We carried out a test sample study to try to identify errors leading to irreproducibility, including incompleteness of peptide sampling, in LC-MS-based proteomics. We distributed a test sample consisting of an equimolar mix of 20 highly purified recombinant human proteins, to 27 laboratories for identification. Each protein contained one or more unique tryptic peptides of 1250 Da to also test for ion selection and sampling in the mass spectrometer. Of the 27 labs, initially only 7 labs reported all 20 proteins correctly, and only 1 lab reported all the tryptic peptides of 1250 Da. Nevertheless, a subsequent centralized analysis of the raw data revealed that all 20 proteins and most of the 1250 Da peptides had in fact been detected by all 27 labs. The centralized analysis allowed us to determine sources of problems encountered in the study, which include missed identifications (false negatives), environmental contamination, database matching, and curation of protein identifications. Improved search engines and databases are likely to increase the fidelity of mass spectrometry-based proteomics.

Premature termination codons (PTCs) are responsible for 10–15% of all inherited disease. PTC suppression during translation offers a promising approach to treat a variety of genetic disorders, yet small molecules that promote PTC read-through have yielded mixed performance in clinical trials. Here we present a high-throughput, cell-based assay to identify anticodon engineered transfer RNAs (ACE-tRNA) which can effectively suppress in-frame PTCs and faithfully encode their cognate amino acid. In total, we identify ACE-tRNA with a high degree of suppression activity targeting the most common human disease-causing nonsense codons. Genome-wide transcriptome ribosome profiling of cells expressing ACE-tRNA at levels which repair PTC indicate that there are limited interactions with translation termination codons. These ACE-tRNAs display high suppression potency in mammalian cells, Xenopus oocytes and mice in vivo, producing PTC repair in multiple genes, including disease causing mutations within cystic fibrosis transmembrane conductance regulator (CFTR).

Extracellular vesicles (EVs) are mediators of intercellular communication under both healthy and pathological conditions, including the induction of pro‐metastatic traits, but it is not yet known how and where functional cargoes of EVs are delivered to their targets in host cell compartments. We have described that after endocytosis, EVs reach Rab7+ late endosomes and a fraction of these enter the nucleoplasmic reticulum and transport EV biomaterials to the host cell nucleoplasm. Their entry therein and docking to outer nuclear membrane occur through a tripartite complex formed by the proteins VAP‐A, ORP3 and Rab7 (VOR complex). Here, we report that the antifungal compound itraconazole (ICZ), but not its main metabolite hydroxy‐ICZ or ketoconazole, disrupts the binding of Rab7 to ORP3–VAP‐A complexes, leading to inhibition of EV‐mediated pro‐metastatic morphological changes including cell migration behaviour of colon cancer cells. With novel, smaller chemical drugs, inhibition of the VOR complex was maintained, although the ICZ moieties responsible for antifungal activity and interference with intracellular cholesterol distribution were removed. Knowing that cancer cells hijack their microenvironment and that EVs derived from them determine the pre‐metastatic niche, small‐sized inhibitors of nuclear transfer of EV cargo into host cells could find cancer therapeutic applications, particularly in combination with direct targeting of cancer cells.

