SDS-PAGE of Arabidopsis plasma membranes.

<p>Arabidopsis plants were grown on ¹⁴N- and ¹⁵N-media, respectively. Leaf and root plasma membranes were isolated and subjected to SDS-PAGE (right), as well as 1/1 mixtures of ¹⁴N-leaf and ¹⁵N-root plasma membranes and vice versa (left).</p

Workflow.

<p>Plasma membrane fractions (or, in one case, soluble protein fractions, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071206#pone-0071206-g004" target="_blank">Fig. 4</a>) were prepared from leaf and root tissue, respectively, obtained from intact Arabidopsis plants hydroponically grown either on a ¹⁴N or a ¹⁵N medium. Left panel: Proteins were separated by SDS-PAGE and protein bands were excised for proteolysis. Peptides were separated by reversed-phase nano-liquid chromatography (RP nano-LC) and analyzed by MALDI-TOF MS/MS for peptide and protein identification, and construction of a peptide library. Right panel: leaf and root plasma membrane preparations were combined at a 1∶1 protein ratio and proteins were separated by SDS-PAGE. Protein bands were excised, digested, and peptides were separated by RP nano-LC and analyzed by MALDI MS. Finally, the relative abundance of individual proteins in leaf and root plasma membranes was determined from the signal intensities of the ¹⁴N/¹⁵N peptide pairs (Compare <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071206#pone-0071206-g004" target="_blank">Fig. 4</a>, below).</p

Immunostaining of marker proteins in Arabidopsis membrane fractions.

<p>Arabidopsis microsomal fractions (MF) obtained from leaves and roots, respectively, were subjected to aqueous two-phase partitioning to produce plasma membrane (PM) fractions. Polypeptides were separated by SDS-PAGE (7 µg of protein per lane), transferred to a blotting membrane, and immunostained with antisera directed against the plasma membrane H⁺-ATPase, subunit II of the mitochondrial cytochrome oxidase (CoxII), and light harvesting complex II (LHCII) of the chloroplast thylakoid membrane. Molecular weights (kDa) are indicated to the left. Note that the antiserum against CoxII also produced a band at a slightly higher molecular weight than the expected 29 kDa.</p

Spectra and q-values for ¹⁴N/¹⁵N-peptide pairs.

<p>Spectra and comparison of q-values for three ¹⁴N/¹⁵N-peptide pairs matching three PIP isoforms. The peptide pairs were from experiments in which either the leaf material was from ¹⁴N plants and the root material was from ¹⁵N plants, or vice versa (compare <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071206#pone-0071206-g005" target="_blank">Figure 5</a>).</p

Q-values for peptides of PIP isoforms.

<p>This is simplified version of the plot mentioned in the Data processing part. A, Q-values for two peptides unique to PIP1;1 and PIP1;2, respectively, and for a peptide shared between these two isoforms. B, Q-values for two peptides unique to PIP2;1 and for three peptides unique to PIP2;2, and for a peptide shared between these two isoforms. Note that PIPs with a monomer molecular mass of about 30 kDa band upon SDS-PAGE both as monomers and dimers, and also in the area in between 30 and 60 kDa. The molecular weights on the y axis are those indicated by the Mw standards used in the SDS-PAGE; the scale is therefore not lineal.</p

Consistency between theoretical and experimental isotope signal envelopes.

<p>Comparisons of intensity ratios for neighboring isotope signals in an experimentally obtained envelope with the corresponding intensity ratios for a theoretical envelope assuming 98.2% ¹⁵N labeling efficiency. Left panel: theoretical isotope pattern for the peptide DVEGPEGFQTR from the aquaporin PIP2;2. Right panel: experimental isotope pattern for the same peptide. The difference from the theoretical ratio was calculated for each pair of signals and only isotopic envelopes that did not differ more than the threshold values preset in the settings file of the processing software were used for further analysis (calculations below the panel).</p

Determination of the range within which reliable quantitative data can be obtained.

<p>A, Soluble leaf protein extracts from plants grown in ¹⁴N - and ¹⁵N -medium, respectively, were mixed at different ratios (1∶9, 2∶8, 3∶7, 5∶5, 7∶3, 8∶2, 9∶1) and subjected to SDS-PAGE. Two bands containing Rubisco large subunit, and fructose-1,6-bisphosphate aldolase (FBPase) were excised from each lane and processed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071206#pone-0071206-g002" target="_blank">Figure 2</a>. B, Experimental data for one of the FBPase peptides (TAAYYQQGAR). C, Average q-values for 11 peptides (6 from Rubisco and 5 from FBPase, in total based on 94 spectra) are summarized (grey columns; bar = standard deviation) and compared to the theoretical values (black columns). The relative abundance of ¹⁴N - and ¹⁵N -labeled peptides is defined as the fraction of ¹⁵N -labeled species of total peptide = the q-value of the peptide (equation at bottom, right. I = Intensity).</p

Integral plasma membrane proteins detected by MS and their relative abundance in leaf and root tissue (q-value).

