Ion mobility separates molecules in the gas-phase based on their physico-chemical properties, providing information about their size as collisional cross-sections. The timsTOF Pro combines trapped ion mobility with a quadrupole, collision cell and a TOF mass analyzer, to probe ions at high speeds with on-the-fly fragmentation. Here, we show that on this platform ion mobility is beneficial for cross-linking MS (XL-MS). Cross-linking reagents covalently link amino acids in proximity, resulting in peptide pairs after proteolytic digestion. These cross-linked peptides are typically present at low abundance in the background of normal peptides, which can partially be resolved by using enrichable cross-linking reagents. Even with a very efficient enrichable cross-linking reagent, like PhoX, the analysis of cross-linked peptides is still hampered by the co-enrichment of peptides connected to a partially hydrolyzed reagent – termed mono-linked peptides. For experiments aiming to uncover protein-protein interactions these are unwanted byproducts. Here, we demonstrate that gas-phase separation by ion mobility enables the separation of mono-linked peptides from cross-linked peptide pairs. A clear partition between these two classes is observed at a CCS of 500 Ų and a monoisotopic mass of 2 kDa, which can be used for targeted precursor selection. A total of 50-70% of the mono-linked peptides are prevented from sequencing, allowing the analysis to focus on sequencing the relevant cross-linked peptide pairs. In applications to both simple proteins and protein mixtures and a complete highly complex lysate this approach provides a substantial increase in detected cross-linked peptides.

Co-fractionation MS (CF-MS) is a technique with potential to characterize endogenous and unmanipulated protein complexes on an unprecedented scale. However this potential has been offset by a lack of guidelines for best-practice CF-MS data collection and analysis. To obtain such guidelines, this study thoroughly evaluates novel and published Saccharomyces cerevisiae CF-MS data sets using very high proteome coverage libraries of yeast gold standard complexes. A new method for identifying gold standard complexes in CF-MS data, Reference Complex Profiling, and the Extending 'Guilt-by-Association' by Degree (EGAD) R package are used for these evaluations, which are verified with concurrent analyses of published human data. By evaluating data collection designs, which involve fractionation of cell lysates, it is found that near-maximum recall of complexes can be achieved with fewer samples than published studies. Distributing sample collection across orthogonal fractionation methods, rather than a single high resolution data set, leads to particularly efficient recall. By evaluating 17 different similarity scoring metrics, which are central to CF-MS data analysis, it is found that two metrics rarely used in past CF-MS studies – Spearman and Kendall correlations – and the recently introduced Co-apex metric frequently maximize recall, whereas a popular metric—Euclidean distance—delivers poor recall. The common practice of integrating external genomic data into CF-MS data analysis is also evaluated, revealing that this practice may improve the precision and recall of known complexes but is generally unsuitable for predicting novel complexes in model organisms. If studying nonmodel organisms using orthologous genomic data, it is found that particular subsets of fractionation profiles (e.g. the lowest abundance quartile) should be excluded to minimize false discovery. These assessments are summarized in a series of universally applicable guidelines for precise, sensitive and efficient CF-MS studies of known complexes, and effective predictions of novel complexes for orthogonal experimental validation.

Renal Cell Carcinoma (RCC) is one of the most commonly diagnosed cancers worldwide with research efforts dramatically improving understanding of the biology of the disease. To investigate the role of the immune system in treatment-naïve clear cell Renal Cell Carcinoma (ccRCC), we interrogated the immune infiltrate in patient-matched ccRCC tumor samples, benign normal adjacent tissue (NAT) and peripheral blood mononuclear cells (PBMCs isolated from whole blood, focusing our attention on the myeloid cell infiltrate. Using flow cytometric, MS, and ExCYT analysis, we discovered unique myeloid populations in PBMCs across patient samples. Furthermore, normal adjacent tissues and ccRCC tissues contained numerous myeloid populations with a unique signature for both tissues. Enrichment of the immune cell (CD45⁺) fraction and subsequent gene expression analysis revealed a number of myeloid-related genes that were differentially expressed. These data provide evidence, for the first time, of an immunosuppressive and pro-tumorigenic role of myeloid cells in early, clinically localized ccRCC. The identification of a number of immune proteins for therapeutic targeting provides a rationale for investigation into the potential efficacy of earlier intervention with single-agent or combination immunotherapy for ccRCC.

