Abnormal (Hydroxy)proline Deuterium Content Redefines Hydrogen Chemical Mass.

Analyzing the δ2H values in individual amino acids of proteins extracted from vertebrates, we unexpectedly found in some samples, notably bone collagen from seals, more than twice as much deuterium in proline and hydroxyproline residues than in seawater. This corresponds to at least 4 times higher δ2H than in any previously reported biogenic sample. We ruled out diet as a plausible mechanism for such anomalous enrichment. This finding puts into question the old adage that "you are what you eat".

Proteomics-Compatible Fourier Transform Isotopic Ratio Mass Spectrometry of Polypeptides.

Measuring the relative abundances of heavy stable isotopes of the elements C, H, N, and O in proteins is of interest in environmental science, archeology, zoology, medicine, and other fields. The isotopic abundance measurements of the fine structure of immonium ions with ultrahigh resolution mass spectrometry obtained in gas-phase fragmentation of polypeptides have previously uncovered anomalous deuterium enrichment in (hydroxy)proline of bone collagen in marine mammals. Here, we provide a detailed description and validation of this approach and demonstrate per mil-range precision of isotopic ratio measurements in aliphatic residues from proteins and cell lysates. The analysis consists of proteomics-type experiment demanding sub-microgram amounts of a protein sample and providing concomitantly protein sequence data allowing one to verify sample purity and establish its identity. A novel software tool protein amino acid-resolved isotopic ratio mass spectrometry (PAIR-MS) is presented for extracting isotopic ratio data from the raw data files acquired on an Orbitrap mass spectrometer.

Haptoglobin Induces a Specific Proteomic Profile and a Mature-Associated Phenotype on Primary Human Monocyte-Derived Dendritic Cells.

Damage-associated molecular patterns (DAMPs) play a critical role in dendritic cells (DCs) ability to trigger a specific and efficient adaptive immune response for different physiological and pathological scenarios. We have previously identified constitutive DAMPs (HMGB1 and Calreticulin) as well as new putative inducible DAMPs such as Haptoglobin (HP), from a therapeutically used heat shock-conditioned melanoma cell lysate (called TRIMEL). Remarkably, HP was shown to be the most abundant protein in the proteomic profile of heat shock-conditioned TRIMEL samples. However, its relative contribution to the observed DCs phenotype has not been fully elucidated. Human DCs were generated from monocytes isolated from PBMC of melanoma patients and healthy donors. DC lineage was induced with rhIL-4 and rhGM-CSF. After additional stimulation with HP, the proteome of these HP-stimulated cells was characterized. In addition, DCs were phenotypically characterized by flow cytometry for canonical maturation markers and cytokine production. Finally, in vitro transmigration capacity was assessed using Transwell plates. Our results showed that the stimulation with HP was associated with the presence of exclusive and higher relative abundance of specific immune-; energy production-; lipid biosynthesis-; and DAMPs-related proteins. Importantly, HP stimulation enhanced the expression of specific DC maturation markers and pro-inflammatory and Th1-associated cytokines, and an in vitro transmigration of primary human DCs. Taken together, these data suggest that HP can be considered as a new inducible DAMP with an important role in in vitro DC activation for cancer immunotherapy.

Anticancer Effect of Deuterium Depleted Water - Redox Disbalance Leads to Oxidative Stress.

Despite the convincing empirical evidence that deuterium depleted water (DDW, 25-125 ppm deuterium) has anticancer effect, the molecular mechanism remains unclear. Here, redox proteomics investigation of the DDW action in A549 cells revealed an increased level of oxidative stress, whereas expression proteomics in combination with thermal profiling uncovered crucial role of mitochondrial proteins. In the proposed scenario, reversal of the normally positive deuterium gradient across the inner membrane leads to an increased export of protons from the matrix to intermembrane space and an increase in the mitochondrial membrane potential, enhancing the production of reactive oxygen species (ROS). The resulting oxidative stress leads to slower growth and can induce apoptosis. However, further deuterium depletion in ambient water triggers a feedback mechanism, which leads to restoration of the redox equilibrium and resumed growth. The DDW-induced oxidative stress, verified by traditional biochemical assays, may be helpful as an adjuvant to ROS-inducing anticancer therapy.

