Reagent for Evaluating Liquid Chromatography–Tandem Mass Spectrometry (LC-MS/MS) Performance in Bottom-Up Proteomic Experiments

We present a novel proteomic standard for assessing liquid chromatography–tandem mass spectrometry (LC-MS/MS) instrument performance, in terms of chromatographic reproducibility and dynamic range within a single LC-MS/MS injection. The peptide mixture standard consists of six peptides that were specifically synthesized to cover a wide range of hydrophobicities (grand average hydropathy (GRAVY) scores of −0.6 to 1.9). A combination of stable isotope labeled amino acids (¹³C and ¹⁵N) were inserted to create five isotopologues. By combining these isotopologues at different ratios, they span four orders of magnitude within each distinct peptide sequence. Each peptide, from lightest to heaviest, increases in abundance by a factor of 10. We evaluate several metrics on our quadrupole orbitrap instrument using the 6 × 5 LC-MS/MS reference mixture spiked into a complex lysate background as a function of dynamic range, including mass measurement accuracy (MMA) and the linear range of quantitation of MS1 and parallel reaction monitoring experiments. Detection and linearity of the instrument routinely spanned three orders of magnitude across the gradient (500 fmol to 0.5 fmol on column) and no systematic trend was observed for MMA of targeted peptides as a function of abundance by analysis of variance analysis (<i>p</i> = 0.17). Detection and linearity of the fifth isotopologue (i.e., 0.05 fmol on column) was dependent on the peptide and instrument scan type (MS1 vs PRM). We foresee that this standard will serve as a powerful method to conduct both intra-instrument performance monitoring/evaluation, technology development, and inter-instrument comparisons

Interaction between the cyclophilin protein and CYDV-RPV and PLRV purified virus.

<p>His-tag cyclophilin vector (CV) and nonvector (CNV) isoforms were expressed <i>in vitro</i> in <i>E. coli</i> and co-immunoprecipitated with CYDV-RPV or PLRV. Co-immunoprecipitated proteins were detected with anti-his antibodies. First lane shows the synthesized cyclophilin protein. Interactions were notable between both isoforms and CYDV-RPV but not between PLRV and the vector isoform.</p

Transmission efficiency is correlated to presence of vectoring cyclophilin allele in field-collected aphid biotypes.

<p>Transmission efficiency is calculated as the number of plants infected with virus out of the number of plants infested with viruliferous aphids (five aphids per plant, 12 plants used). +/- indicates the detection of the vectoring allele. Biotypes NY and H were heterozygous. Biotypes NY, F, G, and H efficiently transmitted CYDV-RPV whereas Biotyoes B, I, and Fl did not transmit at all. Biotypes C, K and Ks transmitted with poor efficiencies.</p

Normalized peak areas from two cyclophilin A peptides show enrichment in co-immunoprecipitation experiments using aphid proteins and purified CYDV-RPV.

<p>Whole insects of genotypes A3, C2 and biotype WY-10A (efficient vector biotype recently collected from a field in Wyoming) were subjected to cryogenic cell lysis and protein extraction. The extracted proteins were co-immonoprecipitated with purified CYDV-RPV using anti-RPV antibodies. Two peptides from cyclophilin were enriched in gentoypes A3, C2 and the field collected biotype WY-10A as compared to the control co-immunoprecipitation with no virus (aphid proteins incubated with beads and antibodies). Peptide FFDMTADGEQLR (2+ precursor <i>m/z</i> 793.369) was higher in intensity than the HTGPGILSMANAGANTNGSQFFTTVK peptide (3+ precursor <i>m/z</i> 912.871). This is not a good indicator of differences in relative abundance between the two peptides, which could result from different ionization efficiencies. However comparison of peak areas for each peptide across the various samples is an accurate way to measure relative abundance of the peptide in each sample. Both peptides showed similar trends in the experimental co-IPs compared to the control. Both peptides were more abundant in the co-IP reactions with virus. Although the peak areas showed an overall lower abundance in biotype WY10-A, which might reflect a lower overall expression of cyclophilin in this biotype as compared to the lab-reared F2 genotypes, this difference was not significant using a Kruskal Wallis test.</p

Effect of sodium sulfite and EDTA on CYDV-RPV virion (arrowhead) attachment to apical plasmalemma and endocytosis into the HG cells of <i>R. padi</i> following membrane acquisition.

