Essentiality of the Escherichia coli YgfZ Protein for the In Vivo Thiomethylation of Ribosomal Protein S12 by the RimO Enzyme.

Enzymes carrying Iron-Sulfur (Fe-S) clusters perform many important cellular functions and their biogenesis require complex protein machinery. In mitochondria, the IBA57 protein is essential and promotes assembly of [4Fe-4S] clusters and their insertion into acceptor proteins. YgfZ is the bacterial homologue of IBA57 but its precise role in Fe-S cluster metabolism is uncharacterized. YgfZ is needed for activity of the radical S-adenosyl methionine [4Fe-4S] cluster enzyme MiaB which thiomethylates some tRNAs. The growth of cells lacking YgfZ is compromised especially at low temperature. The RimO enzyme is homologous to MiaB and thiomethylates a conserved aspartic acid in ribosomal protein S12. To quantitate thiomethylation by RimO, we developed a bottom-up LC-MS2 analysis of total cell extracts. We show here that the in vivo activity of RimO is very low in the absence of YgfZ and independent of growth temperature. We discuss these results in relation to the hypotheses relating to the role of the auxiliary 4Fe-4S cluster in the Radical SAM enzymes that make Carbon-Sulfur bonds.

Intraglandular mesenchymal stem cell treatment induces changes in the salivary proteome of irradiated patients.

BACKGROUND: Hyposalivation and xerostomia (dry mouth), are the leading site-effects to treatment of head and neck cancer. Currently, there are no effective therapies to alleviate radiation-induced hyposalivation. Adipose tissue-derived mesenchymal stem/stromal cells (AT-MSCs) have shown potential for restoring salivary gland function. However, the mode of action is unknown. The purpose of the present study was therefore to characterize the effect of AT-MSC therapy on the salivary proteome in previously irradiated head and neck cancer patients. METHODS: Whole saliva was collected from patients with radiation-induced salivary gland hypofunction (n = 8) at baseline, and 120 days after AT-MSC treatment, and from healthy controls (n = 10). The salivary proteome was characterized with mass spectrometry based proteomics, and data was compared within the AT-MSC group (baseline versus day 120) and between AT-MSC group and healthy controls. Significance levels between groups were determined by using double-sided t-test, and visualized by means of principal component analysis, volcano plots and cluster analysis. RESULTS: Here we show that 140 human proteins are significantly differentially expressed in saliva from patients with radiation-induced hypofunction versus healthy controls. AT-MSC treatment induce a significant impact on the salivary proteome, as 99 proteins are differentially expressed at baseline vs. 120 days after treatment. However, AT-MSC treatment does not restore healthy conditions, as 212 proteins are significantly differentially expressed in saliva 120 days after AT-MSCs treatment, as compared to healthy controls. CONCLUSION: The results indicate an increase in proteins related to tissue regeneration in AT-MSCs treated patients. Our study demonstrates the impact of AT-MSCs on the salivary proteome, thereby providing insight into the potential mode of action of this novel treatment approach.

Currently, there are no effective treatments to ease dry mouth, which is a leading long-term side effect of radiation treatment for head and neck cancer. However, treatment with stem cells has shown potential for restoring function of the salivary glands, which are damaged due to radiation. We compared proteins in saliva of previously radiation-treated patients with healthy non-irradiated persons and found differences in the levels of 140 proteins. After stem cell treatment of irradiated patients, we found changes in the salivary content of proteins related to tissue regeneration. Our study demonstrates the impact of stem cell treatment on proteins in saliva, thereby providing insight into the potential mode of action of this treatment approach for patients with radiation-induced dry mouth. Consequently, this could potentially help to improve treatment of dry mouth in the future.

The Role of Proteomics in Bacterial Response to Antibiotics.

For many years, we have tried to use antibiotics to eliminate the persistence of pathogenic bacteria. However, these infectious agents can recover from antibiotic challenges through various mechanisms, including drug resistance and antibiotic tolerance, and continue to pose a global threat to human health. To design more efficient treatments against bacterial infections, detailed knowledge about the bacterial response to the commonly used antibiotics is required. Proteomics is a well-suited and powerful tool to study molecular response to antimicrobial compounds. Bacterial response profiling from system-level investigations could increase our understanding of bacterial adaptation, the mechanisms behind antibiotic resistance and tolerance development. In this review, we aim to provide an overview of bacterial response to the most common antibiotics with a focus on the identification of dynamic proteome responses, and through published studies, to elucidate the formation mechanism of resistant and tolerant bacterial phenotypes.