Over the past decade, modern methods of MS (MS) have emerged that allow reliable, fast and cost-effective identification of pathogenic microorganisms. Although MALDI-TOF MS has already revolutionized the way microorganisms are identified, recent years have witnessed also substantial progress in the development of liquid chromatography (LC)-MS based proteomics for microbiological applications. For example, LC-tandem MS (LC-MS²) has been proposed for microbial characterization by means of multiple discriminative peptides that enable identification at the species, or sometimes at the strain level. However, such investigations can be laborious and time-consuming, especially if the experimental LC-MS² data are tested against sequence databases covering a broad panel of different microbiological taxa. In this proof of concept study, we present an alternative bottom-up proteomics method for microbial identification. The proposed approach involves efficient extraction of proteins from cultivated microbial cells, digestion by trypsin and LC–MS measurements. Peptide masses are then extracted from MS¹ data and systematically tested against an in silico library of all possible peptide mass data compiled in-house. The library has been computed from the UniProt Knowledgebase covering Swiss-Prot and TrEMBL databases and comprises more than 12,000 strain-specific in silico profiles, each containing tens of thousands of peptide mass entries. Identification analysis involves computation of score values derived from correlation coefficients between experimental and strain-specific in silico peptide mass profiles and compilation of score ranking lists. The taxonomic positions of the microbial samples are then determined by using the best-matching database entries. The suggested method is computationally efficient – less than 2 mins per sample - and has been successfully tested by a test set of 39 LC-MS¹ peak lists obtained from 19 different microbial pathogens. The proposed method is rapid, simple and automatable and we foresee wide application potential for future microbiological applications.

Endometrial carcinoma (EC) is the most common gynecologic malignancy in the United States, with limited effective targeted therapies. Endometrial tumors exhibit frequent alterations in protein kinases, yet only a small fraction of the kinome has been therapeutically explored. To identify kinase therapeutic avenues for EC, we profiled the kinome of endometrial tumors and normal endometrial tissues using Multiplexed Inhibitor Beads and Mass Spectrometry (MIB-MS). Our proteomics analysis identified a network of kinases overexpressed in tumors, including Serine/Arginine-Rich Splicing Factor Kinase 1 (SRPK1). Immunohistochemical (IHC) analysis of endometrial tumors confirmed MIB-MS findings and showed SRPK1 protein levels were highly expressed in endometrioid and uterine serous cancer (USC) histological subtypes. Moreover, querying large-scale genomics studies of EC tumors revealed high expression of SRPK1 correlated with poor survival. Loss-of-function studies targeting SRPK1 in an established USC cell line demonstrated SRPK1 was integral for RNA splicing, as well as cell cycle progression and survival under nutrient deficient conditions. Profiling of USC cells identified a compensatory response to SRPK1 inhibition that involved EGFR and the up-regulation of IGF1R and downstream AKT signaling. Co-targeting SRPK1 and EGFR or IGF1R synergistically enhanced growth inhibition in serous and endometrioid cell lines, representing a promising combination therapy for EC.

AAA+ ATPases constitute a large family of proteins that are involved in a plethora of cellular processes including DNA disassembly, protein degradation and protein complex disassembly. They typically form a hexametric ring-shaped structure with six subunits in a (pseudo) 6-fold symmetry. In a subset of AAA+ ATPases that facilitate protein unfolding and degradation, six subunits cooperate to translocate protein substrates through a central pore in the ring. The number and type of nucleotides in an AAA+ ATPase hexamer is inherently linked to the mechanism that underlies cooperation among subunits and couples ATP hydrolysis with substrate translocation. We conducted a native MS study of a monodispersed form of PAN, an archaeal proteasome AAA+ ATPase, to determine the number of nucleotides bound to each hexamer of the WT protein. We utilized ADP and its analogs (TNP-ADP and mant-ADP), and a nonhydrolyzable ATP analog (AMP-PNP) to study nucleotide site occupancy within the PAN hexamer in ADP- and ATP-binding states, respectively. Throughout all experiments we used a Walker A mutant (PAN^K217A) that is impaired in nucleotide binding as an internal standard to mitigate the effects of residual solvation on mass measurement accuracy and to serve as a reference protein to control for nonspecific nucleotide binding. This approach led to the unambiguous finding that a WT PAN hexamer carried – from expression host – six tightly bound ADP molecules that could be exchanged for ADP and ATP analogs. Although the Walker A mutant did not bind ADP analogs, it did bind AMP-PNP, albeit at multiple stoichiometries. We observed variable levels of hexamer dissociation and an appearance of multimeric species with the over-charged molecular ion distributions across repeated experiments. We posit that these phenomena originated during ESI process at the final stages of ESI droplet evolution.

