The use of protein biomarkers as surrogates for clinical endpoints requires extensive multilevel validation including development of robust and sensitive assays for precise measurement of protein concentration. Multiple reaction monitoring (MRM) is a well-established mass-spectrometric method that can be used for reproducible protein-concentration measurements in biological specimens collected via microsampling. The dried blood spot (DBS) microsampling technique can be performed non-invasively without the expertise of a phlebotomist, and can enhance analyte stability which facilitate the application of this technique in retrospective studies while providing lower storage and shipping costs, because cold-chain logistics can be eliminated. Thus, precise, sensitive, and multiplexed methods for measuring protein concentrations in DBSs can be used for de novo biomarker discovery and for biomarker quantification or verification experiments. To achieve this goal, MRM assays were developed for multiplexed concentration measurement of proteins in DBSs.

The lower limit of quantification (LLOQ) was found to have a median total coefficient of variation (CV) of 18% for 245 proteins, whereas the median LLOQ was 5 fmol of peptide injected on column, and the median inter-day CV over 4 days for measuring endogenous protein concentration was 8%. The majority (88%) of the assays displayed parallelism, whereas the peptide standards remained stable throughout the assay workflow and after exposure to multiple freeze-thaw cycles. For 190 proteins, the measured protein concentrations remained stable in DBS stored at ambient laboratory temperature for up to 2 months. Finally, the developed assays were used to measure the concentration ranges for 200 proteins in twenty same sex, same race and age matched individuals.

Dynamic tyrosine phosphorylation is fundamental to a myriad of cellular processes. However, the inherently low abundance of tyrosine phosphorylation in the proteome and the inefficient enrichment of phosphotyrosine(pTyr)-containing peptides has led to poor pTyr peptide identification and quantitation, critically hindering researchers' ability to elucidate signaling pathways regulated by tyrosine phosphorylation in systems where cellular material is limited. The most popular approaches to wide-scale characterization of the tyrosine phosphoproteome use pTyr enrichment with pan-specific, anti-pTyr antibodies from a large amount of starting material. Methods that decrease the amount of starting material and increase the characterization depth of the tyrosine phosphoproteome while maintaining quantitative accuracy and precision would enable the discovery of tyrosine phosphorylation networks in rarer cell populations. To achieve these goals, the BOOST (Broad-spectrum Optimization Of Selective Triggering) method leveraging the multiplexing capability of tandem mass tags (TMT) and the use of pervanadate (PV) boost channels (cells treated with the broad-spectrum tyrosine phosphatase inhibitor PV) selectively increased the relative abundance of pTyr-containing peptides. After PV boost channels facilitated selective fragmentation of pTyr-containing peptides, TMT reporter ions delivered accurate quantitation of each peptide for the experimental samples while the quantitation from PV boost channels was ignored. This method yielded up to 6.3-fold boost in pTyr quantification depth of statistically significant data derived from contrived ratios, compared with TMT without PV boost channels or intensity-based label-free (LF) quantitation while maintaining quantitative accuracy and precision, allowing quantitation of over 2300 unique pTyr peptides from only 1 mg of T cell receptor-stimulated Jurkat T cells. The BOOST strategy can potentially be applied in analyses of other post-translational modifications where treatments that broadly elevate the levels of those modifications across the proteome are available.

State-of-the-art proteomics-grade mass spectrometers can measure peptide precursors and their fragments with ppm mass accuracy at sequencing speeds of tens of peptides per second with attomolar sensitivity. Here we describe a compact and robust quadrupole-orbitrap mass spectrometer equipped with a front-end High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Interface. The performance of the Orbitrap Exploris 480 mass spectrometer is evaluated in data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes in combination with FAIMS. We demonstrate that different compensation voltages (CVs) for FAIMS are optimal for DDA and DIA, respectively. Combining DIA with FAIMS using single CVs, the instrument surpasses 2500 peptides identified per minute. This enables quantification of >5000 proteins with short online LC gradients delivered by the Evosep One LC system allowing acquisition of 60 samples per day. The raw sensitivity of the instrument is evaluated by analyzing 5 ng of a HeLa digest from which >1000 proteins were reproducibly identified with 5 min LC gradients using DIA-FAIMS. To demonstrate the versatility of the instrument, we recorded an organ-wide map of proteome expression across 12 rat tissues quantified by tandem mass tags and label-free quantification using DIA with FAIMS to a depth of >10,000 proteins.