Comparative Proteomics of Dying and Surviving Cancer Cells Improves the Identification of Drug Targets and Sheds Light on Cell Life/Death Decisions.

Chemotherapeutics cause the detachment and death of adherent cancer cells. When studying the proteome changes to determine the protein target and mechanism of action of anticancer drugs, the still-attached cells are normally used, whereas the detached cells are usually ignored. To test the hypothesis that proteomes of detached cells contain valuable information, we separately analyzed the proteomes of detached and attached HCT-116, A375, and RKO cells treated for 48 h with 5-fluorouracil, methotrexate and paclitaxel. Individually, the proteomic data on attached and detached cells had comparable performance in target and drug mechanism deconvolution, whereas the combined data significantly improved the target ranking for paclitaxel. Comparative analysis of attached versus detached proteomes provided further insight into cell life and death decision making. Six proteins consistently up- or downregulated in the detached versus attached cells regardless of the drug and cell type were discovered; their role in cell death/survival was tested by silencing them with siRNA. Knocking down USP11, CTTN, ACAA2, and EIF4H had anti-proliferative effects, affecting UHRF1 additionally sensitized the cells to the anticancer drugs, while knocking down RNF-40 increased cell survival against the treatments. Therefore, adding detached cells to the expression proteomics analysis of drug-treated cells can significantly increase the analytical value of the approach. The data have been deposited to the ProteomeXchange with identifier PXD007686.

Proteogenomics of Malignant Melanoma Cell Lines: The Effect of Stringency of Exome Data Filtering on Variant Peptide Identification in Shotgun Proteomics.

The identification of genetically encoded variants at the proteome level is an important problem in cancer proteogenomics. The generation of customized protein databases from DNA or RNA sequencing data is a crucial stage of the identification workflow. Genomic data filtering applied at this stage may significantly modify variant search results, yet its effect is generally left out of the scope of proteogenomic studies. In this work, we focused on this impact using data of exome sequencing and LC-MS/MS analyses of six replicates for eight melanoma cell lines processed by a proteogenomics workflow. The main objectives were identifying variant peptides and revealing the role of the genomic data filtering in the variant identification. A series of six confidence thresholds for single nucleotide polymorphisms and indels from the exome data were applied to generate customized sequence databases of different stringency. In the searches against unfiltered databases, between 100 and 160 variant peptides were identified for each of the cell lines using X!Tandem and MS-GF+ search engines. The recovery rate for variant peptides was ∼1%, which is approximately three times lower than that of the wild-type peptides. Using unfiltered genomic databases for variant searches resulted in higher sensitivity and selectivity of the proteogenomic workflow and positively affected the ability to distinguish the cell lines based on variant peptide signatures.

Proteogenomics of Adenosine-to-Inosine RNA Editing in the Fruit Fly.

Adenosine-to-inosine RNA editing is one of the most common types of RNA editing, a posttranscriptional modification made by special enzymes. We present a proteomic study on this phenomenon for Drosophila melanogaster. Three proteome data sets were used in the study: two taken from public repository and the third one obtained here. A customized protein sequence database was generated using results of genome-wide adenosine-to-inosine RNA studies and applied for identifying the edited proteins. The total number of 68 edited peptides belonging to 59 proteins was identified in all data sets. Eight of them being shared between the whole insect, head, and brain proteomes. Seven edited sites belonging to synaptic vesicle and membrane trafficking proteins were selected for validation by orthogonal analysis by Multiple Reaction Monitoring. Five editing events in cpx, Syx1A, Cadps, CG4587, and EndoA were validated in fruit fly brain tissue at the proteome level using isotopically labeled standards. Ratios of unedited-to-edited proteoforms varied from 35:1 ( Syx1A) to 1:2 ( EndoA). Lys-137 to Glu editing of endophilin A may have functional consequences for its interaction to membrane. The work demonstrates the feasibility to identify the RNA editing event at the proteome level using shotgun proteomics and customized edited protein database.

Cytotoxic and Proinflammatory Effects of Metal-Based Nanoparticles on THP-1 Monocytes Characterized by Combined Proteomics Approaches.