This article provides an introduction to Fourier transform-based mass spectrometry (FTMS). The key performance characteristics of FTMS, mass accuracy and resolution, are presented in the view of how they impact the interpretation of measurements in proteomic applications. The theory and principles of operation of two types of mass analyzer, Fourier transform ion cyclotron resonance and Orbitrap, are described. Major benefits as well as limitations of FTMS technology are discussed in the context of practical sample analysis, and illustrated with examples included as figures in this text and in the accompanying slide set. Comparisons highlighting the performance differences between the two mass analyzers are made where deemed useful in assisting the user with choosing the most appropriate technology for his/her application. Recent developments of these high-performing mass spectrometers are mentioned to provide a future outlook.

This article has been withdrawn by the authors. We discovered an error after this manuscript was published as a Paper in Press. Specifically, we learned that the structures of glycans presented for the PD-L1 peptide were drawn and labeled incorrectly. We wish to withdraw this article and submit a corrected version for review.

Complex protein glycosylation occurs through biosynthetic steps in the secretory pathway that create macro- and microheterogeneity of structure and function.  Required for all life forms, glycosylation diversifies and adapts protein interactions with binding partners that underpin interactions at cell surfaces and pericellular and extracellular environments. Because these biological effects arise from heterogeneity of structure and function, it is necessary to measure their changes as part of the quest to understand nature.  Quite often, however, the assumption behind proteomics that post-translational modifications are discrete additions that can be modeled using the genome as a template does not apply to protein glycosylation.  Rather, it is necessary to quantify the glycosylation distribution at each glycosite and to aggregate this information into a population of mature glycoproteins that exist in a given biological system.  To date, mass spectrometric methods for assigning singly glycosylated peptides are well-established.  But it is necessary to quantify glycosylation heterogeneity accurately in order to gauge the alterations that occur during biological processes.  The task is to quantify the glycosylated peptide forms as accurately as possible and then apply appropriate bioinformatics algorithms to the calculation of micro- and macro-similarities.  In this review, we summarize current approaches for protein quantification as they apply to this glycoprotein similarity problem.

This review covers recent developments in glycosaminoglycan (GAG) analysis via mass spectrometry (MS). GAGs participate in a variety of biological functions, including cellular communication, wound healing, and anticoagulation, and are important targets for structural characterization. GAGs exhibit a diverse range of structural features due to the variety of O- and N-sulfation modifications and uronic acid C-5 epimerization that can occur, making their analysis a challenging target. Mass spectrometry approaches to the structure assignment of GAGs have been widely investigated, and new methodologies remain the subject of development. Advances in sample preparation, tandem MS techniques (MS/MS), on-line separations and automated analysis software have advanced the field of GAG analysis. These recent developments have led to remarkable improvements in the precision and time efficiency for the structural characterization of GAGs.

Glycosylation is a prevalent, yet heterogeneous modification with a broad range of implications in molecular biology. This heterogeneity precludes enrichment strategies that can be universally beneficial for all glycan classes. Thus, choice of enrichment strategy has profound implications on experimental outcomes. Here we review common enrichment strategies used in modern mass spectrometry (MS)-based glycoproteomic experiments, including lectins and other affinity chromatographies, hydrophilic interaction chromatography (HILIC) and its derivatives, porous graphitic carbon (PGC), reversible and irreversible chemical coupling strategies, and chemical biology tools that often leverage bioorthogonal handles. Interest in glycoproteomics continues to surge as MS instrumentation and software improve, so this review aims to help equip researchers with necessary information to choose appropriate enrichment strategies that best complement these efforts.

Histone post-translational modifications (PTMs) are one of the main mechanisms of epigenetic regulation. Dysregulation of histone PTMs leads to many human diseases, such as cancer. Due to its high-throughput, accuracy, and flexibility, mass spectrometry (MS) has emerged as a powerful tool in the epigenetic histone modification field, allowing the comprehensive and unbiased analysis of histone PTMs and chromatin-associated factors. Coupled with various techniques from molecular biology, biochemistry, chemical biology and biophysics, MS has been employed to characterize distinct aspects of histone PTMs in the epigenetic regulation of chromatin functions. In this review we will describe advancements in the field of MS that have facilitated the analysis of histone PTMs and chromatin biology.  