We present a technological advancement for the estimation of the affinities of Protein-Protein Interactions (PPIs) in living cells. A novel set of vectors is introduced that enables a quantitative yeast two-hybrid system based on fluorescent fusion proteins. The vectors allow simultaneous quantification of the reaction partners (Bait and Prey) and the reporter at the single-cell level by flow cytometry. We validate the applicability of this system on a small but diverse set of PPIs (eleven protein families from six organisms) with different affinities; the dissociation constants range from 117 pm to 17 μm. After only two hours of reaction, expression of the reporter can be detected even for the weakest PPI. Through a simple gating analysis, it is possible to select only cells with identical expression levels of the reaction partners. As a result of this standardization of expression levels, the mean reporter levels directly reflect the affinities of the studied PPIs. With a set of PPIs with known affinities, it is straightforward to construct an affinity ladder that permits rapid classification of PPIs with thus far unknown affinities. Conventional software can be used for this analysis. To permit automated analysis, we provide a graphical user interface for the Python-based FlowCytometryTools package.

Ion mobility can add a dimension to LC-MS based shotgun proteomics which has the potential to boost proteome coverage, quantification accuracy and dynamic range.  Required for this is suitable software that extracts the information contained in the four-dimensional (4D) data space spanned by m/z, retention time, ion mobility and signal intensity. Here we describe the ion mobility enhanced MaxQuant software, which utilizes the added data dimension. It offers an end to end computational workflow for the identification and quantification of peptides and proteins in LC-IMS-MS/MS shotgun proteomics data. We apply it to trapped ion mobility spectrometry (TIMS) coupled to a quadrupole time-of-flight (QTOF) analyzer. A highly parallelizable 4D feature detection algorithm extracts peaks which are assembled to isotope patterns. Masses are recalibrated with a non-linear m/z, retention time, ion mobility and signal intensity dependent model, based on peptides from the sample. A new matching between runs (MBR) algorithm that utilizes collisional cross section (CCS) values of MS1 features in the matching process significantly gains specificity from the extra dimension. Prerequisite for using CCS values in MBR is a relative alignment of the ion mobility values between the runs. The missing value problem in protein quantification over many samples is greatly reduced by CCS aware MBR.MS1 level label-free quantification is also implemented which proves to be highly precise and accurate on a benchmark dataset with known ground truth. MaxQuant for LC-IMS-MS/MS is part of the basic MaxQuant release and can be downloaded from http://maxquant.org.

Quantitative proteomics by mass spectrometry is widely used in biomarker research and basic biology research for investigation of phenotype level cellular events. Despite the wide application, the methodology for statistical analysis of differentially expressed proteins has not been unified. Various methods such as t-test, linear model and mixed effect models are used to define changes in proteomics experiments. However, none of these methods consider the specific structure of MS-data. Choices between methods, often originally developed for other types of data, are based on compromises between features such as statistical power, general applicability and user friendliness. Furthermore, whether to include proteins identified with one peptide in statistical analysis of differential protein expression varies between studies. Here we present DEqMS, a robust statistical method developed specifically for differential protein expression analysis in mass spectrometry data. In all datasets investigated there is a clear dependence of variance on the number of PSMs or peptides used for protein quantification. DEqMS takes this feature into account when assessing differential protein expression. This allows for a more accurate data-dependent estimation of protein variance and inclusion of single peptide identifications without increasing false discoveries. The method was tested in several datasets including E.coli proteome spike-in data, using both label-free and TMT-labelled quantification. In comparison to previous statistical methods used in quantitative proteomics, DEqMS showed consistently better accuracy in detecting altered protein levels compared to other statistical methods in both label-free and labelled quantitative proteomics data. DEqMS is available as an R package in Bioconductor.

