Reproducible and Consistent Quantification of the Saccharomyces cerevisiae Proteome by SWATH-mass spectrometry

Targeted mass spectrometry by selected reaction monitoring (S/MRM) has proven to be a suitable technique for the consistent and reproducible quantification of proteins across multiple biological samples and a wide dynamic range. This performance profile is an important prerequisite for systems biology and biomedical research. However, the method is limited to the measurements of a few hundred peptides per LC-MS analysis. Recently, we introduced SWATH-MS, a combination of data independent acquisition and targeted data analysis that vastly extends the number of peptides/proteins quantified per sample, while maintaining the favorable performance profile of S/MRM. Here we applied the SWATH-MS technique to quantify changes over time in a large fraction of the proteome expressed in Saccharomyces cerevisiae in response to osmotic stress.We sampled cell cultures in biological triplicates at six time points following the application of osmotic stress and acquired single injection data independent acquisition data sets on a high-resolution 5600 tripleTOF instrument operated in SWATH mode. Proteins were quantified by the targeted extraction and integration of transition signal groups from the SWATH-MS datasets for peptides that are proteotypic for specific yeast proteins. We consistently identified and quantified more than 15,000 peptides and 2500 proteins across the 18 samples. We demonstrate high reproducibility between technical and biological replicates across all time points and protein abundances. In addition, we show that the abundance of hundreds of proteins was significantly regulated upon osmotic shock, and pathway enrichment analysis revealed that the proteins reacting to osmotic shock are mainly involved in the carbohydrate and amino acid metabolism. Overall, this study demonstrates the ability of SWATH-MS to efficiently generate reproducible, consistent, and quantitatively accurate measurements of a large fraction of a proteome across multiple samples.In systems biology and biomedical studies targeted mass spectrometry via selected reaction monitoring (SRM)¹ (also known as multiple reaction monitoring, MRM) has emerged as a powerful technique for the consistent and reproducible quantification of proteins across numerous complex samples (1–6). Optimal sets of precursor/fragment ion pairs, called transitions, uniquely represent a specific peptide. They constitute a definitive mass spectrometric assay for the detection of targeted peptides, and thus the proteins from which they derive, in the complex matrix of trypsinized biological samples (1, 7). Protein quantification is then performed by relating the intensity of the acquired transition signals to suitable reference signals. Most quantification strategies commonly used in proteomics are compatible with this method (8). Recently, the high-throughput development of S/MRM assays has been achieved via the generation of MS/MS spectral libraries from the measurements of thousands of synthetic peptides representing proteotypic peptides (9). Moreover, many experimental and bioinformatics workflows have been developed for assay generation, assay optimization, data evaluation, and the dissemination of optimized S/MRM assays (10–16). In combination, these developments have supported the creation of mass-spectrometric maps of entire proteomes of selected species including Streptococcus pyogenes, Mycobacterium tuberculosis, and Saccharomyces cerevisae (5, 17–19) and the robust use of these resources to quantify specific protein sets across multiple biological samples.Currently, targeted proteomics by S/MRM can be multiplexed to a maximum set of ∼100 proteins that can be measured in a single LC-S/MRM run at optimal quantitative accuracy, limit of detection and dynamic range. The quantification of higher numbers of proteins per run compromises some of the performance parameters of the method because of well understood tradeoffs (8). Attempts have been made to further increase the degree of multiplexing of S/MRM, either by automated adjustment of the scheduled detection windows (20) or by acquiring, in a data-dependent manner, the complete set of precursor-fragment ion pairs of a given assay (21). Alternatively, parallel reaction monitoring (PRM) approach operated on quadrupole-orbitrap mass spectrometer has shown detection and quantification performances similar or better than those obtained in SRM, because of the increased selectivity of the mass analyzer (22–24). These approaches are promising, but their application relies on prior knowledge of the precursor ions that need to be targeted during the data acquisition, and they still are subject of the above-mentioned tradeoffs.Recently, we developed a novel MS strategy that combines data independent acquisition (DIA) of trypsinized protein samples with S/MRM-like, in silico targeted analysis of the acquired complete fragment ion maps (25). We termed the method SWATH-MS, and applied the sequential isolation window acquisition principle (26) to repeatedly cycle, in a single injection, through 32 consecutive 25-Da precursor isolation windows (swaths). The process acquires fragment ion spectra of all precursors in a space defined by the 400–1200 m/z precursor range and a user-specified retention time window. We used the prior information in MS/MS spectral libraries to extract groups of signals that uniquely identify a specific peptide, and to demonstrate that peptides could be identified and quantified over a dynamic range of four orders of magnitude, even when the precursors were not detectable in a survey MS scan. For the 45 proteins involved in the central carbon metabolism of yeast, we demonstrated that the accuracy of quantification was equivalent to that of S/MRM (25). However, because of the lack of adequate software tools at that time, the extensive high-throughput targeted data analysis of the SWATH-MS maps could not be fully demonstrated in that first study.Here we demonstrate the multiplexing capabilities of SWATH-MS for the detection and quantification of significantly larger fractions of a proteome as compared with S/MRM, without compromising reproducibility, consistency, and quantitative accuracy. We describe the large scale deployment of fragment ion spectral libraries and the use of S/MRM-like analysis tools specifically adapted to SWATH-MS data for the detection and quantification of temporal changes of the S. cerevisae proteome in response to osmotic stress.     