The early detection of pancreatic ductal adenocarcinoma is a complex clinical obstacle yet is key to improving the overall likelihood of patient survival. Current and prospective carbohydrate biomarkers CA19-9 and sTRA are sufficient for surveilling disease progression yet are not approved for delineating PDAC from other abdominal cancers and non-cancerous pancreatic pathologies. To further understand these glycan epitopes, an imaging mass spectrometry approach was utilized to assess the N-glycome of the human pancreas and pancreatic cancer in a cohort of PDAC patients represented by tissue microarrays and whole tissue sections. Orthogonally, these same tissues were characterized by multi-round immunofluorescence which defined expression of CA19-9 and sTRA as well as other lectins towards carbohydrate epitopes with the potential to improve PDAC diagnosis. These analyses revealed distinct differences not only in N-glycan spatial localization across both healthy and diseased tissues but importantly between different biomarker-categorized tissue samples. Unique sulfated bi-antennary N-glycans were detected specifically in normal pancreatic islets. N-glycans from CA19-9 expressing tissues tended to be bi-, tri- and tetra-antennary structures with both core and terminal fucose residues and bisecting N-acetylglucosamines. These N-glycans were detected in less abundance in sTRA-expressing tumor tissues, which favored tri- and tetra-antennary structures with polylactosamine extensions. Increased sialylation of N-glycans was detected in all tumor tissues. A candidate new biomarker derived from IMS was further explored by fluorescence staining with selected lectins on the same tissues. The lectins confirmed the expression of the epitopes in cancer cells and revealed different tumor-associated staining patterns between glycans with bisecting GlcNAc and those with terminal GlcNAc. Thus, the combination of lectin-IHC and IMS techniques produces more complete information for tumor classification than the individual analyses alone. These findings potentiate the development of early assessment technologies to rapidly and specifically identify PDAC in the clinic that may directly impact patient outcomes.

Urinary proteomics studies have primarily focused on identifying markers of chronic kidney disease (CKD) progression. Here, we aimed to determine urinary markers of CKD renal parenchymal injury through proteomics analysis in animal kidney tissues and cells and in the urine of patients with CKD. Label-free quantitative proteomics analysis based on liquid chromatography-tandem mass spectrometry was performed on urine samples obtained from 6 normal controls and 9, 11, and 10 patients with CKD stages 1, 3, and 5, respectively, and on kidney tissue samples from a rat CKD model by 5/6 nephrectomy. Tandem mass tag-based quantitative proteomics analysis was performed for primary cultured glomerular endothelial cells (GECs) and proximal tubular epithelial cells (PTECs) before and after inducing 24-h hypoxia injury. Upon hierarchical clustering, out of 858 differentially expressed proteins (DEPs) in the urine of CKD patients, the levels of 416 decreased and 403 increased sequentially according to the disease stage, respectively. Among 2965 DEPs across 5/6 nephrectomized and sham-operated rat kidney tissues, 86 DEPs showed same expression patterns in the urine and kidney tissue. After cross-validation with two external animal proteome datasets, 38 DEPs were organized; only 10 DEPs, including serotransferrin, gelsolin, poly ADP-ribose polymerase 1, neuroblast differentiation-associated protein AHNAK, microtubule-associated protein 4, galectin-1, protein S, thymosin beta-4, myristoylated alanine-rich C-kinase substrate, and vimentin were finalized by screening human GECs and PTECs data. Among these ten potential candidates for universal CKD marker, validation analyses for protein S and galectin-1 were conducted. Galectin-1 was observed to have a significant inverse correlation with renal function as well as higher expression in glomerulus with chronic injury than protein S. This constitutes the first multi-sample proteomics study for identifying key renal-expressed proteins associated with CKD progression. The discovered proteins represent potential markers of chronic renal cell and tissue damage and candidate contributors to CKD pathophysiology.

Members' Christmas drinks 6 December 2022 — 6:00PM TO 8:00PM Anonymous (not verified) 9 November 2022 Chatham House

Join us at 10 St James’s Square for a chance to raise a glass with fellow Chatham House members and staff.

This evening is a special opportunity to meet fellow Chatham House members and staff around the Christmas tree.

Please note this reception is open to members of Chatham House only. Regrettably, we are unable to register non-member guests.

President-elect Donald Trump announced Tuesday that Elon Musk and Vivek Ramaswamy will lead his administration's new Department of Government Efficiency, or DOGE, to end "government waste" and "slash excess regulations."

Ukraine launched a massive drone attack on Moscow overnight, the largest on the Russian capital since the Russian invasion more than two years ago, forcing three airports to divert traffic and causing casualties.

Brian Thomas Jr. and Jameson Williams are among UPI senior sports writer Alex Butler's four must-start wide receivers for Week 10 of the fantasy football campaign.