Thorough characterization of toxic effects of nanoparticles (NP) is desirable due to the increasing risk of potential environmental contamination by NP. In the current study, we combined three recently developed proteomics approaches to assess the effects of Au, CuO, and CdTe NP on the innate immune system. The human monocyte cell line THP-1 was employed as a model. The anticancer drugs camptothecin and doxorubicin were used as positive controls for cell death, and lipopolysaccharide was chosen as a positive control for proinflammatory activation. Despite equivalent overall toxicity effect (50 ± 10% dead cells), the three NP induced distinctly different proteomics signatures, with the strongest effect being induced by CdTe NP, followed by CuO and gold NP. The CdTe toxicity mechanism involves down-regulation of topoisomerases. The effect of CuO NP is most reminiscent of oxidative stress and involves up-regulation of proteins involved in heat response. The gold NP induced up-regulation of the inflammatory mediator, NF-κB, and its inhibitor TIPE2 was identified as a direct target of gold NP. Furthermore, gold NP triggered activation of NF-κB as evidenced by phosphorylation of the p65 subunit. Overall, the combined proteomics approach described here can be used to characterize the effects of NP on immune cells.

DeMix workflow for efficient identification of cofragmented peptides in high resolution data-dependent tandem mass spectrometry.

Based on conventional data-dependent acquisition strategy of shotgun proteomics, we present a new workflow DeMix, which significantly increases the efficiency of peptide identification for in-depth shotgun analysis of complex proteomes. Capitalizing on the high resolution and mass accuracy of Orbitrap-based tandem mass spectrometry, we developed a simple deconvolution method of "cloning" chimeric tandem spectra for cofragmented peptides. Additional to a database search, a simple rescoring scheme utilizes mass accuracy and converts the unwanted cofragmenting events into a surprising advantage of multiplexing. With the combination of cloning and rescoring, we obtained on average nine peptide-spectrum matches per second on a Q-Exactive workbench, whereas the actual MS/MS acquisition rate was close to seven spectra per second. This efficiency boost to 1.24 identified peptides per MS/MS spectrum enabled analysis of over 5000 human proteins in single-dimensional LC-MS/MS shotgun experiments with an only two-hour gradient. These findings suggest a change in the dominant "one MS/MS spectrum - one peptide" paradigm for data acquisition and analysis in shotgun data-dependent proteomics. DeMix also demonstrated higher robustness than conventional approaches in terms of lower variation among the results of consecutive LC-MS/MS runs.

Exome-driven characterization of the cancer cell lines at the proteome level: the NCI-60 case study.

Cancer genome deviates significantly from the reference human genome, and thus a search against standard genome databases in cancer cell proteomics fails to identify cancer-specific protein variants. The goal of this Article is to combine high-throughput exome data [Abaan et al. Cancer Res. 2013] and shotgun proteomics analysis [Modhaddas Gholami et al. Cell Rep. 2013] for cancer cell lines from NCI-60 panel to demonstrate further that the cell lines can be effectively recognized using identified variant peptides. To achieve this goal, we generated a database containing mutant protein sequences of NCI-60 panel of cell lines. The proteome data were searched using Mascot and X!Tandem search engines against databases of both reference and mutant protein sequences. The identification quality was further controlled by calculating a fraction of variant peptides encoded by the own exome sequence for each cell line. We found that up to 92.2% peptides identified by both search engines are encoded by the own exome. Further, we used the identified variant peptides for cell line recognition. The results of the study demonstrate that proteome data supported by exome sequence information can be effectively used for distinguishing between different types of cancer cell lines.

Proteomic Discovery of RNA-Protein Molecular Clamps Using a Thermal Shift Assay with ATP and RNA (TSAR).