Author Correction: The dental proteome of Homo antecessor.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The dental proteome of Homo antecessor.

The phylogenetic relationships between hominins of the Early Pleistocene epoch in Eurasia, such as Homo antecessor, and hominins that appear later in the fossil record during the Middle Pleistocene epoch, such as Homo sapiens, are highly debated1-5. For the oldest remains, the molecular study of these relationships is hindered by the degradation of ancient DNA. However, recent research has demonstrated that the analysis of ancient proteins can address this challenge6-8. Here we present the dental enamel proteomes of H. antecessor from Atapuerca (Spain)9,10 and Homo erectus from Dmanisi (Georgia)1, two key fossil assemblages that have a central role in models of Pleistocene hominin morphology, dispersal and divergence. We provide evidence that H. antecessor is a close sister lineage to subsequent Middle and Late Pleistocene hominins, including modern humans, Neanderthals and Denisovans. This placement implies that the modern-like face of H. antecessor-that is, similar to that of modern humans-may have a considerably deep ancestry in the genus Homo, and that the cranial morphology of Neanderthals represents a derived form. By recovering AMELY-specific peptide sequences, we also conclude that the H. antecessor molar fragment from Atapuerca that we analysed belonged to a male individual. Finally, these H. antecessor and H. erectus fossils preserve evidence of enamel proteome phosphorylation and proteolytic digestion that occurred in vivo during tooth formation. Our results provide important insights into the evolutionary relationships between H. antecessor and other hominin groups, and pave the way for future studies using enamel proteomes to investigate hominin biology across the existence of the genus Homo.

Early Pleistocene enamel proteome from Dmanisi resolves Stephanorhinus phylogeny.

The sequencing of ancient DNA has enabled the reconstruction of speciation, migration and admixture events for extinct taxa1. However, the irreversible post-mortem degradation2 of ancient DNA has so far limited its recovery-outside permafrost areas-to specimens that are not older than approximately 0.5 million years (Myr)3. By contrast, tandem mass spectrometry has enabled the sequencing of approximately 1.5-Myr-old collagen type I4, and suggested the presence of protein residues in fossils of the Cretaceous period5-although with limited phylogenetic use6. In the absence of molecular evidence, the speciation of several extinct species of the Early and Middle Pleistocene epoch remains contentious. Here we address the phylogenetic relationships of the Eurasian Rhinocerotidae of the Pleistocene epoch7-9, using the proteome of dental enamel from a Stephanorhinus tooth that is approximately 1.77-Myr old, recovered from the archaeological site of Dmanisi (South Caucasus, Georgia)10. Molecular phylogenetic analyses place this Stephanorhinus as a sister group to the clade formed by the woolly rhinoceros (Coelodonta antiquitatis) and Merck's rhinoceros (Stephanorhinus kirchbergensis). We show that Coelodonta evolved from an early Stephanorhinus lineage, and that this latter genus includes at least two distinct evolutionary lines. The genus Stephanorhinus is therefore currently paraphyletic, and its systematic revision is needed. We demonstrate that sequencing the proteome of Early Pleistocene dental enamel overcomes the limitations of phylogenetic inference based on ancient collagen or DNA. Our approach also provides additional information about the sex and taxonomic assignment of other specimens from Dmanisi. Our findings reveal that proteomic investigation of ancient dental enamel-which is the hardest tissue in vertebrates11, and is highly abundant in the fossil record-can push the reconstruction of molecular evolution further back into the Early Pleistocene epoch, beyond the currently known limits of ancient DNA preservation.

Sweet google O' mine-The importance of online search engines for MS-facilitated, database-independent identification of peptide-encoded book prefaces: A EUPA YPIC challenge entry.

In the recent year, we felt like we were not truly showing our full potential in our PhD projects, and so we were very happy and excited when YPIC announced the ultimate proteomics challenge. This gave us the opportunity of showing off and procrastinating at the same time:) The challenge was to identify the amino acid sequence of 19 synthetic peptides made up from an English text and then find the book that it came from. For this task we chose to run on an Orbitrap Fusion™ Lumos™ Tribrid™ Mass Spectrometer with two different sensitive MS2 resolutions, each with both HCD and CID fragmentation consecutively. This strategy was chosen because we speculated that multiple MS2 scans at high quality would be beneficial over lower resolution, speed and quantity in the relatively sparse sample. The resulting chromatogram did not reveal 19 sharp distinct peaks and it was not clear to us where to start a manual spectra interpretation. We instead used the de novo option in the MaxQuant software and the resulting output gave us two phrases with words that were specific enough to be searched in the magic Google search engine. Google gave us the name of a very famous physicist, namely Sir Joseph John Thomson, and a reference to his book "Rays of positive electricity" from 1913. We then converted the paragraph we believed to be the right one into a FASTA format and used it with MaxQuant to do a database search. This resulted in 16 perfectly FASTA search-identified peptide sequences, one with a missing PTM and one found as a truncated version. The remaining one was identified within the MaxQuant de novo sequencing results. We thus show in this study that our workflow combining de novo spectra analysis algorithms with an online search engine is ideally suited for all applications where users want to decipher peptide-encoded prefaces of 20th century science books.

