Stress granules (SGs) harbour translationally stalled messenger ribonucleoproteins and play important roles in regulating gene expression and cell fate. Here we show that neddylation promotes SG assembly in response to arsenite-induced oxidative stress. Inhibition or depletion of key components of the neddylation machinery concomitantly inhibits stress-induced polysome disassembly and SG assembly. Affinity purification and subsequent mass-spectrometric analysis of Nedd8-conjugated proteins from translationally stalled ribosomal fractions identified ribosomal proteins, translation factors and RNA-binding proteins (RBPs), including SRSF3, a previously known SG regulator. We show that SRSF3 is selectively neddylated at Lys85 in response to arsenite. A non-neddylatable SRSF3 (K85R) mutant do not prevent arsenite-induced polysome disassembly, but fails to support the SG assembly, suggesting that the neddylation pathway plays an important role in SG assembly.

Voltage-gated ion channels underlie electrical activity of neurons and are dynamically regulated by diverse cell signaling pathways that alter their phosphorylation state. Recent global mass spectrometric–based analyses of the mouse brain phosphoproteome have yielded a treasure trove of new data as to the extent and nature of phosphorylation of numerous ion channel principal or α subunits in mammalian brain. Here we compile and review data on 347 phosphorylation sites (261 unique) on 42 different voltage-gated ion channel α subunits that were identified in these recent studies. Researchers in the ion channel field can now begin to explore the role of these novel in vivo phosphorylation sites in the dynamic regulation of the localization, activity, and expression of brain ion channels through multisite phosphorylation of their principal subunits.

Voltage-gated sodium and potassium channels underlie electrical activity of neurons, and are dynamically regulated by diverse cell signaling pathways that ultimately exert their effects by altering the phosphorylation state of channel subunits. Recent mass spectrometric-based studies have led to a new appreciation of the extent and nature of phosphorylation of these ion channels in mammalian brain. This has allowed for new insights into how neurons dynamically regulate the localization, activity and expression through multisite ion channel phosphorylation.

Objectives A MALDI-TOF MS-based identification method for KPC-producing Enterobacterales was developed. Methods The molecular mass of the intact KPC-2 polypeptide was estimated for blaKPC-2 transformants using MALDI Microflex and the exact mass was confirmed by LC and a high-resolution MS/MS system. A total of 1181 clinical Enterobacterales strains, including 369 KPC producers and 812 KPC non-producers, were used to set up the methodology and the results were compared with those from PCR analyses. For external validation, a total of 458 Enterobacterales clinical isolates from a general hospital between December 2018 and April 2019 were used. Results The exact molecular mass of the intact KPC-2 protein was 28 718.13 Da and KPC peaks were observed at m/z 28 708.87–28 728.34 using MALDI Microflex. Most of the KPC-2 (99.1%, 335/338) and KPC-3 (100%, 6/6) producers presented a clear peak via this method, while 12.0% (3/25) of the KPC-4 producers had a peak of weak intensity associated with low levels of gene expression. It took less than 20 min for the entire assay to be performed with colonies on an agar plate. External validation showed that the analytical sensitivity and specificity of the method compared with PCR were 100% (59/59) and 99.50% (397/399), respectively. Conclusions The MALDI-TOF MS-based method for directly detecting the intact KPC protein is applicable to routine tests in clinical microbiology laboratories, supported by its speed, low cost and excellent sensitivity and specificity.

Purpose Clinical mass spectrometry requires a simple step process for sample preparation. This study aims to optimize the method for isolating periplasmic protein from Gram‐negative bacteria and apply to clinical mass spectrometry. Experimental Design The Klebsiella pneumoniae carbapenemase (KPC)‐producing E. coli standard cells were used for optimizing the osmotic shock (OS) lysis method. The supernatant from OS lysis was analysed by LC‐MS/MS and MALDI‐TOF MS. The effectiveness of the OS lysis method for KPC‐2‐producing Enterobacteriaceae clinical isolates were then confirmed by MALDI‐TOF MS. Results The optimized OS lysis using KPC‐2 producing E. coli standard cells showed a high yield of KPC‐2 protein and enriches periplasmic proteins. Compared with other lysis methods, the detection sensitivity of KPC‐2 protein significantly increased in MALDI‐TOF MS analysis. Nineteen clinical isolates were validated by MALDI‐TOF MS using the OS method, which also showed higher detection sensitivity compared to other lysis method (e.g., 1.5% n‐octyl‐β‐D‐glucopyranoside) (p < 0.001). Conclusions and Clinical Relevance This study provides a straightforward, rapid, affordable, and detergent‐free method for the analysis of periplasmic proteins from Enterobacteriaceae clinical isolates. This approach may contribute to MS‐based clinical diagnostics.