Phosphorylation is one of the most relevant and ubiquitous post-translational modifications. Despite its relevance, the analysis of protein phosphorylation has been revealed as one of the most ...challenging tasks due to its highly dynamic nature and low stoichiometry. However, the development and introduction of new analytical methods are modifying rapidly and substantially this field. Especially important has been the introduction of more sensitive and specific methods for phosphoprotein and phosphopeptide purification as well as the use of more sensitive and accurate MS-based analytical methods. The integration of both approaches has enabled large-scale phosphoproteome studies to be performed, an unimaginable task few years ago. Additionally, methods originally developed for differential proteomics have been adapted making the study of the highly dynamic nature of protein phosphorylation feasible. This review aims at offering an overview on the most frequently used methods in phosphoprotein and phosphopeptide enrichment as well as on the most recent MS-based analysis strategies. Current strategies for quantitative phosphoproteomics and the study of the dynamics of protein phosphorylation are highlighted.
Cancer cells possess aberrant proteomes that can arise by the disruption of genes involved in physiological protein degradation. Here we demonstrate the presence of promoter CpG island ...hypermethylation-linked inactivation of DERL3 (Derlin-3), a key gene in the endoplasmic reticulum-associated protein degradation pathway, in human tumours. The restoration of in vitro and in vivo DERL3 activity highlights the tumour suppressor features of the gene. Using the stable isotopic labelling of amino acids in cell culture workflow for differential proteome analysis, we identify SLC2A1 (glucose transporter 1, GLUT1) as a downstream target of DERL3. Most importantly, SLC2A1 overexpression mediated by DERL3 epigenetic loss contributes to the Warburg effect in the studied cells and pinpoints a subset of human tumours with greater vulnerability to drugs targeting glycolysis.
Persistent organic pollutants (POPs) bioaccumulate in biota, have long residence times in the environment, and potential for long range atmospheric transport. Here, we show the first measurements of ...legacy POPs in the atmosphere of the Antarctic Plateau from 73° South to the South Pole. Samples were taken using passive samplers. The amount of polychlorinated biphenyls (as ∑26PCBs) per sample ranged from 0.8 ng to 26 ng. The mass per sample of hexachlorobenzene (HCB) and γ-hexachlorocyclohexane (γ-HCH) in the gas-phase ranged from 0.67 ng to 2.7 ng and from non-detected to 2.6 ng, respectively. The lowest amounts of POPs were observed at the South Pole. This work shows that POPs have also reached the remotest region of Earth from primary sources. The assessment of the air mass back trajectories and current knowledge of atmospheric circulation over the Antarctic continent suggests that POPs reach the Antarctic Plateau by subduction of air masses from the free troposphere.
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•The occurrence of POPs in the Antarctic Plateau is demonstrated for the first time.•The mass per sample of PCBs, HCHs and HCB show a minimum at the South Pole.•POPs reach the Antarctic Plateau by atmospheric transport from the free troposphere.
Objective To describe the goals of the Proteomics Standards Initiative (PSI) of the Human Proteome Organization, the methods that the PSI has employed to create data standards, the resulting output ...of the PSI, lessons learned from the PSI’s evolution, and future directions and synergies for the group.
Materials and Methods The PSI has 5 categories of deliverables that have guided the group. These are minimum information guidelines, data formats, controlled vocabularies, resources and software tools, and dissemination activities. These deliverables are produced via the leadership and working group organization of the initiative, driven by frequent workshops and ongoing communication within the working groups. Official standards are subjected to a rigorous document process that includes several levels of peer review prior to release.
Results We have produced and published minimum information guidelines describing what information should be provided when making data public, either via public repositories or other means. The PSI has produced a series of standard formats covering mass spectrometer input, mass spectrometer output, results of informatics analysis (both qualitative and quantitative analyses), reports of molecular interaction data, and gel electrophoresis analyses. We have produced controlled vocabularies that ensure that concepts are uniformly annotated in the formats and engaged in extensive software development and dissemination efforts so that the standards can efficiently be used by the community.