疣柄牛肝菌属一中国新记录种

报道了采自内蒙古呼伦贝尔市的中国疣柄牛肝菌属1个新记录种,即假褐疣柄牛肝菌Leccinum pseudoscabrum(Kallenb.)utara.。主要特征为菌肉伤后变红到紫褐色,再到黑褐色,基部菌丝体手触后呈紫褐色;具泡状菌丝,呈栅栏状。比较和讨论了该种与相近种在伤后不同的变色反应及菌丝形态。研究标本存放于吉林农业大学菌物标本馆(HMJAU)。     

La TEP-TDM au 18FDG a-t-elle un intérêt dans la stadification ganglionnaire des mélanomes malins cutanés cervicofaciaux bénéficiant de la technique du ganglion sentinelle ? À propos de 22 cas

Background and objectivesIn N0 cutaneous head and neck melanoma, sentinel lymph node biopsy (SLNB) is less reliable and accurate than in trunk or extremities melanoma (false negative cases and spotting failure). The aim of our study was to assess the utility of PET-CT ¹⁸FDG in a specific group of N0 patients, combined with SLNB.Patients and methodsTwenty-two patients with N0 cutaneous head and neck melanoma were retrospectively reviewed. All of them had underwent PET-CT and SLNB before surgery. Average follow-up time was 17 months (1–44).ResultsAt least one sentinel lymph node (SLN) was identified in 20 patients. Ten patients (50%) had metastatic SLN. Among these 10 N+ patients, PET-CT was positive for occult nodal metastases for only two patients. During follow-up, two patients had cervical nodal recurrence, whereas SLNB was negative. PET-CT was also negative for these two patients. SLNB and PET-CT sensitivity were respectively 83 and 18%. PET-CT specificity was 84% (regarding neck sides).ConclusionIn this specific population with N0 cutaneous head and neck melanoma, PET-CT sensitivity is too low and failed to detect occult nodal metastases in two patients with false negative SLNB. Consequently, PET-CT seems to be not useful for nodal staging N0 cutaneous head and neck melanomas, in which SLNB is the most accurate technique.     