The presentation of post-translationally modified (PTM) peptides by cell surface HLA molecules has the potential to increase the diversity of targets for surveilling T cells. Whilst immunopeptidomics studies routinely identify thousands of HLA-bound peptides from cell lines and tissue samples, in-depth analyses of the proportion and nature of peptides bearing one or more PTMs remains challenging. Here we have analyzed HLA-bound peptides from a variety of allotypes and assessed the distribution of mass spectrometry-detected PTMs, finding deamidation of asparagine or glutamine to be highly prevalent. Given that asparagine deamidation may arise either spontaneously or through enzymatic reaction, we assessed allele-specific and global motifs flanking the modified residues. Notably, we found that the N-linked glycosylation motif NX(S/T) was highly abundant across asparagine-deamidated HLA-bound peptides. This finding, demonstrated previously for a handful of deamidated T cell epitopes, implicates a more global role for the retrograde transport of nascently N-glycosylated polypeptides from the ER and their subsequent degradation within the cytosol to form HLA-ligand precursors. Chemical inhibition of Peptide:N-Glycanase (PNGase), the endoglycosidase responsible for the removal of glycans from misfolded and retrotranslocated glycoproteins, greatly reduced presentation of this subset of deamidated HLA-bound peptides. Importantly, there was no impact of PNGase inhibition on peptides not containing a consensus NX(S/T) motif. This indicates that a large proportion of HLA-I bound asparagine deamidated peptides are generated from formerly glycosylated proteins that have undergone deglycosylation via the ER-associated protein degradation (ERAD) pathway. The information herein will help train deamidation prediction models for HLA-peptide repertoires and aid in the design of novel T cell therapeutic targets derived from glycoprotein antigens.

Site-specific recombinases, such as Cre, are a widely used tool for genetic lineage tracing in the fields of developmental biology, neural science, stem cell biology, and regenerative medicine. However, nonspecific cell labeling by some genetic Cre tools remains a technical limitation of this recombination system, which has resulted in data misinterpretation and led to many controversies in the scientific community. In the past decade, to enhance the specificity and precision of genetic targeting, researchers have used two or more orthogonal recombinases simultaneously for labeling cell lineages. Here, we review the history of cell-tracing strategies and then elaborate on the working principle and application of a recently developed dual genetic lineage-tracing approach for cell fate studies. We place an emphasis on discussing the technical strengths and caveats of different methods, with the goal to develop more specific and efficient tracing technologies for cell fate mapping. Our review also provides several examples for how to use different types of DNA recombinase–mediated lineage-tracing strategies to improve the resolution of the cell fate mapping in order to probe and explore cell fate–related biological phenomena in the life sciences.

Obioma Ezekobe is a GP in an urgent care centre in Central Middlesex Hospital. She believes that the public need to be educated about the use of NHS resources, and be taught when it is appropriate to seek care. If you would like to contribute to this collection, please email a brief audio recording to voices@bmj.com or phone +44 (20) 3058 7427...

Invitation Only Research Event

Research Event

Invitation Only Research Event

Event participants

Dr Ashwini Swain, Fellow, CUTS Institute for Regulation and Competition
Glada Lahn, Senior Research Fellow, Energy, Environment and Resources, Chatham House
Dr Gareth Price, Senior Research Fellow, Asia Programme, Chatham House

Research Event

Event participants

Dr David Zweig, Chair Professor, Division of Social Science, Hong Kong University of Science and Technology
Chair: Dr Michal Meidan, Associate Fellow, Asia Programme, Chatham House

Invitation Only Research Event

Event participants

Moïse Katumbi Chapwe, Governor, Katanga Province, Democratic Republic of the Congo

4 August 2015

A new paper finds that while natural resources may provide low-income countries with a significant development opportunity, the prevailing extractives-led growth agenda is in urgent need of re-evaluation.

28 January 2016

How China responds to the challenges of resource security and sustainability, working with others, will help define its reputation as a responsible actor on the world stage in the next decade, according to a new paper.

• It is time to upgrade global resource governance
• Meaningful progress cannot be achieved without China
• China will need to be both innovative and pragmatic in its approach
• New modes of cooperation are needed
• Changes in China’s economy present opportunities and risks

Invitation Only Research Event

With immigration increasingly visible in the news and the political space in the United States and internationally, getting access to accurate, high-quality data is essential to understand immigration’s demographic effects and impacts on the economy, education and labor systems, and communities. This event marks the release of the Immigration Data Matters guide.

At this release of an updated version of the popular Immigration Data Matters guide, presenters discuss how to navigate and access the increasing number of data sources on immigration and immigrants in the United States and internationally. 

OBJECTIVE

To compare medical resource use, costs, and health utilities for 14,752 patients with type 2 diabetes who were randomized to once-weekly exenatide (EQW) or placebo in addition to usual diabetes care in the Exenatide Study of Cardiovascular Event Lowering (EXSCEL).

The ADA’s Council on Dental Practice, in collaboration with the New Dentist Committee and several external dental consultants, has developed the new Guidelines for Practice Success module on Managing Pregnancy, a resource that combines best practices with practical information to guide new dentists through the process of managing a new chapter in the lives and careers.