Uncompetitive inhibition is an effective strategy for suppressing dysregulated enzymes and their substrates, but discovery of suitable ligands depends on often-unavailable structural knowledge and serendipity. Hence, despite surging interest in mass spectrometry-based target identification, proteomic studies of substrate-dependent target engagement remain sparse. Herein, we describe the Thermal Shift Assay with ATP and RNA (TSAR) as a template for proteome-wide discovery of substrate-dependent ligand binding. Using proteomic thermal shift assays, we show that simple biochemical additives can facilitate detection of target engagement in native cell lysates. We apply our approach to rocaglates, a family of molecules that specifically clamp RNA to eukaryotic translation initiation factor 4A (eIF4A), DEAD-box helicase 3X (DDX3X), and potentially other members of the DEAD-box (DDX) family of RNA helicases. To identify unexpected interactions, we optimized a target class-specific thermal denaturation window and evaluated ATP analog and RNA probe dependencies for key rocaglate-DDX interactions. We report novel DDX targets of the rocaglate clamping spectrum, confirm that DDX3X is a common target of several widely studied analogs, and provide structural insights into divergent DDX3X affinities between synthetic rocaglates. We independently validate novel targets of high-profile rocaglates, including the clinical candidate Zotatifin (eFT226), using limited proteolysis-mass spectrometry and fluorescence polarization experiments. Taken together, our study provides a model for screening uncompetitive inhibitors using a systematic chemical-proteomics approach to uncover actionable DDX targets, clearing a path towards characterization of novel molecular clamps and associated RNA helicase targets.

Chromosome-centric approach to overcoming bottlenecks in the Human Proteome Project.

The international Human Proteome Project (HPP), a logical continuation of the Human Genome Project, was launched on 23 September 2010 in Sydney, Australia. In accordance with the gene-centric approach, the goals of the HPP are to prepare an inventory of all human proteins and decipher the network of cellular protein interactions. The greater complexity of the proteome in comparison to the genome gives rise to three bottlenecks in the implementation of the HPP. The main bottleneck is the insufficient sensitivity of proteomic technologies, hampering the detection of proteins with low- and ultra-low copy numbers. The second bottleneck is related to poor reproducibility of proteomic methods and the lack of a so-called 'gold' standard. The last bottleneck is the dynamic nature of the proteome: its instability over time. The authors here discuss approaches to overcome these bottlenecks in order to improve the success of the HPP.

A mass spectrometry-based non-radioactive differential radial capillary action of ligand assay (DRaCALA) to assess ligand binding to proteins.

Binding of ligands to macromolecules changes their physicochemical and enzymatic characteristics. Cyclic di-GMP is a second messenger involved in motility/sessility and acute/chronic infection life style transition. Although the GGDEF domain, predominantly a diguanylate cyclase, represents one of the most abundant bacterial domain superfamilies, the number of cyclic di-GMP receptors falls short. To facilitate screening for cyclic di-nucleotide binding proteins, we describe a non-radioactive, matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF)-based modification of the widely applied differential radial capillary action of ligand assay (DRaCALA). The results of this assay suggest that the diguanylate cyclase/phosphodiesterase variant YciRFec101 , but not selected catalytic mutants, bind cyclic di-GMP. HIGHLIGHTS: Cyclic di-nucleotides are ubiquitous second messengers in bacteria. However, few receptors have been identified. Previous screening of cell lysates by differential radial capillary action of ligand assay (DRaCALA) using radioactive ligand identified cyclic di-nucleotide binding proteins. A MALDI-TOF-based DRaCALA was developed to detect cyclic di-nucleotide binding as a non-radioactive alternative. Known cyclic di-GMP binding proteins were verified and potential cyclic di-GMP binding proteins were identified.

Protein domain-dependent vesiculation of Lipoprotein A, a protein that is important in cell wall synthesis and fitness of the human respiratory pathogen Haemophilus influenzae.

The human pathogen Haemophilus influenzae causes respiratory tract infections and is commonly associated with prolonged carriage in patients with chronic obstructive pulmonary disease. Production of outer membrane vesicles (OMVs) is a ubiquitous phenomenon observed in Gram-negative bacteria including H. influenzae. OMVs play an important role in various interactions with the human host; from neutralization of antibodies and complement activation to spread of antimicrobial resistance. Upon vesiculation certain proteins are found in OMVs and some proteins are retained at the cell membrane. The mechanism for this phenomenon is not fully elucidated. We employed mass spectrometry to study vesiculation and the fate of proteins in the outer membrane. Functional groups of proteins were differentially distributed on the cell surface and in OMVs. Despite its supposedly periplasmic and outer membrane location, we found that the peptidoglycan synthase-activator Lipoprotein A (LpoA) was accumulated in OMVs relative to membrane fractions. A mutant devoid of LpoA lost its fitness as revealed by growth and electron microscopy. Furthermore, high-pressure liquid chromatography disclosed a lower concentration (55%) of peptidoglycan in the LpoA-deficient H. influenzae compared to the parent wild type bacterium. Using an LpoA-mNeonGreen fusion protein and fluorescence microscopy, we observed that LpoA was enriched in "foci" in the cell envelope, and further located in the septum during cell division. To define the fate of LpoA, C-terminally truncated LpoA-variants were constructed, and we found that the LpoA C-terminal domain promoted optimal transportation to the OMVs as revealed by flow cytometry. Taken together, our study highlights the importance of LpoA for H. influenzae peptidoglycan biogenesis and provides novel insights into cell wall integrity and OMV production.

