Tracking Pathogen Infections by Time‐Resolved Chemical Proteomics

Studying the dynamic interaction between host cells and pathogen is vital but remains technically challenging. We describe herein a time‐resolved chemical proteomics strategy enabling host and pathogen temporal interaction profiling (HAPTIP) for tracking the entry of a pathogen into the host cell. A novel multifunctional chemical proteomics probe was introduced to label living bacteria followed by in vivo crosslinking of bacteria proteins to their interacting host‐cell proteins at different time points initiated by UV for label‐free quantitative proteomics analysis. We observed over 400 specific interacting proteins crosslinked with the probe during the formation of Salmonella‐containing vacuole (SCV). This novel chemical proteomics approach provides a temporal interaction profile of host and pathogen in high throughput and would facilitate better understanding of the infection process at the molecular level.     

化学生物学新前沿——化学蛋白质组学

随着包括人类在内的主要模式生物的基因组计划的完成,生命科学的研究重心转向蛋白质组的研究--在对应基因组的整体蛋白质水平上系统研究调控细胞生命活动的蛋白质.化学蛋白质组学是化学生物学在后基因组时代的最新发展:化学蛋白质组学利用化学小分子为工具和手段,以基于靶蛋白质功能的新战略探测体内蛋白质组,是新一代的功能蛋白质组学.本文综述了化学蛋白质组学的最新进展、有关技术及其在生物医学和药物研发等方面的应用,并对化学蛋白质组学的发展趋势和前景进行了讨论.     

Clinical Amyloid Typing by Proteomics: Performance Evaluation and Data Sharing between Two Centres

Amyloidosis is a relatively rare human disease caused by the deposition of abnormal protein fibres in the extracellular space of various tissues, impairing their normal function. Proteomic analysis of patients’ biopsies, developed by Dogan and colleagues at the Mayo Clinic, has become crucial for clinical diagnosis and for identifying the amyloid type. Currently, the proteomic approach is routinely used at National Amyloidosis Centre (NAC, London, UK) and Istituto di Tecnologie Biomediche-Consiglio Nazionale delle Ricerche (ITB-CNR, Milan, Italy). Both centres are members of the European Proteomics Amyloid Network (EPAN), which was established with the aim of sharing and discussing best practice in the application of amyloid proteomics. One of the EPAN’s activities was to evaluate the quality and the confidence of the results achieved using different software and algorithms for protein identification. In this paper, we report the comparison of proteomics results obtained by sharing NAC proteomics data with the ITB-CNR centre. Mass spectrometric raw data were analysed using different software platforms including Mascot, Scaffold, Proteome Discoverer, Sequest and bespoke algorithms developed for an accurate and immediate amyloid protein identification. Our study showed a high concordance of the obtained results, suggesting a good accuracy of the different bioinformatics tools used in the respective centres. In conclusion, inter-centre data exchange is a worthwhile approach for testing and validating the performance of software platforms and the accuracy of results, and is particularly important where the proteomics data contribute to a clinical diagnosis.     

Development of a PDEδ‐Targeting PROTACs that Impair Lipid Metabolism

The prenyl‐protein chaperone PDEδ modulates the localization of lipidated proteins in the cell, but current knowledge about its biological function is limited. Small‐molecule inhibitors that target the PDEδ prenyl‐binding site have proven invaluable in the analysis of biological processes mediated by PDEδ, like KRas cellular trafficking. However, allosteric inhibitor release from PDEδ by the Arl2/3 GTPases limits their application. We describe the development of new proteolysis‐targeting chimeras (PROTACs) that efficiently and selectively reduce PDEδ levels in cells through induced proteasomal degradation. Application of the PDEδ PROTACs increased sterol regulatory element binding protein (SREBP)‐mediated gene expression of enzymes involved in lipid metabolism, which was accompanied by elevated levels of cholesterol precursors. This finding for the first time demonstrates that PDEδ function plays a role in the regulation of enzymes of the mevalonate pathway.     

Simple correction improving long‐term reproducibility of HPLC–MS

Chromatographic peak areas in long series of high‐performance liquid chromatography–MS experiments often vary, which decrease reproducibility and may cause bias in the results. It was found that the sensitivity of various components change differently; in our case, variability is in the order of 20–40%, and it is most likely due to changing conditions in electrospray ionization (ESI). The most often used peak area correction methods do not take this effect into account. The change in peak areas can be well described by a polynomial function; we found that a fourth‐order polynomial is most often suitable. We suggest a simple correction algorithm based on polynomial fitting. When the experiments were inherently well reproducible, this correction improved reproducibility from 12% to 3% (on average for various components). When random errors were larger, this improvement was less significant (15% to 12% in nano‐ESI) but nevertheless essential in order to avoid possible bias in the results. Copyright © 2015 John Wiley & Sons, Ltd.     

Over 10 000 Peptide Identifications from the HeLa Proteome by Using Single‐Shot Capillary Zone Electrophoresis Combined with Tandem Mass Spectrometry

Capillary zone electrophoresis (CZE)–tandem mass spectrometry (MS/MS) has recently attracted attention as a tool for shotgun proteomics. However, its performance for this analysis has so far fallen far below that of reversed‐phase liquid chromatography (RPLC)–MS/MS. The use of a CZE method with a wide separation window (up to 90 min) and high peak capacity (ca. 300) is reported. This method was coupled to an Orbitrap Fusion mass spectrometer through an electrokinetically pumped sheath‐flow interface for the analysis of complex proteome digests. Single‐shot CZE–MS/MS lead to the identification of over 10 000 peptides and 2100 proteins from a HeLa cell proteome digest in approximately 100 min. This performance is nearly an order of magnitude better than earlier CZE studies and is within a factor of two to four of the state‐of‐the‐art nano ultrahigh‐pressure LC system.     