Physicists have developed a deep understanding of the fundamental laws of nature through careful observations, experiments, and precise measurements. However, what if artificial intelligence (AI) could uncover governing laws of […]

The post Archetype AI’s Newton Model Masters Physics From Raw Data appeared first on HPCwire.

A Rhode Island man with a passion for caffeine earned a Guinness World Record for his collection of 1,019 energy drink cans.

MTV's Europe Music Awards included a heartfelt tribute to late musician Liam Payne.

President-elect Donald Trump announced Tuesday that Elon Musk and Vivek Ramaswamy will lead his administration's new Department of Government Efficiency, or DOGE, to end "government waste" and "slash excess regulations."

The first step to saving nature is the rewilding of our own minds, says conservationist and former Patagonia CEO Kristine McDivitt Tompkins. With an unwavering commitment to protecting ecosystems, she and her late husband Douglas Tompkins created vast conservation parks across South America that allowed ancient flora and fauna to flourish once again. Now, she's carrying that legacy and mission forward with a bold plan to connect parks across geographic boundaries, creating a system of continental-scale wildlife corridors — before it's too late.

Marc Tucker reviews David Driscoll's new book, 'Commitment and Common Sense', and describes how the Massachusetts reforms are comparable to those in top performing education systems around the world.

Massachusetts' new performance assessment for teacher candidates helps boost readiness.

Relying on newly available data under ESSA, a local advocacy group found several districts that spend more money on wealthy students than poor students, despite the state's intentions.

The state, which earned a B-plus, led the nation in K-12 achievement rankings and outperformed other states in several key academic indicators, but fell short on funding equity.

This Quality Counts 2019 Highlights Report captures all the data you need to assess your state's performance on key educational outcomes.

The state's "Influence 100" project includes a leadership development program that will give aspiring district leaders a hands-on opportunity to work through an equity issue in their home districts.

Massachusetts is the fifth state to join the Innovative Assessment Demonstration Authority created through the Every Student Succeeds Act, which allows states to experiment with new forms of testing.

This Quality Counts 2020 Highlights Report captures all the data you need to assess your state's performance on key educational outcomes.

The work of Diné artist DY Begay, now on view at the National Museum of the American Indian, blends tradition and modernity

Fish biomass faces steep falls by end of century under high-emissions scenario

Tod Machover, an MIT professor whose classroom produced Guitar Hero and Rock Band, talks about a future in which everyone can make music

In 1536, Thomas Cromwell spotted an opportunity to enrich his master, Henry VIII, and further increase his own standing: the dissolution of the monasteries and claiming their wealth for the Crown.

Aziz Ansari | Smithsonian Magazine’s 2016 American Ingenuity Award Winner for Performing Arts The actor, comedian and author is being honored for his starring role as Dev Shah in “Master of None,” the Netflix series that he created with Alan Yang. Like the character he plays, Ansari is the son of Indian immigrant parents, and his smart, surprising take on life, love, technology and cultural identity in the United States has helped make the show “the year’s best comedy straight out of the gate,” as the New York Times put it. Among Ansari’s other accomplishments are his unforgettable portrayal of the loopy Tom Haverford on NBC’s “Parks and Recreation,” his best-selling book about dating in the internet age, Modern Romance (co-authored with Eric Klinenberg), and his blockbuster stand-up act that sold out Madison Square Garden. Read more about Ansari’s work: http://smithmag.co/jvdAaL | #IngenuityAwards And more about the American Ingenuity Awards: http://smithmag.co/77xPqy

National Portrait Gallery historian David C. Ward discusses images of Abraham Lincoln that document his life in the White House (Anika Gupta, Beth Py-Lieberman, Jesse Rhodes and Ryan Reed). Read more at http://www.smithsonianmag.com/history-archaeology/life-of-lincoln.html

Television reporter Shannon LaNier is a direct descendant of the third President of the United States. (Credit: Drew Gardner)

From the inventor's New Jersey film studio, the delight of cats doing things on film was discovered early in 1894

Read more about Thomas Jefferson: http://j.mp/w07Y8G At the turn of the 18th century, Americans learned what their leaders looked like through paintings and drawings, explains a historian at the National Portrait Gallery

Some of the brightest minds in the world gathered at Smithsonian's "The Future is Here" conference to discuss the great triumphs and future innovations in science and technology http://www.smithsonianmag.com/ideas-innovations/The-Future-is-Here.html

Jeffersons respect for the enlightenment ideals of memory, reason and imagination shaped how he organized his library (Video: Molly Roberts). Read more at https://www.smithsonianmag.com/history/on-the-hunt-for-jeffersons-lost-books-38566672/