<p>Proteins are grouped according to function and all annotation is via the database TAIR.</p>a<p>Predicted transmembrane domains determined by Phobius (Kall et al., 2004).</p>b<p>Number of unique peptides first identified by MS/MS and then detected in reliable ¹⁴N/¹⁵N spectra used for determination of the q-value for that specific protein. All these spectra have fulfilled all criteria for that specific peptide/protein, including correct position of the protein on the SDS-gel, consistent elution of the peptide in the nano-LC gradient, and correct peptide molecular mass as well as isotopic pattern.</p>c<p>The q-value is a measure of the distribution of the peptide between root and leaf plasma membranes: A q-value of 1 means that the peptide is found in roots only; 0, in leaves only. SD is the standard deviation for the q-value of the protein based on all unique peptides for that specific protein.</p>d<p>Number of MS spectra containing reliable data for the ¹⁴N/¹⁵N peptide pair(s) used to determine the q-value.</p>e<p>Genevestigator data for mRNA distribution between Arabidopsis leaf rosettes and roots converted to a simple letter code: L, mRNA found in leaves only, R, in roots only; LR, about equally distributed between leaves and roots; Lr, mainly in leaves; lR, mainly in roots.</p>*<p>The three q-values for SYP71 were obtained with two unique peptides found in three neighboring segments of the SDS gel (see text for discussion).</p

Unique and shared peptides from Arabidopsis PIP (plasma membrane Intrinsic protein) aquaporin isoforms and their relative abundance in leaf and root plasma membranes (q-value).

a<p>Peptide sequences for which reliable ¹⁴N/¹⁵N spectra could be obtained.</p>b<p>The q-value is a measure of the distribution of the peptide between root and leaf plasma membranes: A q-value of 1 means that the peptide is found in roots only; 0, in leaves only. SD is the standard deviation for the q-value of the peptide based on all spectra containing the ¹⁴N/¹⁵N peptide pair for that specific peptide.</p>c<p>Number of MS spectra containing reliable data for the ¹⁴N/¹⁵N peptide pair used to determine the q-value. All these spectra have fulfilled all criteria for that specific peptide/protein, including correct position of the protein on the SDS-gel, consistent elution of the peptide in the nano-LC gradient, and correct peptide molecular mass as well as isotopic pattern.</p>d<p>Genevestigator data for mRNA distribution between Arabidopsis leaf rosettes and roots converted to a simple letter code: L, mRNA found in leaves only, R, in roots only; LR, about equally distributed between leaves and roots; Lr, mainly in leaves; lR, mainly in roots.</p

Disaggregation of gold nanoparticles by <i>Daphnia magna</i>

<p>The use of manufactured nanomaterials is rapidly increasing, while our understanding of the consequences of releasing these materials into the environment is still limited and many questions remain, for example, how do nanoparticles affect living organisms in the wild? How do organisms adapt and protect themselves from exposure to foreign materials? How does the environment affect the performance of nanoparticles, including their surface properties? In an effort to address these crucial questions, our main aim has been to probe the effects of aquatic organisms on nanoparticle aggregation. We have, therefore, carried out a systematic study with the purpose to disentangle the effects of the freshwater zooplankter, <i>Daphnia magna</i>, on the surface properties, stability, and aggregation properties of gold (Au) nanoparticles under different aqueous conditions as well as identified the proteins bound to the nanoparticle surface. We show that Au nanoparticles aggregate in pure tap water, but to a lesser extent in water that either contains <i>Daphnia</i> or has been pre-conditioned with <i>Daphnia</i>. Moreover, we show that proteins generated by <i>Daphnia</i> bind to the Au nanoparticles and create a modified surface that renders them less prone to aggregation. We conclude that the surrounding milieu, as well as the surface properties of the original Au particles, are important factors in determining how the nanoparticles are affected by biological metabolism. In a broader context, our results show how nanoparticles released into a natural ecosystem become chemically and physically altered through the dynamic interactions between particles and organisms, either through biological metabolism or through the interactions with biomolecules excreted by organisms into the environment.</p