30Premature termination codons (PTCs) are responsible for 10-15% of all inherited disease. PTC 31 suppression during translation offers a promising approach to treat a variety of genetic disorders, yet 32 small molecules that promote PTC read-through have yielded mixed performance in clinical trials. 33We present a high-throughput, cell-based assay to identify anticodon engineered transfer RNAs 34 (ACE-tRNA) which can effectively suppress in-frame PTCs and faithfully encode their cognate amino 35 acid. In total, we identified ACE-tRNA with a high degree of suppression activity targeting the most 36 common human disease-causing nonsense codons. Genome-wide transcriptome ribosome 37 profiling of cells expressing ACE-tRNA at levels which repair PTC indicate that there are limited 38 interactions with translation termination codons. These ACE-tRNAs display high suppression 39 potency in mammalian cells, Xenopus oocytes and mice in vivo, producing PTC repair in multiple 40 genes, including disease causing mutations within the cystic fibrosis transmembrane conductance 41 regulator (CFTR). 42 43 44 Amino acid codons most vulnerable to PTC conversion are those with a single nucleotide 56 substitution from a stop codon: tryptophan, tyrosine, cysteine, glutamic acid, lysine, glutamine, 57 serine, leucine, arginine, and glycine (Supplemental Figure 1). As such, PTCs represent a unique 58 constellation of diseases which afflict over 30 million people worldwide, accounting for 10-15% of all 59 genetic diseases 13 . 60 61 Small molecules, such as aminoglycosides 14 , dipeptides 15 , and oxadiazoles 16 , promote the "read-62 through" or "suppression" of nonsense mutations. These compounds are effective in model 63 organisms 17, 18 , mammalian cell lines 19 and some animal disease models 16, 20 . However, this 64 approach results in the encoding of a near-cognate amino acid 21 , effectively generating a missense 65 mutation at the PTC, which itself may have deleterious effects on protein folding, trafficking, and 66 function. Furthermore, aminoglycosides are oto-and nephrotoxic 22 , and the first-in-class oxadiazole, 67Ataluren, displayed unexpectedly low efficacy in patient populations (ACT DMD Phase 3 clinical trial, 68 NCT01826487; ACT CF, NCT02139306), thus limiting their utility as PTC therapeutics. Recent and 69 ongoing advances in CRISPR/Cas9-mediated genome editing provides potentially a permanent 70 solution for diseases resulting from nonsense mutations. However, aspects of this technology 71 impart hurdles for its rapid use as a therapeutic 23, 24 , and these challenges are not limited to the 72 requirement of "precision" or "personalized" diagnostics for each mutation based on the context of 73 each patient's genetic variability. 74 75We sought to identify a PTC repair approach that displays the versatility of small molecules and the 76 precision of gene editing. We investigated tRNAs to fulfill these criteria, whereby their anticodons 77 have been engineered via mutagenesis to recognize and suppress UGA, UAA or U...

Both the matrix selected and the laser fluence play important roles in MALDI-quadrupole/ time of flight (QqTOF) fragmentation processes. "Hot" matrices, such as ␣-cyano-4-hydroxycinnamic acid (HCCA), can increase fragmentation in MS spectra. Higher laser fluence also increases fragmentation. Typical peptide fragment ions observed in the QqTOF are a, b, and y ion series, which resemble low-energy CID product ions. This fragmentation may occur in the high-pressure region before the first mass-analyzing quadrupole. Fragment ions can be selected by the first quadrupole (Q1), and further sequenced by conventional MS/MS. This allows pseudo-MS 3 experiments to be performed.For peptides of higher molecular weight, pseudo-MS 3 can extend the mass range beyond what is usually accessible for sequencing, by allowing one to sequence a fragment ion of lower molecular weight instead of the full-length peptide. Peptides that predominantly show a single product ion after MS/MS yield improved sequence information when this technique is applied. This method was applied to the analysis of an in vitro phosphorylated peptide, where the intact enzymatically-generated peptide showed poor dissociation via MS/MS. Sequencing a fragment ion from the phosphopeptide enabled the phosphorylation site to be unambiguously determined. A fter the matrix-assisted laser desorption/ionization (MALDI) technique was first developed [1][2][3][4][5], there were extensive studies on the selection of matrices optimized for various types of lasers [6 -9]. Although most researchers are now primarily using 337 nm nitrogen lasers and only a few selected matrices, there has been much discussion of MALDI matrices with regard to mechanisms of ion formation [10 -24].In-source decay (ISD [25,26]) and post-source decay (PSD [27]) in MALDI/TOF-MS have been studied for the purpose of determining the mechanism of MALDI ionization, and have led to the classification of matrices such as ␣-cyano-4-hydroxycinnamic acid (HCCA) and 2,5-dihydroxybenzoic acid (DHB) as "hot" or "cold" based originally on the amount of glycoprotein fragmentation they induce [28].The differences in fragmentation observed with various matrices have been attributed to differences in initial velocities of the ions generated [29 -31], with higher initial velocities leading to cooling and less fragmentation. It has been noted that, for a given matrix, all analyte ions below 25 kDa appear to have the same initial velocity irrespective of mass, but the initial velocity depends on the matrix [31]. An early hypothesis that the internal energy and velocity of both analyte and matrix are a function of matrix sublimation temperatures has found empirical support [32]. Zenobi and co-workers found, however, that the internal energy distributions of analyte ions did not correlate with matrix sublimation temperatures, but instead depended on gas-phase proton transfer reactions [20,21,23]. Increasing laser fluence has been shown to increase analyte ion dissociation via in-source decay (ISD) [33][34][35][36][37][38...