Quantitative metaproteomics of medieval dental calculus reveals individual oral health status.

The composition of ancient oral microbiomes has recently become accessible owing to advanced biomolecular methods such as metagenomics and metaproteomics, but the utility of metaproteomics for such analyses is less explored. Here, we use quantitative metaproteomics to characterize the dental calculus associated with the remains of 21 humans retrieved during the archeological excavation of the medieval (ca. 1100-1450 CE) cemetery of Tjærby, Denmark. We identify 3671 protein groups, covering 220 bacterial species and 81 genera across all medieval samples. The metaproteome profiles of bacterial and human proteins suggest two distinct groups of archeological remains corresponding to health-predisposed and oral disease-susceptible individuals, which is supported by comparison to the calculus metaproteomes of healthy living individuals. Notably, the groupings identified by metaproteomics are not apparent from the bioarchaeological analysis, illustrating that quantitative metaproteomics has the potential to provide additional levels of molecular information about the oral health status of individuals from archeological contexts.

Ancient proteins from ceramic vessels at Çatalhöyük West reveal the hidden cuisine of early farmers.

The analysis of lipids (fats, oils and waxes) absorbed within archaeological pottery has revolutionized the study of past diets and culinary practices. However, this technique can lack taxonomic and tissue specificity and is often unable to disentangle signatures resulting from the mixing of different food products. Here, we extract ancient proteins from ceramic vessels from the West Mound of the key early farming site of Çatalhöyük in Anatolia, revealing that this community processed mixes of cereals, pulses, dairy and meat products, and that particular vessels may have been reserved for specialized foods (e.g., cow milk and milk whey). Moreover, we demonstrate that dietary proteins can persist on archaeological artefacts for at least 8000 years, and that this approach can reveal past culinary practices with more taxonomic and tissue-specific clarity than has been possible with previous biomolecular techniques.

Proteomic profiling of archaeological human bone.

Ancient protein analysis provides clues to human life and diseases from ancient times. Here, we performed shotgun proteomics of human archeological bones for the first time, using rib bones from the Hitotsubashi site (AD 1657-1683) in Tokyo, called Edo in ancient times. The output data obtained were analysed using Gene Ontology and label-free quantification. We detected leucocyte-derived proteins, possibly originating from the bone marrow of the rib. Particularly prevalent and relatively high expression of eosinophil peroxidase suggests the influence of infectious diseases. This scenario is plausible, considering the overcrowding and unhygienic living conditions of the Edo city described in the historical literature. We also observed age-dependent differences in proteome profiles, particularly for proteins involved in developmental processes. Among them, alpha-2-HS-glycoprotein demonstrated a strong negative correlation with age. These results suggest that analysis of ancient proteins could provide a useful indicator of stress, disease, starvation, obesity and other kinds of physiological and pathological information.

Phosphoproteomics of Primary Cells Reveals Druggable Kinase Signatures in Ovarian Cancer.

Our understanding of the molecular determinants of cancer is still inadequate because of cancer heterogeneity. Here, using epithelial ovarian cancer (EOC) as a model system, we analyzed a minute amount of patient-derived epithelial cells from either healthy or cancerous tissues by single-shot mass-spectrometry-based phosphoproteomics. Using a multi-disciplinary approach, we demonstrated that primary cells recapitulate tissue complexity and represent a valuable source of differentially expressed proteins and phosphorylation sites that discriminate cancer from healthy cells. Furthermore, we uncovered kinase signatures associated with EOC. In particular, CDK7 targets were characterized in both EOC primary cells and ovarian cancer cell lines. We showed that CDK7 controls cell proliferation and that pharmacological inhibition of CDK7 selectively represses EOC cell proliferation. Our approach defines the molecular landscape of EOC, paving the way for efficient therapeutic approaches for patients. Finally, we highlight the potential of phosphoproteomics to identify clinically relevant and druggable pathways in cancer.

