Combined Statistical Analyses of Peptide Intensities and Peptide Occurrences Improves Identification of Significant Peptides from MS-Based Proteomics Data

Liquid chromatography−mass spectrometry-based (LC−MS) proteomics uses peak intensities of proteolytic peptides to infer the differential abundance of peptides/proteins. However, substantial run-to-run variability in intensities and observations (presence/absence) of peptides makes data analysis quite challenging. The missing observations in LC−MS proteomics data are difficult to address with traditional imputation-based approaches because the mechanisms by which data are missing are unknown a priori. Data can be missing due to random mechanisms such as experimental error or nonrandom mechanisms such as a true biological effect. We present a statistical approach that uses a test of independence known as a G-test to test the null hypothesis of independence between the number of missing values across experimental groups. We pair the G-test results, evaluating independence of missing data (IMD) with an analysis of variance (ANOVA) that uses only means and variances computed from the observed data. Each peptide is therefore represented by two statistical confidence metrics, one for qualitative differential observation and one for quantitative differential intensity. We use three LC−MS data sets to demonstrate the robustness and sensitivity of the IMD−ANOVA approach

Whole cell lipidomics reveal an altered lipid composition in DENV infected cells.

<p>Panel A and B represent an average expression (fold change) of the total number of individual lipids significantly expressed (p<0.05) per lipid class at 36 and 60 hr post-infection, respectively. The fold changes represent DENV-infected cells or UV-DENV exposed cells compared to the mock control. A lack of cones indicates that the expression level of those specific lipids were not significant (p<0.05). Panels C–H are representative lipid molecular species from specific lipid classes significantly regulated at the two different time points. The data are plotted as the integrated LC-MS peak abundance, in log 2 scale with standard deviation. PC, phosphatidylcholine; PE, phosphatidylethanolamine; SM, sphingomyelin; CER, ceramide; CER-PE, ceramide phosphoethanolamine; Lyso, lysophospholipids. See supplementary <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002584#ppat.1002584.s004" target="_blank">table S1</a> for a complete list of lipid features detected in this study. Four replicates were included in the lipidomic analyses. The error bars represent standard deviation of the mean. The blue dashed line separates species that remain elevated at both time points (36 and 60 hr) from species that are only elevated at the 36 hr time point. Infections were carried out using an MOI of 20 in C6/36 cells. Significantly expressed lipids species are shown denoted with an asterisk (*).</p

Bioactive sphingolipid species are differentially regulated in replication complex membranes isolated from DENV-infected mosquito cells.

<p>Multiple Reaction Monitoring (MRM) analysis of sphingolipids species differentially regulated in DENV-infected cells (MOI 20) or UV-DENV exposed cells compared to the mock control (see also supplementary <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002584#ppat.1002584.s006" target="_blank">table S3</a>). The data represent fold changes observed in three subcellular fractions that were analyzed in this study; 16K, replication complex membranes; CE, cytoplasmic extracts following removal of replication complex membranes and nuclei; N, nuclear fraction. Panels A–C represent ceramide, sphingomyelin and monohexosylceramide species respectively. The dashed line highlights values equal to the mock. The data represent three independent experiments. The error bars represent standard deviation of the mean.</p

Newly synthesized lipids and viral RNA in subcellular fractions show a dynamic distribution with time of infection.

<p><b>A.</b> A pulse-chase analysis of ¹⁴C-acetate incorporation into newly synthesized lipids. The results show total labeled lipid in post-nuclear supernatants of C6/36 cells infected with DENV for 36 and 60 hr at an MOI of 20. <b>B.</b> The same pulse-chase analysis showing ¹⁴C-acetate incorporation into newly synthesized lipids in subcellular fractions (16K and CE) at 36 and 60 hr post-infection. <b>C.</b> The ratio of viral RNA genome copies per labeled lipid in subcellular fractions (described in B) at 36 and 60 hr post-infection. 16K, membrane fraction (pellet) following centrifugation of post-nuclear supernatants at 16, 000× g. CE, cytoplasmic extract following centrifugation of post-nuclear supernatants at 16,000× g. cpm, counts per minute.</p

Several lipid classes are differentially regulated in replication complex membranes isolated from DENV infected cells.

<p>Panel A shows fold changes for each lipid class significantly (p<0.05) regulated in membrane fractions isolated from DENV infected (red) or UV-DENV exposed (blue) cells compared to the mock control. Infection was carried out using an MOI of 20. Panels B–D show fold changes for individual molecular species within each lipid class that are regulated. PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; MG, monoacylglycerol; DG, diacylglycerol; PA, phosphatidic acid. See supplementary <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002584#ppat.1002584.s005" target="_blank">table S2</a> for a complete list of lipid features detected in this analysis and supplementary <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002584#ppat-1002584-g001" target="_blank">figure 1</a> for a heat map representation of the data. Three replicates were included in the lipidomic analyses.</p

Dengue virus infection perturbs lipid homestasis in infected mosquito cells.