Mechanistic Insights into a CDK9 Inhibitor Via Orthogonal Proteomics Methods.

Cyclin-dependent-kinases (CDKs) are members of the serine/threonine kinase family and are highly regulated by cyclins, a family of regulatory subunits that bind to CDKs. CDK9 represents one of the most studied examples of these transcriptional CDKs. CDK9 forms a heterodimeric complex with its regulatory subunit cyclins T1, T2 and K to form the positive transcription elongation factor b (P-TEFb). This complex regulates transcription via the phosphorylation of RNA polymerase II (RNAPolII) on Ser-2, facilitating promoter clearance and transcription elongation and thus remains an attractive therapeutic target. Herein, we have utilized classical affinity purification chemical proteomics, kinobeads assay, compressed CEllular Thermal Shift Assay (CETSA)-MS and Limited Proteolysis (LiP) to study the selectivity, target engagement and downstream mechanistic insights of a CDK9 tool compound. The above experiments highlight the value of quantitative mass spectrometry approaches to drug discovery, specifically proteome wide target identification and selectivity profiling. The approaches utilized in this study unanimously indicated that the CDK family of kinases are the main target of the compound of interest, with CDK9, showing the highest target affinity with remarkable consistency across approaches. We aim to provide guidance to the scientific community on the available chemical biology/proteomic tools to study advanced lead molecules and to highlight pros and cons of each technology while describing our findings in the context of the CDKs biology.

CETSA MS Profiling for a Comparative Assessment of FDA-Approved Antivirals Repurposed for COVID-19 Therapy Identifies TRIP13 as a Remdesivir Off-Target.

The reuse of preexisting small molecules for a novel emerging disease threat is a rapid measure to discover unknown applications for previously validated therapies. A pertinent and recent example where such a strategy could be employed is in the fight against coronavirus disease 2019 (COVID-19). Therapies designed or discovered to target viral proteins also have off-target effects on the host proteome when employed in a complex physiological environment. This study aims to assess these host cell targets for a panel of FDA-approved antiviral compounds including remdesivir, using the cellular thermal shift assay (CETSA) coupled with mass spectrometry (CETSA MS) in noninfected cells. CETSA MS is a powerful method to delineate direct and indirect interactions between small molecules and protein targets in intact cells. Biologically active compounds can induce changes in thermal stability, in their primary binding partners, and in proteins that in turn interact with the direct targets. Such engagement of host targets by antiviral drugs may contribute to the clinical effect against the virus but can also constitute a liability. We present here a comparative study of CETSA molecular target engagement fingerprints of antiviral drugs to better understand the link between off-targets and efficacy.

A Tale of Two Tails: Efficient Profiling of Protein Degraders by Specific Functional and Target Engagement Readouts.

Targeted protein degradation represents an area of great interest, potentially offering improvements with respect to dosing, side effects, drug resistance, and reaching "undruggable" proteins compared with traditional small-molecule therapeutics. A major challenge in the design and characterization of degraders acting as molecular glues is that binding of the molecule to the protein of interest (PoI) is not needed for efficient and selective protein degradation; instead, one needs to understand the interaction with the responsible ligase. Similarly, for proteasome targeting chimeras (PROTACs), understanding the binding characteristics of the PoI alone is not sufficient. Therefore, simultaneously assessing the binding to both PoI and the E3 ligase as well as the resulting degradation profile is of great value. The cellular thermal shift assay (CETSA) is an unbiased cell-based method, designed to investigate the interaction of compounds with their cellular protein targets by measuring compound-induced changes in protein thermal stability. In combination with mass spectrometry (MS), CETSA can simultaneously evaluate compound-induced changes in the stability of thousands of proteins. We have used CETSA MS to profile a number of protein degraders, including molecular glues (e.g., immunomodulatory drugs) and PROTACs, to understand mode of action and to deconvolute off-target effects in intact cells. Within the same experiment, we were able to monitor both target engagement by observing changes in protein thermal stability as well as efficacy by simultaneous assessment of protein abundances. This allowed us to correlate target engagement (i.e., binding to the PoI and ligases) and functional readout (i.e., degrader induced protein degradation).