With the coronavirus disease now deemed a pandemic by the World Health Organization, the American Dental Association updated its webpage on the disease March 11 to include a link to frequently asked questions from member dentists covering topics such as personal protective equipment and patient communications.

The ADA is sharing with members the CDC guidelines on the use of personal protective equipment on its website dedicated to COVID-19.

Beginning March 25, the ADA is offering members two free e-books a week. Until March 31, members can access “Managing the Regulatory Environment: Best Practices” and “A Dentist’s Guide to the Law: 228 Things Every Dentist Should Know” free of charge.

Noting that we’re living through a time of uncertainty and it’s normal to feel stressed and anxious, the Center for Professional Success today posted free resources on stress management to help dentists navigate this time of uncertainty amid the COVID-19 pandemic.

The mission of Medip Academy Resources site is to provide teaching and learning materials to medical students (UG and PG) and medical professionals. Medip Academy Resources is a platform for sharing University Examination Papers, Medical Educational Materials, Practical Guides, MCQs, Problem solving etc. The resources available on this site are easily searchable and free to download. URL: http://www.medipacademy.com/resources Email: resources@medipacademy.com How to add a resource? Please share your useful resource by email to resources@medipacademy.com Happy Sharing! Dr. Bhaven Kataria Department of Pharmacology, GMERS Medical College, Sola Ahmedabad, Gujarat, India

As states work to build high-quality early childhood systems and implement the federal Workforce Innovation and Opportunity Act (WIOA) and Every Student Succeeds Act (ESSA), having detailed knowledge of the characteristics of immigrant parents can help maximize the effectiveness of programs that seek to improve child and family outcomes, as this commentary explains.

WASHINGTON — The Migration Policy Institute (MPI) today published the annual update to its data-rich article, Frequently Requested Statistics on Immigrants and Immigration in the United States, offering readers a wealth of information that can help inform understanding about an issue that is the subject of much conversation.

WASHINGTON – Even as 1.5 million immigrants and refugees are already employed in the U.S. health care system as doctors, registered nurses and pharmacists, another 263,000 foreign-born health care graduates are on the sidelines during the COVID-19 pandemic—many of them because of difficulties getting their credentials accepted by employers and licensing bodies.

In a time of critical shortages of U.S. health-care workers during the COVID-19 pandemic, retired doctors are being called back to work and medical students are graduating on a fast track. There is another important pool that could be tapped: Immigrants and refugees who have college degrees in health fields but are working in low-skilled jobs or out of work. MPI estimates 263,000 immigrants are experiencing skill underutilization and could be a valuable resource.

Police in northeast Ohio have placed a school resource officer on unpaid leave for activating a Taser to wake up a sleeping student.

Governors in several natural resource-dependent states said recently they will have to continue to cut public education funding because prices for oil and coal have not rebounded.

These tools encourage school administrators to gather as much information as possible about the students who will be entering kindergarten and the early-learning offerings in their communities.

The Knowledge Sharing for the Development of Learning Resources tutorial provides a professional step forward, a learning experience that leads to recognition that your leadership is well founded as well as ensuring that participants in the development of learning resources recognize they are contributing to an exceptional achievement.

"This report is the final report for the Orange-bellied Parrot Habitat Management and Restoration Project carried out on King Island. The report relates to the activity period starting 30th of June 2007 and finishing on the 25th of May 2008."--Introduction.

This report provides an overview of the implementation of the Water plan (Mary Basin) 2006 and summarises the findings of the assessments undertaken for the last five years. The plan supports urban water supply and a variety of industries including significant agricultural production, a growing tourism industry and fisheries.

Two Aboriginal Elders, one of whom had been removed from her family as a child, and the other who was affected by siblings and other family members being removed, provided consent to share their personal stories specifically for this resource, with the vision that people would be better informed and communication improved when delivering care. To respect their privacy, their stories have been de-identified.

The Regional Recycling Transport Assistance Package provides funding to support resource recovery and recycling in regional Queensland, helping fund the costs of transporting recyclable material from regional Queensland to facilities where it can be recovered or processed and turned into new products. Details regarding eligible applicants, projects and costs are provided in these guidelines.

Pollock, David.

Governors in several natural resource-dependent states said recently they will have to continue to cut public education funding because prices for oil and coal have not rebounded.