MethylQuant: A Tool for Sensitive Validation of Enzyme-Mediated Protein Methylation Sites from Heavy-Methyl SILAC Data

The study of post-translational methylation is hampered by the fact that large-scale LC–MS/MS experiments produce high methylpeptide false discovery rates (FDRs). The use of heavy-methyl stable isotope labeling by amino acids in cell culture (heavy-methyl SILAC) can drastically reduce these FDRs; however, this approach is limited by a lack of heavy-methyl SILAC compatible software. To fill this gap, we recently developed MethylQuant. Here, using an updated version of MethylQuant, we demonstrate its methylpeptide validation and quantification capabilities and provide guidelines for its best use. Using reference heavy-methyl SILAC data sets, we show that MethylQuant predicts with statistical significance the true or false positive status of methylpeptides in samples of varying complexity, degree of methylpeptide enrichment, and heavy to light mixing ratios. We introduce methylpeptide confidence indicators, MethylQuant Confidence and MethylQuant Score, and demonstrate their strong performance in complex samples characterized by a lack of methylpeptide enrichment. For these challenging data sets, MethylQuant identifies 882 of 1165 true positive methylpeptide spectrum matches (i.e., >75% sensitivity) at high specificity (<2% FDR) and achieves near-perfect specificity at 41% sensitivity. We also demonstrate that MethylQuant produces high accuracy relative quantification data that are tolerant of interference from coeluting peptide ions. Together MethylQuant’s capabilities provide a path toward routine, accurate characterizations of the methylproteome using heavy-methyl SILAC

High temperature results in reduced <i>Wolbachia</i> densities and maternal leakage.

<p>(<b>A</b>) Larvae from the <i>w</i>AlbA, <i>w</i>AlbB, <i>w</i>Au, and <i>w</i>Mel strains were reared at constant 27°C (C) or with temperature fluctuating between 27–37°C (12hours:12hours) (H) and assessed for <i>Wolbachia</i> density by qPCR upon adult emergence. Each point represents a pool of 3 adult mosquitoes. The centre of a box plot shows median <i>Wolbachia</i> density, edges show upper and lower quartiles, and whiskers indicate upper and lower extremes. Statistical analyses were performed using a two-tailed Student’s t-test. (<b>B</b>) Females reared under larval temperature cycling conditions were allowed to recover upon emergence at a constant 27°C and were crossed to wild-type males with infection rates in resulting progeny assessed (1 Gen). Females reared under heat treatment were mated with wild-type males, and resulting progeny were also reared under high temperature conditions—resulting in two consecutive generations of high temperature treatment. Infection rates were then assessed in the pupae resulting from the second round of larval heating (2 Gen). Error bars show binomial 95% confidence intervals.</p

Generation of a <i>w</i>Au – <i>w</i>AlbB superinfection.

<p>(<b>A</b>) Crosses between <i>w</i>Au<i>w</i>AlbB and wild-type lines. Eggs are from crosses of 20 males and 20 females. Numbers show percentage hatch rates with total numbers of eggs counted in parentheses. (<b>B</b>) Total <i>Wolbachia</i> densities measured by qPCR in <i>w</i>AlbB, <i>w</i>Au, and <i>w</i>Au<i>w</i>AlbB carrying <i>Aedes aegypti</i> females at ten days post adult eclosion. Each bar represents 10 biological replicates, with pools of 5 females per replicate. Error bars show SD. (<b>C</b>) <i>w</i>Au and <i>w</i>AlbB strain-specific densities in the ovaries of <i>w</i>AlbB, <i>w</i>Au<i>w</i>AlbB, and <i>w</i>Au carrying <i>Ae</i>. <i>aegypti</i>. Each bar represents the average densities from 5 biological replicates each containing ovaries of 10 adult females. Error bars show SD. Statistical analysis was performed using a one-way ANOVA. (<b>D</b>) Fluorescent <i>in situ</i> hybridization showing distributions of <i>w</i>Au (green) and <i>w</i>AlbB (red) in ovaries of the <i>w</i>Au, <i>w</i>AlbB, <i>w</i>Au<i>w</i>AlbB and wild-type (wt) lines. For all images ovaries were treated with both red and green probes. A no-probe control showing some green auto-fluorescence in wild-type ovaries is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006815#ppat.1006815.s004" target="_blank">S4 Fig</a>. Blue stain is DAPI.</p

Crosses between <i>Wolbachia</i>-infected lines.

<p>Crosses between <i>Wolbachia</i>-infected lines.</p

Virus inhibition in <i>Wolbachia</i>-infected <i>Aedes aegypti</i> lines.

<p>(<b>A</b>) Semliki Forest virus (SFV) genome copies per host cell following thoracic injection into <i>Wolbachia</i>-infected lines and wild-type <i>Ae</i>. <i>aegypti</i>. Females were left for 10 days prior to total RNA extraction and virus quantification by qPCR. Levels of target RNA sequences were normalized against the RPS17 house-keeping gene. 17, 16, 18, 17 and 17 females were PCR’d for the <i>w</i>AlbA, <i>w</i>Mel, <i>w</i>Au, <i>w</i>AlbB and wt, respectively. Statistical analysis was performed using a one-way ANOVA with a Dunnett’s post-hoc test. Dengue-2 (DENV) (<b>B</b> and <b>C</b>) and Zika (ZIKV) (<b>D</b> and <b>E</b>) viruses were orally administered to 5-day old females. After an incubation period of 12 days, females were salivated (Zika only) and salivary glands and abdomens dissected. Viral RNA in salivary glands (SG) and abdomens were quantified by reverse-transcriptase qPCR, with viral RNA levels normalized to host RNA using the <i>RpS17</i> house-keeping gene. A value of zero for normalized virus levels, indicates no amplification for virus cDNA in that sample. Zika viral titers in saliva were quantified by fluorescent focus assay with results show focus forming units (FFU). Proportions underneath each graph indicate the infection rate for a given strain. Statistical analyses for panels B, C, D and E were performed using a one-tailed Fisher’s exact test comparing rates of virus-positive to virus-negative samples. Black lines indicate median of non-zero values.</p

<i>Wolbachia</i> densities and tropism in <i>Aedes</i> mosquitoes.

<p>(<b>A</b>) Total <i>Wolbachia</i> densities were measured by qPCR in <i>w</i>AlbA, <i>w</i>AlbB, <i>w</i>Au, and <i>w</i>Mel carrying <i>Aedes aegypti</i> females at varying time points post adult eclosion. Each box represents 10 biological replicates, with pools of 5 females per replicate. The centre of a box plot shows median <i>Wolbachia</i> density, edges show upper and lower quartiles, and whiskers indicate upper and lower extremes. (<b>B</b>) Total <i>Wolbachia</i> densities in dissected tissues measured by qPCR. Each bar represents the average density of 5 biological replicates. For each of the tissue-specific replicates 5 biological replicates of 5 sets of salivary glands, 5 midguts, or 5 ovary pairs were assessed. Error bars show SD. Statistical analyses were performed using a two-tailed Student’s t-test.</p