System-wide identification and prioritization of enzyme substrates by thermal analysis.

Despite the immense importance of enzyme-substrate reactions, there is a lack of general and unbiased tools for identifying and prioritizing substrate proteins that are modified by the enzyme on the structural level. Here we describe a high-throughput unbiased proteomics method called System-wide Identification and prioritization of Enzyme Substrates by Thermal Analysis (SIESTA). The approach assumes that the enzymatic post-translational modification of substrate proteins is likely to change their thermal stability. In our proof-of-concept studies, SIESTA successfully identifies several known and novel substrate candidates for selenoprotein thioredoxin reductase 1, protein kinase B (AKT1) and poly-(ADP-ribose) polymerase-10 systems. Wider application of SIESTA can enhance our understanding of the role of enzymes in homeostasis and disease, opening opportunities to investigate the effect of post-translational modifications on signal transduction and facilitate drug discovery.

Changes in the plasma microvesicle proteome during the ovarian hyperstimulation phase of assisted reproductive technology.

The incidence of pulmonary and venous thromboembolism is increased during the first trimester of pregnancies after assisted reproductive technology (ART) compared to spontaneous conception. We previously found that haemostatic plasma variables changed but within normal limits during controlled ovarian hyperstimulation (COH) concomitant with a major increase in plasma microvesicles (MVs) and markers indicating cell activation. We now explored the proteome of these MVs. Thirty-one women undergoing ART were blood sampled at down-regulation (DR) of oestrogen and at high level stimulation (HLS) with its 10-100-fold increased oestrogen level. Samples were analysed by liquid chromatography and tandem mass spectrometry to identify and quantify the proteome. We identified 306 proteins in the MVs and 72 had changed significantly at HLS compared to DR and more than 20% of them were associated with haemostasis. Thus, proteins related to both haemostasis and complement activation altered in plasma MVs in parallel with MV activation during COH. This needs to be further explored in the clinical context.

Histone Purification Combined with High-Resolution Mass Spectrometry to Examine Histone Post-Translational Modifications and Histone Variants in Caenorhabditis elegans.

Histones are the major proteinaceous component of chromatin in eukaryotic cells and an important part of the epigenome, affecting most DNA-related events, including transcription, DNA replication, and chromosome segregation. The properties of histones are greatly influenced by their post-translational modifications (PTMs), over 200 of which are known today. Given this large number, researchers need sophisticated methods to study histone PTMs comprehensively. In particular, mass spectrometry (MS)-based approaches have gained popularity, allowing for the quantification of dozens of histone PTMs at once. Using these approaches, even the study of co-occurring PTMs and the discovery of novel PTMs become feasible. The success of MS-based approaches relies substantially on obtaining pure and well-preserved histones for analysis, which can be difficult depending on the source material. Caenorhabditis elegans has been a popular model organism to study the epigenome, but isolation of pure histones from these animals has been challenging. Here, we address this issue, presenting a method for efficient isolation of pure histone proteins from C. elegans at good yield. Further, we describe an MS pipeline optimized for accurate relative quantification of histone PTMs from C. elegans. We alkylate and tryptically digest the histones, analyze them by bottom-up MS, and then evaluate the resulting data by a C. elegans-adapted version of the software EpiProfile 2.0. Finally, we show the utility of this pipeline by determining differences in histone PTMs between C. elegans strains that age at different rates and thereby achieve very different lifespans. © 2020 The Authors. Basic Protocol 1: Large-scale growth and harvesting of synchronized C. elegans Basic Protocol 2: Nuclear preparation, histone extraction, and histone purification Basic Protocol 3: Bottom-up mass spectrometry analysis of histone PTMs and histone variants.