Protein sequences bound to mineral surfaces persist into deep time.

Proteins persist longer in the fossil record than DNA, but the longevity, survival mechanisms and substrates remain contested. Here, we demonstrate the role of mineral binding in preserving the protein sequence in ostrich (Struthionidae) eggshell, including from the palaeontological sites of Laetoli (3.8 Ma) and Olduvai Gorge (1.3 Ma) in Tanzania. By tracking protein diagenesis back in time we find consistent patterns of preservation, demonstrating authenticity of the surviving sequences. Molecular dynamics simulations of struthiocalcin-1 and -2, the dominant proteins within the eggshell, reveal that distinct domains bind to the mineral surface. It is the domain with the strongest calculated binding energy to the calcite surface that is selectively preserved. Thermal age calculations demonstrate that the Laetoli and Olduvai peptides are 50 times older than any previously authenticated sequence (equivalent to ~16 Ma at a constant 10°C).

Metaproteomics of saliva identifies human protein markers specific for individuals with periodontitis and dental caries compared to orally healthy controls.

BACKGROUND: The composition of the salivary microbiota has been reported to differentiate between patients with periodontitis, dental caries and orally healthy individuals. To identify characteristics of diseased and healthy saliva we thus wanted to compare saliva metaproteomes from patients with periodontitis and dental caries to healthy individuals. METHODS: Stimulated saliva samples were collected from 10 patients with periodontitis, 10 patients with dental caries and 10 orally healthy individuals. The proteins in the saliva samples were subjected to denaturing buffer and digested enzymatically with LysC and trypsin. The resulting peptide mixtures were cleaned up by solid-phase extraction and separated online with 2 h gradients by nano-scale C18 reversed-phase chromatography connected to a mass spectrometer through an electrospray source. The eluting peptides were analyzed on a tandem mass spectrometer operated in data-dependent acquisition mode. RESULTS: We identified a total of 35,664 unique peptides from 4,161 different proteins, of which 1,946 and 2,090 were of bacterial and human origin, respectively. The human protein profiles displayed significant overexpression of the complement system and inflammatory markers in periodontitis and dental caries compared to healthy controls. Bacterial proteome profiles and functional annotation were very similar in health and disease. CONCLUSIONS: Overexpression of proteins related to the complement system and inflammation seems to correlate with oral disease status. Similar bacterial proteomes in healthy and diseased individuals suggests that the salivary microbiota predominantly thrives in a planktonic state expressing no disease-associated characteristics of metabolic activity.

miR-625-3p regulates oxaliplatin resistance by targeting MAP2K6-p38 signalling in human colorectal adenocarcinoma cells.

Oxaliplatin resistance in colorectal cancers (CRC) is a major medical problem, and predictive markers are urgently needed. Recently, miR-625-3p was reported as a promising predictive marker. Herein, we show that miR-625-3p functionally induces oxaliplatin resistance in CRC cells, and identify the signalling networks affected by miR-625-3p. We show that the p38 MAPK activator MAP2K6 is a direct target of miR-625-3p, and, accordingly, is downregulated in non-responder patients of oxaliplatin therapy. miR-625-3p-mediated resistance is reversed by anti-miR-625-3p treatment and ectopic expression of a miR-625-3p insensitive MAP2K6 variant. In addition, reduction of p38 signalling by using siRNAs, chemical inhibitors or expression of a dominant-negative MAP2K6 protein induces resistance to oxaliplatin. Transcriptome, proteome and phosphoproteome profiles confirm inactivation of MAP2K6-p38 signalling as one likely mechanism of oxaliplatin resistance. Our study shows that miR-625-3p induces oxaliplatin resistance by abrogating MAP2K6-p38-regulated apoptosis and cell cycle control networks, and corroborates the predictive power of miR-625-3p.

Conformation-specific anti-Mad2 monoclonal antibodies for the dissection of checkpoint signaling.

