Higher-order structural organization of the mitochondrial proteome charted by in situ cross-linking mass spectrometry

Mitochondria are densely packed with proteins, of which most are involved physically or more transiently in protein-protein interactions (PPIs). Mitochondria host among others all enzymes of the Krebs cycle and the oxidative phosphorylation (OXPHOS) pathway and are foremost associated with cellular bioenergetics (1, 2). However, mitochondria are also important contributors to apoptotic cell death (3) and contain their own genome (4) indicating that they play additionally an eminent role in processes beyond bioenergetics (5). Despite intense efforts in identifying and characterizing mitochondrial protein complexes by structural biology and proteomics techniques, many PPIs have remained elusive. Several of these (membrane embedded) PPIs are less stable in-vitro hampering their characterization by most contemporary methods in structural biology. Particularly in these cases, cross-linking mass spectrometry (XL-MS) has proven valuable for the in-depth characterization of mitochondrial protein complexes in situ. Here, we highlight experimental strategies for the analysis of proteome-wide protein-protein interactions in mitochondria using XL-MS. We showcase the ability of in situ XL-MS as a tool to map sub-organelle interactions and topologies, and aid in refining structural models of protein complexes. We describe some of the most recent technological advances in XL-MS that may benefit the in situ characterization of PPIs even further, especially when combined with electron microscopy and structural modelling

Native mass spectrometry:what is in the name?

Electrospray ionization mass spectrometry (ESI-MS) is nowadays one of the cornerstones of biomolecular mass spectrometry and proteomics. Advances in sample preparation and mass analyzers have enabled researchers to extract much more information from biological samples than just the molecular weight. In particular, relevant for structural biology, noncovalent protein–protein and protein–ligand complexes can now also be analyzed by MS. For these types of analyses, assemblies need to be retained in their native quaternary state in the gas phase. This initial small niche of biomolecular mass spectrometry, nowadays often referred to as “native MS,” has come to maturation over the last two decades, with dozens of laboratories using it to study mostly protein assemblies, but also DNA and RNA-protein assemblies, with the goal to define structure–function relationships. In this perspective, we describe the origins of and (re)define the term native MS, portraying in detail what we meant by “native MS,” when the term was coined and also describing what it does (according to us) not entail. Additionally, we describe a few examples highlighting what native MS is, showing its successes to date while illustrating the wide scope this technology has in solving complex biological questions. [Figure: see text

The background from single electromagnetic subcascades for a stereo system of air Cherenkov telescopes

The MAGIC experiment, a very large Imaging Air Cherenkov Telescope (IACT) with sensitivity to low energy (E < 100 GeV) VHE gamma rays, has been operated since 2004. It has been found that the gamma/hadron separation in IACTs becomes much more difficult below 100 GeV [Albert et al 2008] A system of two large telescopes may eventually be triggered by hadronic events containing Cherenkov light from only one electromagnetic subcascade or two gamma subcascades, which are products of the single pi^0 decay. This is a possible reason for the deterioration of the experiment's sensitivity below 100 GeV. In this paper a system of two MAGIC telescopes working in stereoscopic mode is studied using Monte Carlo simulations. The detected images have similar shapes to that of primary gamma-rays and they have small sizes (mainly below 400 photoelectrons (p.e.)) which correspond to an energy of primary gamma-rays below 100 GeV. The background from single or two electromagnetic subcascdes is concentrated at energies below 200 GeV. Finally the number of background events is compared to the number of VHE gamma-ray excess events from the Crab Nebula. The investigated background survives simple cuts for sizes below 250 p.e. and thus the experiment's sensitivity deteriorates at lower energies.Comment: 15 pages, 7 figures, published in Journ.of Phys.

Активизация инвестиционной деятельности коммерческими банками

В статье рассмотрены основные вопросы активизации инвестиционной деятельности коммерческими банками Украины.У статті розглядаються основні питання активізації інвестиційної діяльності комерційними банками України.The main questions of the investment activity by the commercial banks of Ukraine were descried in the article

Агро- и микроклиматическая оценка условий формирования урожайности винограда

Проблема агроклиматического обеспечения аграрного сектора экономики остается важнейшей задачей агрометеорологов и направлена на оценку агроклиматических ресурсов территорий с целью оптимизации размещения сельскохозяйственных культур как условия повышения продуктивности и стабильности отрасли. Актуальность исследований в этом направлении обусловлена отсутствием информации о реально достижимой урожайности отдельных сельскохозяйственных культур как в региональном разрезе, так и на локальном уровне.Проблема агрокліматічеського забезпечення аграрного сектора економіки залишається найважливішою задачею агрометеорології і направлена на оцінку агрокліматічеськіх ресурсів територій з метою оптимізації розміщення сільськогосподарських культур як умови підвищення продуктивності і стабільності галузі. Актуальність досліджень в цьому напрямі обумовлена відсутністю інформації про реально досяжну врожайність окремих сільськогосподарських культур як в регіональному розрізі, так і на локальному рівні

