Rosa Rakownikow Jersie-Christensen

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 β‐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.

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). DOI: http://dx.doi.org/10.7554/eLife.17092.001

Phosphoproteomics of Primary Cells Reveals Druggable Kinase Signatures in Ovarian Cancer

Summary 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.

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).

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.

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.

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

ABSTRACT 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.

Early Pleistocene enamel proteome sequences from Dmanisi resolve Stephanorhinus phylogeny.

Ancient DNA (aDNA) sequencing has enabled unprecedented reconstruction of speciation, migration, and admixture events for extinct taxa1. Outside the permafrost, however, irreversible aDNA post-mortem degradation2 has so far limited aDNA recovery within the ˜0.5 million years (Ma) time range3. Tandem mass spectrometry (MS)-based collagen type I (COL1) sequencing provides direct access to older biomolecular information4, though with limited phylogenetic use. In the absence of molecular evidence, the speciation of several Early and Middle Pleistocene extinct species remain contentious. In this study, we address the phylogenetic relationships of the Eurasian Pleistocene Rhinocerotidae5-7 using ˜1.77 million years (Ma) old dental enamel proteome sequences of a Stephanorhinus specimen from the Dmanisi archaeological site in Georgia (South Caucasus)8. Molecular phylogenetic analyses place the Dmanisi Stephanorhinus as a sister group to the woolly (Coelodonta antiquitatis) and Merck’s rhinoceros (S. kirchbergensis) clade. We show that Coelodonta evolved from an early Stephanorhinus lineage and that this genus includes at least two distinct evolutionary lines. As such, the genus Stephanorhinus is currently paraphyletic and its systematic revision is therefore needed. We demonstrate that Early Pleistocene dental enamel proteome sequencing overcomes the limits of ancient collagen- and aDNA-based phylogenetic inference, and also provides additional information about the sex and taxonomic assignment of the specimens analysed. Dental enamel, the hardest tissue in vertebrates, is highly abundant in the fossil record. Our findings reveal that palaeoproteomic investigation of this material can push biomolecular investigation further back into the Early Pleistocene.