylceramide in response to palmitate and high concentrations of glucose in INS-1 cells.

<p>Cells were incubated with 0.4 mM palmitate in the presence of 5 mM (G5) or 30 mM (G30) glucose for 12 h. Levels of N-acyl chain lengths of Cer, SM and GlcCer were determined by LC–MS/MS. Levels of S1P in INS-1 cells were also determined by LC-MS/MS measurement. Results are expressed as pmol/nmol of phospholipids (PL) for Cer and SM and as fmol/nmol PL for GlcCer and S1P and are means ± S.D. for three independent experiments. *p<0.05 vs G5 except for S1P *p<0.05 vs G30.</p

Palmitate and glucose impairs ceramide flow from the ER to the Golgi apparatus in INS-1 cells.

<p>INS-1 cells grown on a glass coverslip were pretreated 30 min ± 0.1 µM thapsigargin. At the end of the pretreatment, the cells were treated with or without palmitate in the presence of 5 mM or 30 mM glucose for 12 h and then incubated with a) 2.5 µM BODIPY-C₅Cer or b) 2.5 µM NBD-C₆Cer as BSA complex 1∶1 (m/m) in DMEM for 30 min at 4°C; labeled cells were incubated at 37°C for 30 min and analyzed. All images were processed and printed identically.</p