The Impact of Commonly Used Alkylating Agents on Artifactual Peptide Modification

Iodoacetamide is by far the most commonly used agent for alkylation of cysteine during sample preparation for proteomics. An alternative, 2-chloroacetamide, has recently been suggested to reduce the alkylation of residues other than cysteine, such as the N-terminus, Asp, Glu, Lys, Ser, Thr, and Tyr. Here we show that although 2-chloroacetamide reduces the level of off-target alkylation, it exhibits a range of adverse effects. The most significant of these is methionine oxidation, which increases to a maximum of 40% of all Met-containing peptides, compared with 2–5% with iodoacetamide. Increases were also observed for mono- and dioxidized tryptophan. No additional differences between the alkylating reagents were observed for a range of other post-translational modifications and digestion parameters. The deleterious effects were observed for 2-chloroacetamide from three separate suppliers. The adverse impact of 2-chloroacetamide on methionine oxidation suggests that it is not the ideal alkylating reagent for proteomics

The Impact of Commonly Used Alkylating Agents on Artifactual Peptide Modification

Iodoacetamide is by far the most commonly used agent for alkylation of cysteine during sample preparation for proteomics. An alternative, 2-chloroacetamide, has recently been suggested to reduce the alkylation of residues other than cysteine, such as the N-terminus, Asp, Glu, Lys, Ser, Thr, and Tyr. Here we show that although 2-chloroacetamide reduces the level of off-target alkylation, it exhibits a range of adverse effects. The most significant of these is methionine oxidation, which increases to a maximum of 40% of all Met-containing peptides, compared with 2–5% with iodoacetamide. Increases were also observed for mono- and dioxidized tryptophan. No additional differences between the alkylating reagents were observed for a range of other post-translational modifications and digestion parameters. The deleterious effects were observed for 2-chloroacetamide from three separate suppliers. The adverse impact of 2-chloroacetamide on methionine oxidation suggests that it is not the ideal alkylating reagent for proteomics

Effect of salt on the stimulation of dynamin I middle domain splice variants by GST-SNX9 SH3 and GST-intersectin I SH3A.

<p>Statistical analysis by two-way ANOVA followed by Bonferroni’s post-test shows GST-SNX9 SH3 <b>(A)</b> and GST-intersectin I SH3A (B) elicit a greater rate of GTPase activity in the presence of high salt for both splice variants. All data shown is ± S.E.M. with n ≥ 2 (*, p < 0.05 and **, p < 0.01).</p

Stimulation of the dynamin GTPase activity by SH3 domains is determined by its middle domain.

<p>Effect of GST-SH3 domains (40 μg/ml) on the GTPase activity rate of purified recombinant human dynamin I middle domain splice variants ab and bb (hudynI a and b, both at 100 nM / 10 μg/ml) determined using ELIPA. The assays were performed in the presence of low salt (30 mM NaCl) <b>(A)</b> or high salt (150 mM NaCl) <b>(B)</b>. All data is shown as means ± S.E.M. and n ≥ 2. <b>(C)</b> The relative binding of dynamin to individual GST-SH3 domains (~12 μg) was compared by performing pull-down analysis using purified recombinant full-length hudynI a and b splice variants (~3 μg). The pull-down was performed in the presence of low or high salt for both splice variants. Dynamin was resolved on 10% SDS-acrylamide gels and a Coomassie Blue stained gel is shown. The images are representative of n = 2 independent experiments.</p

Rank order analysis of the stimulation of sheep dynamin I activity (Fig 1B) and assembly (Fig 1C) by 13 GST-SH3 domains.

<p>The GST-SH3 domains are listed in descending order of their stimulatory effect on endogenous sheep brain dynamin I activity and assembly under low salt conditions. Both parameters are represented as fold change relative to dynamin basal (no SH3) control. Those exhibiting poor correlation between activity and assembly are indicated in bold.</p><p>Rank order analysis of the stimulation of sheep dynamin I activity (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144609#pone.0144609.g001" target="_blank">Fig 1B</a>) and assembly (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144609#pone.0144609.g001" target="_blank">Fig 1C</a>) by 13 GST-SH3 domains.</p

Lack of correlation between SH3 binding, stimulation and assembly of dynamin.

<p><b>(A)</b> The relative binding of dynamin to individual GST-SH3 domains (~12 μg) was compared by performing pull-down analysis using purified sheep dynamin I (~3 μg) in the presence of low or high salt. The pull-down was resolved on 10% SDS-acrylamide gels and a Coomassie Blue stained gel is shown. The image are representative of n = 3 independent experiments. <b>(B)</b> The effect of purified recombinant GST-SH3 domains (40 μg/ml) on stimulation of purified sheep brain dynamin I (100 nM / 10 μg/ml) GTPase activity using ELIPA in the presence of low (30 mM NaCl) or high (150 mM NaCl) salt. All data is mean ± S.E.M. and n ≥ 3. The inset shows a zoom of the low and high salt values for GST-SNX9 SH3. A two-tailed Student t-test showed significance (*, p < 0.05). <b>(C)</b> Relative ability of 13 GST-SH3 domains to oligomerise dynamin using a sedimentation assay. GST-SH3 domain stimulated dynamin I assembly obtained from densitometric analysis of the dynamin pellet (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144609#pone.0144609.s002" target="_blank">S2 Fig</a>). The data is representative of n = 3 independent experiments performed in duplicate and is expressed as fold change relative to dynamin basal (no SH3) control.</p

Rank order analysis of the stimulation of the activity of endogenous sheep dynamin I (from Fig 1B) and single splice variants human dynamin Iab and Ibb (Fig 2A) by 13 GST-SH3 domains.