Long-term dynamics of aboveground fungal communities in a subalpine Norway spruce forest under elevated nitrogen input

As anthropogenic N deposition has been suspected to be the main reason for the decline of macromycetous sporocarp production in forest ecosystems, various N-fertilization experiments were started in the mid 1990s. The dynamics of ectomycorrhizal (root-inhabiting) and terricolous saprobic (litter-inhabiting) fungal communities were studied by exhaustive sporocarp inventories in a substitution Norway spruce (Picea abies) forest in two 256-m² plots sampled for periods of 1 week at 1-m² resolution between 1994 and 2007. N was added to the soil twice per year in one plot from the fourth year onwards. The effects of N input and time on aboveground fungal communities were assessed using redundancy analysis, principal response curves and non-parametric multivariate ANOVA. Results of this long-term experiment revealed that both ectomycorrhizal and saprobic fungal communities responded to an increase in soil N input. The ectomycorrhizal community reacted by a fast decrease in sporocarp production and in species richness, whereas the saprobic community was less affected. The response was highly species specific, especially for the saprobic community. The difference in species composition between control and fertilized plots was significant after 1 year of N addition for ectomycorrhizal fungi and only after 3 years for saprobic fungi. An aging effect affected sporocarp production in the whole area. For both communities, this unidirectional drift in species composition was as important as the treatment effect. This result highlights the importance of considering the respective role of treatment and year effects in long-term field experiments on fungal communities.     

Accommodation of tmRNA–SmpB into stalled ribosomes: A cryo-EM study

In eubacteria, translation of defective messenger RNAs (mRNAs) produces truncated polypeptides that stall on the ribosome. A quality control mechanism referred to as trans-translation is performed by transfer-messenger RNA (tmRNA), a specialized RNA acting as both a tRNA and an mRNA, associated with small protein B (SmpB). So far, a clear view of the structural movements of both the protein and RNA necessary to perform accommodation is still lacking. By using a construct containing the tRNA-like domain as well as the extended helix H2 of tmRNA, we present a cryo-electron microscopy study of the process of accommodation. The structure suggests how tmRNA and SmpB move into the ribosome decoding site after the release of EF-Tu·GDP. While two SmpB molecules are bound per ribosome in a preaccommodated state, our results show that during accommodation the SmpB protein interacting with the small subunit decoding site stays in place while the one interacting with the large subunit moves away. Relative to canonical translation, an additional movement is observed due to the rotation of H2. This suggests that the larger movement required to resume translation on a tmRNA internal open reading frame starts during accommodation.     

流式细胞术研究细胞凋亡的方法与技术

细胞发生凋亡时,会伴随着一系列形态学、生物化学及分子生物学性质的变化,包括细胞皱缩,核染色质凝聚,细胞膜通透性改变,Caspases激活,线粒体跨膜电位降低,膜磷酯酰丝氨酸外化,胞质Ca2+浓度升高,DNA片段化及含量变化等特点.应用流式细胞术进行细胞凋亡的研究,对于探讨胚胎发育、衰老以及研究肿瘤的发生、发展和转化等病理生理过程和病毒感染及免疫等具有十分重要的意义.本文就细胞凋亡的特征、基于细胞膜功能的流式细胞术检测方法和基于细胞器功能的流式细胞术检测方法等关键性问题进行了阐述.     

Major changes in the cell wall during silique development in Arabidopsis thaliana

Fruit development is a highly complex process, which involves major changes in plant metabolism leading to cell growth and differentiation. Changes in cell wall composition and structure play a major role in modulating cell growth. We investigated the changes in cell wall composition and the activities of associated enzymes during the dry fruit development of the model plant Arabidopsis thaliana. Silique development is characterized by several specific phases leading to fruit dehiscence and seed dispersal. We showed that early phases of silique growth were characterized by specific changes in non-cellulosic sugar content (rhamnose, arabinose, xylose, galactose and galacturonic acid). Xyloglucan oligosaccharide mass profiling further showed a strong increase in O-acetylated xyloglucans over the course of silique development, which could suggest a decreased capacity of xyloglucans to be associated with each other or to cellulose. The degree of methylesterification, mediated by the activity of pectin methylesterases (PMEs), decreased over the course of silique growth and dehiscence. The major changes in cell wall composition revealed by our analysis suggest that it could be major determinants in modulating cell wall rheology leading to growth or growth arrest.