The mechanistic target of rapamycin (mTOR) coordinates eukaryotic cell growth and metabolism with environmental inputs, including nutrients and growth factors. Extensive research over the past two decades has established a central role for mTOR in regulating many fundamental cell processes, from protein synthesis to autophagy, and deregulated mTOR signaling is implicated in the progression of cancer and diabetes, as well as the aging process. Here, we review recent advances in our understanding of mTOR function, regulation, and importance in mammalian physiology. We also highlight how the mTOR signaling network contributes to human disease and discuss the current and future prospects for therapeutically targeting mTOR in the clinic.

mTOR Signaling in Growth, Metabolism, and Disease

Proteomic and Metabolomic Characterization of COVID-19 Patient Sera.

Here we present an optimized workflow for global proteome and phosphoproteome analysis of tissues or cell lines that uses isobaric tags (TMT (tandem mass tags)-10) for multiplexed analysis and relative quantification, and provides 3× higher throughput than iTRAQ (isobaric tags for absolute and relative quantification)-4-based methods with high intra- and inter-laboratory reproducibility. The workflow was systematically characterized and benchmarked across three independent laboratories using two distinct breast cancer subtypes from patient-derived xenograft models to enable assessment of proteome and phosphoproteome depth and quantitative reproducibility. Each plex consisted of ten samples, each being 300 μg of peptide derived from 0.88. The maximum deviation for the phosphoproteome coverage was 37,000 quantified phosphosites per sample and differential quantification correlations of r > 0.72. The full procedure, including sample processing and data generation, can be completed within 10 d for ten tissue samples, and 100 samples can be analyzed in ~4 months using a single LC-MS/MS instrument. The high quality, depth, and reproducibility of the data obtained both within and across laboratories should enable new biological insights to be obtained from mass spectrometry-based proteomics analyses of cells and tissues together with proteogenomic data integration.

Reproducible workflow for multiplexed deep-scale proteome and phosphoproteome analysis of tumor tissues by liquid chromatography-mass spectrometry

Ever present hurdles for the discovery of new drugs for cancer therapy have necessitated the development of the alternative strategy of drug repurposing, the development of old drugs for new therapeutic purposes. This strategy with a cost-effective way offers a rare opportunity for the treatment of human neoplastic disease, facilitating rapid clinical translation. With an increased understanding of the hallmarks of cancer and the development of various data-driven approaches, drug repurposing further promotes the holistic productivity of drug discovery and reasonably focuses on target-defined antineoplastic compounds. The “treasure trove” of non-oncology drugs should not be ignored since they could target not only known but also hitherto unknown vulnerabilities of cancer. Indeed, different from targeted drugs, these old generic drugs, usually used in a multi-target strategy may bring benefit to patients. In this review, aiming to demonstrate the full potential of drug repurposing, we present various promising repurposed non-oncology drugs for clinical cancer management and classify these candidates into their proposed administration for either mono- or drug combination therapy. We also summarize approaches used for drug repurposing and discuss the main barriers to its uptake.

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Overcoming cancer therapeutic bottleneck by drug repurposing

Multi-organ proteomic landscape of COVID-19 autopsies.

Isobaric labeling empowers proteome-wide expression measurements simultaneously across multiple samples. Here an expanded set of 16 isobaric reagents based on an isobutyl-proline immonium ion reporter structure (TMTpro) is presented. These reagents have similar characteristics to existing tandem mass tag reagents but with increased fragmentation efficiency and signal. In a proteome-scale example dataset, we compared eight common cell lines with and without Torin1 treatment with three replicates, quantifying more than 8,800 proteins (mean of 7.5 peptides per protein) per replicate with an analysis time of only 1.1 h per proteome. Finally, we modified the thermal stability assay to examine proteome-wide melting shifts after treatment with DMSO, 1 or 20 µM staurosporine with five replicates. This assay identified and dose-stratified staurosporine binding to 228 cellular kinases in just one, 18-h experiment. TMTpro reagents allow complex experimental designs—all with essentially no missing values across the 16 samples and no loss in quantitative integrity. A set of isobaric labeling reagents called TMTpro enables deep quantitative comparisons of proteome measurements across 16 samples.

TMTpro reagents: a set of isobaric labeling mass tags enables simultaneous proteome-wide measurements across 16 samples.

We present a novel tandem mass tag solid-phase amino labeling (TMT-SPAL) protocol using reversible immobilization of peptides onto octadecyl-derivatized (C18) solid supports. This method can reduce the number of steps required in complex protocols, saving time and potentially reducing sample loss. In our global phosphopeptide profiling workflow (SysQuant), we can cut 24 h from the protocol while increasing peptide identifications (20%) and reducing side reactions. Solid-phase labeling with TMTs does require some modification to typical labeling conditions, particularly pH. It has been found that complete labeling equivalent to standard basic pH solution-phase labeling for small and large samples can be achieved on C18 resins under slightly acidic buffer conditions. Improved labeling behavior on C18 compared to that with standard basic pH solution-phase labeling is demonstrated. We analyzed our samples for histidine, serine, threonine, and tyrosine labeling to determine the degree of overlabeling and obser...

Low-pH Solid-Phase Amino Labeling of Complex Peptide Digests with TMTs Improves Peptide Identification Rates for Multiplexed Global Phosphopeptide Analysis

Provided is a method for characterising a polypeptide, which method comprises the steps of: (a) optionnally reducing cysteine disulphide bridges in the polypeptide to form free thiols, and capping the free thiols; (b) cleaving the polypeptide with a sequence specific cleavage reagent to form peptide fragments; (c) optionally deactivating the cleavage reagent; (d) capping one or more e-amino groups that are present with a lysine reactive agent; (e) analysing peptide fragments by mass spectrometry to form a mass fingerprint for the polypeptide; and (f) determining the identity of the polypeptide from the mass fingerprint.

Method for characterizing polypeptides

The invention describes compounds useful for labelling molecules of interest (i.e. analytes), particularly biomolecules such as peptides, proteins, oligonucleotides and nucleic acids, and also methods for analysing, detecting and/or isolating these labelled molecules using mass spectrometry. The compound in one aspect is a mass marker for labelling of an analyte detectable by mass spectrometry such as neutral loss mass spectroscopy, in which the mass marker comprises a neutral loss mass modifier linked via a first collision cleavable linker to a reactive group having reactive functionality for attachment to the analyte. The neutral loss mass modifier upon cleavage from the analyte during mass spectroscopy is uncharged.

Mass markers and methods

Provided is a method for characterising an analyte by matrix assisted laser desorption ionisation (MALDI) mass spectrometry, which method comprises: (a) labelling the analyte with a light-absorbing label that absorbs light at a pre-determined frequency, to form a labelled analyte; (b) embedding the labelled analyte in a matrix formed from at least one compound that absorbs light, to form an embedded labelled analyte; (c) desorbing the embedded labelled analyte by exposing it to light having the pre-determined frequency, to form a desorbed analyte; and (d) detecting the desorbed analyte by mass spectrometry to characterise the analyte.

Andrew Hugin Thompson

Papers

TMTpro reagents: a set of isobaric labeling mass tags enables simultaneous proteome-wide measurements across 16 samples.

Low-pH Solid-Phase Amino Labeling of Complex Peptide Digests with TMTs Improves Peptide Identification Rates for Multiplexed Global Phosphopeptide Analysis

Method for characterizing polypeptides

Mass markers and methods

Method for characterising analytes