The spindle assembly checkpoint (SAC) ensures accurate chromosome segregation during mitosis by delaying the activation of the anaphase-promoting complex/cyclosome (APC/C) in response to unattached kinetochores. The Mad2 protein is essential for a functional checkpoint because it binds directly to Cdc20, the mitotic co-activator of the APC/C, thereby inhibiting progression into anaphase. Mad2 exists in at least 2 different conformations, open-Mad2 (O-Mad2) and closed-Mad2 (C-Mad2), with the latter representing the active form that is able to bind Cdc20. Our ability to dissect Mad2 biology in vivo is limited by the absence of monoclonal antibodies (mAbs) useful for recognizing the different conformations of Mad2. Here, we describe and extensively characterize mAbs specific for either O-Mad2 or C-Mad2, as well as a pan-Mad2 antibody, and use these to investigate the different Mad2 complexes present in mitotic cells. Our antibodies validate current Mad2 models but also suggest that O-Mad2 can associate with checkpoint complexes, most likely through dimerization with C-Mad2. Furthermore, we investigate the makeup of checkpoint complexes bound to the APC/C, which indicate the presence of both Cdc20-BubR1-Bub3 and Mad2-Cdc20-BubR1-Bub3 complexes, with Cdc20 being ubiquitinated in both. Thus, our defined mAbs provide insight into checkpoint signaling and provide useful tools for future research on Mad2 function and regulation.

Simple and Reproducible Sample Preparation for Single-Shot Phosphoproteomics with High Sensitivity.

The traditional sample preparation workflow for mass spectrometry (MS)-based phosphoproteomics is time consuming and usually requires multiple steps, e.g., lysis, protein precipitation, reduction, alkylation, digestion, fractionation, and phosphopeptide enrichment. Each step can introduce chemical artifacts, in vitro protein and peptide modifications, and contaminations. Those often result in sample loss and affect the sensitivity, dynamic range and accuracy of the mass spectrometric analysis. Here we describe a simple and reproducible phosphoproteomics protocol, where lysis, denaturation, reduction, and alkylation are performed in a single step, thus reducing sample loss and increasing reproducibility. Moreover, unlike standard cell lysis procedures the cell harvesting is performed at high temperatures (99 °C) and without detergents and subsequent need for protein precipitation. Phosphopeptides are enriched using TiO2 beads and the orbitrap mass spectrometer is operated in a sensitive mode with higher energy collisional dissociation (HCD).

Rapid and deep proteomes by faster sequencing on a benchtop quadrupole ultra-high-field Orbitrap mass spectrometer.

Shotgun proteomics is a powerful technology for global analysis of proteins and their post-translational modifications. Here, we investigate the faster sequencing speed of the latest Q Exactive HF mass spectrometer, which features an ultra-high-field Orbitrap mass analyzer. Proteome coverage is evaluated by four different acquisition methods and benchmarked across three generations of Q Exactive instruments (ProteomeXchange data set PXD001305). We find the ultra-high-field Orbitrap mass analyzer to be capable of attaining a sequencing speed above 20 Hz, and it routinely exceeds 10 peptide spectrum matches per second or up to 600 new peptides sequenced per gradient minute. We identify 4400 proteins from 1 µg of HeLa digest using a 1 h gradient, which is an approximately 30% improvement compared to that with previous instrumentation. In addition, we show that very deep proteome coverage can be achieved in less than 24 h of analysis time by offline high-pH reversed-phase peptide fractionation, from which we identify more than 140,000 unique peptide sequences. This is comparable to state-of-the-art multiday, multienzyme efforts. Finally, the acquisition methods are evaluated for single-shot phosphoproteomics, where we identify 7600 unique HeLa phosphopeptides in one gradient hour and find the quality of fragmentation spectra to be more important than quantity for accurate site assignment.

Proteome reference map of Lactobacillus acidophilus NCFM and quantitative proteomics towards understanding the prebiotic action of lactitol.

Lactobacillus acidophilus NCFM is a probiotic bacterium adapted to survive in the gastrointestinal tract and with potential health benefits to the host. Lactitol is a synthetic sugar alcohol used as a sugar replacement in low calorie foods and selectively stimulating growth of L. acidophilus NCFM. In the present study the whole-cell extract proteome of L. acidophilus NCFM grown on glucose until late exponential phase was resolved by 2-DE (pH 3-7). A total of 275 unique proteins assigned to various physiological processes were identified from 650 spots. Differential 2-DE (DIGE) (pH 4-7) of L. acidophilus NCFM grown on glucose and lactitol, revealed 68 spots with modified relative intensity. Thirty-two unique proteins were identified in 41 of these spots changing 1.6-12.7-fold in relative abundance by adaptation of L. acidophilus NCFM to growth on lactitol. These proteins included ß-galactosidase small subunit, galactokinase, galactose-1-phosphate uridylyltransferase and UDP-glucose-4-epimerase, which all are potentially involved in lactitol metabolism. This first comprehensive proteome analysis of L. acidophilus NCFM provides insights into protein abundance changes elicited by the prebiotic lactitol.