Інформаційно-аналітична підтримка прийняття управлінських рішень

Розглянуто питання інформаційно-аналітичної підтримки прийняття управлінських рішень, застосування сучасних інформаційних технологій і організації інформаційного забезпечення аналітичної обробки інформації у корпоративних організаціях.Рассмотрены вопросы информационно-аналитической поддержки принятия управленческих решений, применения современных информационных технологий и организации информационного обеспечения аналитической обработки информации в корпоративных организациях.The questions of information-analytical support of administrative decision-making, application of modern information technologies and organization of information maintenance of analytical processing of the information in corporate organizations are considered

Підготовка адвокатом позовної заяви до суду

Аналізуються питання, пов’язані із підготовкою адвокатом позовної заяви до суду.Анализируется вопросы связаны с подготовкой адвокатом искового заявления в суд.The question connected with preparation of the point of claim to the court by the lawyer is analysed

Direct Monitoring of Protein O-GlcNAcylation by High-Resolution Native Mass Spectrometry

O-GlcNAcylation is one of the most abundant metazoan nuclear-cytoplasmic post-translational modifications. Proteins modified by O-GlcNAc play key cellular roles in signaling, transcription, metabolism, and cell division. Mechanistic studies on protein O-GlcNAcylation are hampered by the lack of methods that can simultaneously quantify O-GlcNAcylation, determine its stoichiometry, and monitor O-GlcNAcylation kinetics. Here, we demonstrate that high-resolution native mass spectrometry can be employed to monitor the small mass shifts induced by modification by O-GlcNAc on two known protein substrates, CK2α and TAB1, without the need for radioactive labeling or chemoenzymatic tagging using large mass tags. Limited proteolysis enabled further localization of the O-GlcNAc sites. In peptide-centric MS analysis, the O-GlcNAc moiety is known to be easily lost. In contrast, we demonstrate that the O-GlcNAc is retained under native MS conditions, enabling precise quantitative analysis of stoichiometry and O-GlcNAcylation kinetics. Together, the data highlight that high resolution native MS may provide an alternative tool to monitor kinetics on one of the most labile of protein post-translational modifications, in an efficient, reliable, and quantitative manner

Recent advancements in mass spectrometry-based tools to investigate newly synthesized proteins

Tight regulation of protein translation drives the proteome to undergo changes under influence of extracellular or intracellular signals. Despite mass spectrometry-based proteomics being an excellent method to study differences in protein abundance in complex proteomes, analyzing minute or rapid changes in protein synthesis and abundance remains challenging. Therefore, several dedicated techniques to directly detect and quantify newly synthesized proteins have been developed, notably puromycin-based, bio-orthogonal noncanonical amino acid tagging-based, and stable isotope labeling by amino acids in cell culture-based methods, combined with mass spectrometry. These techniques have enabled the investigation of perturbations, stress, or stimuli on protein synthesis. Improvements of these methods are still necessary to overcome various remaining limitations. Recent improvements include enhanced enrichment approaches and combinations with various stable isotope labeling techniques, which allow for more accurate analysis and comparison between conditions on shorter timeframes and in more challenging systems. Here, we aim to review the current state in this field

Targeted LC-ESI-MS2 characterization of human milk oligosaccharide diversity at 6 to 16 weeks post-partum reveals clear staging effects and distinctive milk groups

Many molecular components in human milk (HM), such as human milk oligosaccharides (HMOs), assist in the healthy development of infants. It has been hypothesized that the functional benefits of HM may be highly dependent on the abundance and individual fine structures of contained HMOs and that distinctive HM groups can be defined by their HMO profiles. However, the structural diversity and abundances of individual HMOs may also vary between milk donors and at different stages of lactations. Improvements in efficiency and selectivity of quantitative HMO analysis are essential to further expand our understanding about the impact of HMO variations on healthy early life development. Hence, we applied here a targeted, highly selective, and semi-quantitative LC-ESI-MS2 approach by analyzing 2 × 30 mature human milk samples collected at 6 and 16 weeks post-partum. The analytical approach covered the most abundant HMOs up to hexasaccharides and, for the first time, also assigned blood group A and B tetrasaccharides. Principal component analysis (PCA) was employed and allowed for automatic grouping and assignment of human milk samples to four human milk groups which are related to the maternal Secretor (Se) and Lewis (Le) genotypes. We found that HMO diversity varied significantly between these four HM groups. Variations were driven by HMOs being either dependent or independent of maternal genetic Se and Le status. We found preliminary evidence for an additional HM subgroup within the Se- and Le-positive HM group I. Furthermore, the abundances of 6 distinct HMO structures (including 6'-SL and 3-FL) changed significantly with progression of lactation. Graphical abstract