Conclusion In its first dozen years of operation, the PSI has produced many standards that have accelerated the field of proteomics by facilitating data exchange and deposition to data repositories. We look to the future to continue developing standards for new proteomics technologies and workflows and mechanisms for integration with other omics data types. Our products facilitate the translation of genomics and proteomics findings to clinical and biological phenotypes. The PSI website can be accessed at http://www.psidev.info.
The range of heterogeneous approaches available for quantifying protein abundance via mass spectrometry (MS)11The abbreviations used are:CVcontrolled vocabularyiTRAQisobaric tag for relative and ...absolute quantitationLCliquid chromatographyMIAPEMinimum Information about a Proteomics ExperimentMSmass spectrometryPSIProteomics Standards InitiativeSILACstable isotope labeling by amino acids in cell cultureXSDXML Schema Definition. leads to considerable challenges in modeling, archiving, exchanging, or submitting experimental data sets as supplemental material to journals. To date, there has been no widely accepted format for capturing the evidence trail of how quantitative analysis has been performed by software, for transferring data between software packages, or for submitting to public databases. In the context of the Proteomics Standards Initiative, we have developed the mzQuantML data standard. The standard can represent quantitative data about regions in two-dimensional retention time versus mass/charge space (called features), peptides, and proteins and protein groups (where there is ambiguity regarding peptide-to-protein inference), and it offers limited support for small molecule (metabolomic) data. The format has structures for representing replicate MS runs, grouping of replicates (for example, as study variables), and capturing the parameters used by software packages to arrive at these values. The format has the capability to reference other standards such as mzML and mzIdentML, and thus the evidence trail for the MS workflow as a whole can now be described. Several software implementations are available, and we encourage other bioinformatics groups to use mzQuantML as an input, internal, or output format for quantitative software and for structuring local repositories. All project resources are available in the public domain from the HUPO Proteomics Standards Initiative http://www.psidev.info/mzquantml.
In order to identify new regulators of the phosphate (Pi) starvation signaling pathway in plants, we analyzed variation in the abundance of nuclear-enriched proteins isolated from Arabidopsis roots ...that depends on Pi supply. We used 2-D fluorescence difference gel electrophoresis and MALDI-TOF/TOF techniques for proteome separation, visualization and relative protein abundance quantification and identification. Pi-controlled proteins identified in our analysis included components of the chromatin remodeling, DNA replication, and mRNA splicing machineries. In addition, by combining Pi starvation conditions with proteasome inhibitor treatments, we characterized the role of the ubiquitin–proteasome system, a major mechanism for targeted protein degradation in eukaryotes, in the control of the stability of Pi-responsive proteins. Among Pi-responsive proteins, the histone chaperone NAP1;2 was selected for further characterization, and was shown to display differential nucleo-cytoplasmic accumulation in response to Pi deprivation. We also found that mutants affecting three members of the NAP1 family accumulate lower Pi levels and display reduced expression of Pi starvation-inducible genes, reflecting a potential regulatory role for these chromatin-remodeling proteins in Pi homeostasis.
In this study, we explore the feasibility of nuclear proteomics to identify regulatory proteins and ubiquitin–proteasome targets within a specific stress signaling pathway in plants, in our case phosphate starvation signaling in Arabidopsis. It will be of interest for researchers involved in the dissection of any signaling pathway in plants, in particular those with an interest in the ubiquitin–proteasome functions, and for the plant nutrition community.
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•Nuclear proteome variation under phosphate (Pi) starvation is analyzed in Arabidopsis.•New potential regulators of phosphate stress signaling are identified.•Proteins regulated by both Pi supply and the ubiquitin–proteasome system are found.•Histone chaperone NAP1;2 is a potential novel regulator of Pi homeostasis.
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