Peptide prefractionation is essential for proteomic approaches employing multiple‐reaction monitoring of fruit proteomic research

Off‐gel? IEF has become a popular tool in proteomics research to fractionate peptides or proteins. We conducted a detailed investigation on the fruit proteomics of apple, banana, and strawberry fruit employing Off‐gel? electrophoresis (OGE) as a crucial step to improve the proteome coverage and quantitative proteomic workflows including multiple‐reaction monitoring (MRM). We provide technical details concerning the application of Off‐gel?IEF, nano‐LC–MS detection, and MRM optimization and analysis. Our results demonstrated that the application of OGE is an effective method for peptide fractionation and increased significantly the number of proteins identified by at least ten times, with more total peptides detected and collected. Furthermore, we developed a protocol combining OGE and MRM studies to identify and quantitatively investigate monodehydroascorbate reductase, a key enzyme in the redox and antioxidant system of apple fruit during fruit ripening. Using this method, the quantitative changes in this protein during ripening and in response to ethylene treatment was investigated. Our results provide direct and comprehensive evidence demonstrating the benefits of OGE and its application for both shotgun and quantitative proteomics research.     

ANOVA–principal component analysis and ANOVA–simultaneous component analysis: a comparison

ANOVA–simultaneous component analysis (ASCA) is a recently developed tool to analyze multivariate data. In this paper, we enhance the explorative capability of ASCA by introducing a projection of the observations on the principal component subspace to visualize the variation among the measurements. We compare the significance of experimental effects for ASCA and ANOVA–principal component analysis (PCA), a similar tool to explore multivariate data, by using permutation tests. Furthermore, we quantify the quality of the loadings estimate obtained with ASCA and compare this with the loadings estimate obtained with ANOVA–PCA. Copyright © 2011 John Wiley & Sons, Ltd.     

High-resolution separations of protein isoforms with liquid chromatography time-of-flight mass spectrometry using polymer monolithic capillary columns

The separation of intact proteins, including protein isoforms arising from various amino-acid modifications, employing a poly(styrene-co-divinylbenzene) monolithic capillary column in high-performance liquid chromatography coupled on-line to a time-of-flight mass spectrometer (MS) is described. Using a 250 mm × 0.2 mm monolithic capillary column high-sensitivity separations yielding peak capacities of >600 were achieved with a 2h linear gradient and formic acid added in the mobile phase as ion-pairing agent. The combination of high-resolution chromatography with high-accuracy MS allowed to distinguish protein isoforms that differ only in their oxidation and biotinylation state allowing the separation between structural isoforms. Finally, the potential to separate proteins isoforms due to glycosylation is discussed.     

A Small‐Molecule Probe for Selective Profiling and Imaging of Monoamine Oxidase B Activities in Models of Parkinson’s Disease

The design of the first dual‐purpose activity‐based probe of monoamine oxidase B (MAO‐B) is reported. This probe is highly selective towards MAO‐B, even at high MAO‐A expression levels, and could sensitively report endogenous MAO‐B activities by both in situ proteome profiling and live‐cell bioimaging. With a built‐in imaging module as part of the probe design, the probe was able to accomplish what all previously reported MAO‐B imaging probes failed to do thus far: the live‐cell imaging of MAO‐B activities without encountering diffusion problems.     

Stilbene Derivatives from Gnetum montanum Markgr. f. megalocarpum Markgr.

Three new stilbene derivatives, gnetumelin A ( 1 ), gnetumelin B ( 2 ), and gnetumelin C ( 3 ), along with the nine known stilbene derivatives 4 – 12 were isolated from Gnetum montanum Markgr . f. megalocarpum Markgr. Their structures were determined by spectroscopic analysis and comparison of the data with reported ones.     

Glucose/Ribitol Dehydrogenase and 16.9 kDa Class I Heat Shock Protein 1 as Novel Wheat Allergens in Baker’s Respiratory Allergy

Wheat allergens are responsible for symptoms in 60–70% of bakers with work-related allergy, and knowledge, at the molecular level, of this disorder is progressively accumulating. The aim of the present study is to investigate the panel of wheat IgE positivity in allergic Italian bakers, evaluating a possible contribution of novel wheat allergens included in the water/salt soluble fraction. The water/salt-soluble wheat flour proteins from the Italian wheat cultivar Bolero were separated by using 1-DE and 2-DE gel electrophoresis. IgE-binding proteins were detected using the pooled sera of 26 wheat allergic bakers by immunoblotting and directly recognized in Coomassie stained gel. After a preparative electrophoretic step, two enriched fractions were furtherly separated in 2-DE allowing for detection, by Coomassie, of three different proteins in the range of 21–27 kDa that were recognized by the pooled baker’s IgE. Recovered spots were analyzed by nanoHPLC Chip tandem mass spectrometry (MS/MS). The immunodetected spots in 2D were subjected to mass spectrometry (MS) analysis identifying two new allergenic proteins: a glucose/ribitol dehydrogenase and a 16.9 kDa class I heat shock protein 1. Mass spectrometer testing of flour proteins of the wheat cultivars utilized by allergic bakers improves the identification of until now unknown occupational wheat allergens.