<p>The lipidomic analyses of dengue virus infected C6/36 mosquito cells suggest several metabolic pathways that may be significantly up regulated during infection. The grey dashed line highlights specific pathways of interest. Black arrows highlight reactions suggested by the lipidomic data and grey arrows represent reactions not observed in the data. Metabolites highlighted in boxes (solid line) are up regulated (white) or down regulated (grey) in DENV infected mosquito cells. <b>1.</b> Through the recruitment and activation of FAS, DENV stimulates <i>de novo</i> phospholipid biosynthesis in the replication complex <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002584#ppat.1002584-Heaton1" target="_blank">[20]</a>. <b>2.</b> Inhibition of this process with C75 disrupts the cellular lipid repertoire in mosquito cells to be unfavorable for virus replication. <b>3.</b> The lipidomic analyses reveal an up regulation of fatty acids such as palmitic (C16) and stearic (C18) acid. These fatty acids are intermediates in the biosynthesis of phospholipids, which is up regulated during DENV infection. Interestingly, in DENV infected cells the prevalent phospholipids primarily consist of C16 and C18 unsaturated acyl chains. Very long chain fatty acids are not significantly up regulated during infection. <b>4.</b> FAS activity also stimulates <i>de novo</i> sphingolipid biosynthesis. In the lipidomic analyses, the up regulation of intermediates such as N-palmitoylesphingosine suggests sphingolipid biosynthesis is activated during DENV infection. Specifically, SM and CER are enriched in DENV infected cells. Alternately, the up regulation in CER (and DG) during infection could result from the degradation of SM through the activity of sphingomyelinases (Smase). The resulting CER and DG could be redirected into several signaling pathways or be utilized for <i>de novo</i> phospholipid biosynthesis. The glycopshingolipids, GlcCER and GalCER are down regulated during DENV infection, which suggest that they are catabolized to produce CER. <b>5.</b> Lipidomic analyses also suggest the up regulation of triacylglycerol catabolism (Lipolysis) in DENV infected cells. This pathway results in the generation of MG, DG and palmitic acid. These intermediates are all up regulated in DENV infected cells and could be utilized for downstream signaling or <i>de novo</i> phospholipid biosynthesis. It has also been shown that TG catabolism is necessary for mitochondrial β-oxidation during DENV infection <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002584#ppat.1002584-Zaitseva1" target="_blank">[50]</a>. <b>6.</b> Elevated levels of LPC in DENV infected cells also suggest activation of PC hydrolysis by PLA₂. This enzyme is activated during DENV infection. The elevated levels of other phospholipids such as PA, PI, PE, PG as well as PC suggest that the CDG-DG pathway for phospholipid biosynthesis could also be activated. FAS, fatty acid synthase; DENV, dengue virus; C75, inhibitor of FAS; SM, sphingomyelin; CER, ceramide; MG, monoacylglycerol; DG, diacylglycerol; TG, triacylglycerol; LPC, lysophosphatidylcholine; PLA₂, phospholipase A₂; PA, phosphatidic acid; PI, phosphatidylinositol; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PC, phosphatidylcholine.</p

Transmission electron microscopy shows cell wall thickening during lipid accumulation.

<p>Both (A) confocal laser scanning microscopy and TEM show thickened cell wall morphology in later time points. Cells were stained for cell wall (blue) and lipid bodies (red) in (A). CW indicates the cell wall in (B).</p

Lipid spectrum accumulated in batch culture is dynamic.

<p>Intracellular FAMEs and intact lipids were extracted at 12 hour intervals. (A) Extractable FAMEs accumulate to a maximum level at 48 h and drop in concentration as extracellular glucose is depleted. (B) Intact lipid z-scores were clustered using a Euclidian approach. PE, phosphatidylethanolamine; PC, phosphatidylcholine; TG, triacylglycerol; DG, diacylglycerol; CR, ceramide; FA, free fatty acid; UN, unknown headgroup. Underlines indicate intact lipids identified in negative ESI mode while lack of an underline indicates those identified in positive ESI mode. *Intact lipids that changed significantly during the time course by ANOVA (p < 0.01).</p

Lipid accumulation over the course of five days during <i>Y</i>. <i>lipolytica</i> batch culture.

<p>(A) Wild-type <i>Y</i>. <i>lipolytica</i> (ATCC 20460^TM) was grown in YNB-R medium for five days with samples collected at 12 h intervals. Extracellular glucose was exhausted by 72 h. (B) The volume of lipid bodies was calculated from z-stack images and binned into percentiles to indicate the size distribution. (C) Scanning laser confocal microscopy of cells stained for neutral lipids (red) and cell wall (blue) reveal lipid bodies making up much of the intracellular space. Scale bar: 5 μm. (D) Helium ion microscopy reveals detailed cell surface structure. Arrowhead denotes typical bud scarring, arrowhead with asterisks highlights unusual bud scar morphology and arrows show areas of lumpy cell wall characteristic of cells of 36 h and older age. Scale bar: 1 μm.</p

The Lipid repertoire of DENV infected mosquito cells is unfavorable for replication in the presence of C75.

<p>The specific lipid classes differentially regulated by C75 treatment of cells are shown. Panel A represents the fold changes of lipid classes expressed in DENV infected cells (MOI 3) compared to C75 treated DENV infected cells. Panels B–D show fold changes for individual molecular species within each lipid class that are regulated. PI, phosphatidylinositol; PG, phosphatidylglycerol; MG, monoacylglycerol; DG, diacylglycerol; PA, phosphatidic acid. See supplementary <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002584#ppat.1002584.s006" target="_blank">table S3</a> for a complete list of lipid features detected in this analysis and supplementary <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002584#ppat-1002584-g002" target="_blank">figure 2</a> for a heat map representation of the data. Three replicates were included in the lipidomic analyses.</p