<p>The rate of dynamin GTPase activity (μmoles Pi/mg of dyn/min) shown was generated under low salt assay conditions. The GST-SH3 domains are listed in descending order of their stimulatory effect across the three dynamins on sheep brain dynamin I. Those exhibiting poor correlation with sheep dynamin I, either in terms of stimulation or rank order, are indicated in bold.</p><p>Rank order analysis of the stimulation of the activity of endogenous sheep dynamin I (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144609#pone.0144609.g001" target="_blank">Fig 1B</a>) and single splice variants human dynamin Iab and Ibb (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144609#pone.0144609.g002" target="_blank">Fig 2A</a>) by 13 GST-SH3 domains.</p

Sorting Nexin 9 Recruits Clathrin Heavy Chain to the Mitotic Spindle for Chromosome Alignment and Segregation

<div><p>Sorting nexin 9 (SNX9) and clathrin heavy chain (CHC) each have roles in mitosis during metaphase. Since the two proteins directly interact for their other cellular function in endocytosis we investigated whether they also interact for metaphase and operate on the same pathway. We report that SNX9 and CHC functionally interact during metaphase in a specific molecular pathway that contributes to stabilization of mitotic spindle kinetochore (K)-fibres for chromosome alignment and segregation. This function is independent of their endocytic role. SNX9 residues in the clathrin-binding low complexity domain are required for CHC association and for targeting both CHC and transforming acidic coiled-coil protein 3 (TACC3) to the mitotic spindle. Mutation of these sites to serine increases the metaphase plate width, indicating inefficient chromosome congression. Therefore SNX9 and CHC function in the same molecular pathway for chromosome alignment and segregation, which is dependent on their direct association.</p> </div

SNX9 is required for efficient recruitment of CHC and TACC3 to the mitotic spindle.

<p>(A) Representative microscopy images illustrating the localization of CHC during interphase (Int) and the indicated mitotic stages in untreated cells and cells depleted of SNX9, SNX18 or SNX33 by siRNA. Met, metaphase. Ana, anaphase. Cyto, cytokinesis. Scale bars represent 10 µm. (B–C) The graphs represents the fluorescence intensity ratio of CHC at the mitotic spindle over the whole cell (B) and the overall fluorescence intensity of CHC within the whole cell (C) during metaphase (mean ± S.E.M., n > 16 per sample). ns, not significant; ***, <i>p</i> < 0.001 (One-way ANOVA). (D) Representative microscopy images demonstrating the localization of SNX9 (upper panels) in untreated and CHC-depleted HeLa cells during interphase, and metaphase. DNA shown in lower panels. Scale bars represent 10 µm. (E–F) Untreated, SNX9 and CHC-depleted metaphase HeLa cells were stained for TACC3. Representative microscopy images of TACC3 localization (upper panels) in these cells are shown in D. DNA shown in lower panels. Graphs represents the amount of TACC3 in each cell, expressed as (F) ratio of fluorescence intensity at the mitotic spindle compared to the whole cell and (G) the overall fluorescence intensity of TACC3 within the whole cell (mean ± S.E.M., n = > 6).</p

CHC interaction with the LC domain of SNX9 is required for efficient recruitment of mitotic spindle components.

<p>(A) A schematic diagram illustrating the domain structure of SNX9 and the amino acids in part of the low complexity sequence (LC domain). SNX9 contains an Src-homology 3 (SH3) domain at the N terminus followed by a low complexity (LC) domain, a phox-homology (PX) domain that is flanked by Yoke (Y) domains. A C-terminal Bin/Amphiphysin/Rvs (BAR) domain is located at the C-termini. Single and double mutations in LC1 (LC1 and LC1W1) and LC2 (LC2 and LC2W1) regions are shown whereby the tryptophan (W) residues (underlined) were mutated to serine (S). (B) GST alone, full length wild-type GST-SNX9 (WT) and GST-SNX9 harbouring LC1, LC1W1, LC2 and LC2W1 mutants coupled to glutathione-Sepharose were incubated with lysates from asynchronously growing HeLa cells and immunoblotted for CHC, dynII and dynII^S764. Lower panel shows amount of GST fusion protein in each sample (10%) as determined by Coomassie Blue staining. Lysate (2.5%) was also immunoblotted for the above mentioned proteins to reveal input. (C) The amount of CHC bound to the GST-SNX9 proteins indicated in B were quantified by densitometry analyses of immunoblots. Graph illustrates the relative amount of CHC bound to mutant GST-SNX9 (mean ± S.E.M. from 3–4 independent experiments) compared to wild-type GST-SNX9. (D–G) Metaphase-synchronised HeLa cells expressing GFP alone or GFP-SNX9-WT, LC1 or LC2 mutants were stained for CHC and TACC3. Representative microscopy images of the localization of CHC (D) and TACC3 (F) in these cells are shown. Scale bars represent 10 µm. The graphs represent the fluorescence intensity ratio of CHC (E) and TACC3 (G) at the mitotic spindle over the whole cell. (H) The graph illustrates the width of the metaphase plate of those cells analysed in E and G. Data shown in graphs (E, G and H) represent mean ± S.E.M. from at least two independent experiments. n = 15-30 cells per sample in each experiment. ns, not significant; *, <i>p</i> < 0.05; **, <i>p</i> < 0.01 (One-way ANOVA).</p