<p>(A and B) no sulfite or EDTA (buffer only) virus is internalized into cells of <i>R. padi</i> HG and can be found in tubular vesicles; (C and D) EDTA only treatment shows no effect of EDTA on acquisition of virions into the aphid HG which is consistent with the transmission data presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048177#pone-0048177-t001" target="_blank">Tables 1</a> & <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048177#pone-0048177-t002" target="_blank">2</a>; (E-F) sodium sulfite treatment prevents attachment and acquisition of virus into cells of the HG. APL, apical plasmalemma; HG, HG; L, lumen; TV, tubular vesicle; T, tubule; R, ribosome.</p

Discovery and Targeted LC-MS/MS of Purified Polerovirus Reveals Differences in the Virus-Host Interactome Associated with Altered Aphid Transmission

<div><p>Circulative transmission of viruses in the <em>Luteoviridae</em>, such as cereal yellow dwarf virus (CYDV), requires a series of precisely orchestrated interactions between virus, plant, and aphid proteins. Natural selection has favored these viruses to be retained in the phloem to facilitate acquisition and transmission by aphids. We show that treatment of infected oat tissue homogenate with sodium sulfite reduces transmission of the purified virus by aphids. Transmission electron microscopy data indicated no gross change in virion morphology due to treatments. However, treated virions were not acquired by aphids through the hindgut epithelial cells and were not transmitted when injected directly into the hemocoel. Analysis of virus preparations using nanoflow liquid chromatography coupled to tandem mass spectrometry revealed a number of host plant proteins co-purifying with viruses, some of which were lost following sodium sulfite treatment. Using targeted mass spectrometry, we show data suggesting that several of the virus-associated host plant proteins accumulated to higher levels in aphids that were fed on CYDV-infected plants compared to healthy plants. We propose two hypotheses to explain these observations, and these are not mutually exclusive: (a) that sodium sulfite treatment disrupts critical virion-host protein interactions required for aphid transmission, or (b) that host infection with CYDV modulates phloem protein expression in a way that is favorable for virus uptake by aphids. Importantly, the genes coding for the plant proteins associated with virus may be examined as targets in breeding cereal crops for new modes of virus resistance that disrupt phloem-virus or aphid-virus interactions.</p> </div

Negative stained grids, coated with CYDV-RPV coat protein antibody, of purified virus from each virus preparation after purification and virus recovered from membranes fed on by <i>R. padi</i> for a 24 h AAP.

<p>Virion morphology was similar within each group and a representative picture for each is shown. Transmissible virions after purification (A) and after membrane feeding (B) look morphologically similar. Non-transmissible virions after purification (C) and after membrane feeding (D) look morphologically similar to each other and are indistinguishable from transmissible virions in shape and size. Scale bars = 100 nm.</p

Summary of <i>R. padi</i> transmission and virion detection following membrane feeding of different cereal yellow dwarf virus<i>-</i>RPV preparations used for RT-PCR and TEM evaluations.

a<p>Purified virus that was used for RT-PCR and TEM analysis was tested for transmissibility using aphid transmission assays. Number of plants infected/total number of plants inoculated; total percent infection indicated.</p>b<p>Number of aphids with detectable amount of viral RNA/total number of aphids tested.</p>c<p>Number of aphids with virus in the HG cell/total number of aphids evaluated.</p

RT-PCR of total RNA extracted from <i>R. padi</i> aphids that fed on different virus preparations was performed to detect RPV using RPV coat protein primers, amplifying a 614 bp product.

<p>Aphids were collected after initially feeding for a 24 h AAP (A) and after 3 d IAP (B). Three independent replicates using RNA collected from small pools of aphids are shown for each treatment.</p

Mapping differential cellular protein response of mouse alveolar epithelial cells to multi-walled carbon nanotubes as a function of atomic layer deposition coating

<p>Carbon nanotubes (CNTs), a prototypical engineered nanomaterial, have been increasingly manufactured for a variety of novel applications over the past two decades. However, since CNTs possess fiber-like shape and cause pulmonary fibrosis in rodents, there is concern that mass production of CNTs will lead to occupational exposure and associated pulmonary diseases. The aim of this study was to use contemporary proteomics to investigate the mechanisms of cellular response in E10 mouse alveolar epithelial cells <i>in vitro</i> after exposure to multi-walled CNTs (MWCNTs) that were functionalized by atomic layer deposition (ALD). ALD is a method used to generate highly uniform and conformal nanoscale thin-film coatings of metals to enhance novel conductive properties of CNTs. We hypothesized that specific types of metal oxide coatings applied to the surface of MWCNTs by ALD would determine distinct proteomic profiles in mouse alveolar epithelial cells <i>in vitro</i> that could be used to predict oxidative stress and pulmonary inflammation. Uncoated (U)-MWCNTs were functionalized by ALD with zinc oxide (ZnO) to yield Z-MWCNTs or aluminum oxide (Al₂O₃) to yield A-MWCNTs. Significant differential protein expression was found in the following critical pathways: mTOR/eIF4/p70S6K signaling and Nrf-2 mediated oxidative stress response increased following exposure to Z-MWCNTs, interleukin-1 signaling increased following U-MWCNT exposure, and inhibition of angiogenesis by thrombospondin-1, oxidative phosphorylation, and mitochondrial dysfunction increased following A-MWCNT exposure. This study demonstrates that specific types of metal oxide thin film coatings applied by ALD produce distinct cellular and biochemical responses related to lung inflammation and fibrosis compared to uncoated MWCNT exposure <i>in vitro</i>.</p