Benchmarking sample preparation/digestion protocols reveals tube‐gel being a fast and repeatable method for quantitative proteomics

Sample preparation, typically by in‐solution or in‐gel approaches, has a strong influence on the accuracy and robustness of quantitative proteomics workflows. The major benefit of in‐gel procedures is their compatibility with detergents (such as SDS) for protein solubilization. However, SDS‐PAGE is a time‐consuming approach. Tube‐gel (TG) preparation circumvents this drawback as it involves directly trapping the sample in a polyacrylamide gel matrix without electrophoresis. We report here the first global label‐free quantitative comparison between TG, stacking gel (SG), and basic liquid digestion (LD). A series of UPS1 standard mixtures (at 0.5, 1, 2.5, 5, 10, and 25 fmol) were spiked in a complex yeast lysate background. TG preparation allowed more yeast proteins to be identified than did the SG and LD approaches, with mean numbers of 1979, 1788, and 1323 proteins identified, respectively. Furthermore, the TG method proved equivalent to SG and superior to LD in terms of the repeatability of the subsequent experiments, with mean CV for yeast protein label‐free quantifications of 7, 9, and 10%. Finally, known variant UPS1 proteins were successfully detected in the TG‐prepared sample within a complex background with high sensitivity. All the data from this study are accessible on ProteomeXchange (PXD003841).     

How fast is fast? Eco‐evolutionary dynamics and rates of change in populations and phenotypes

It is increasingly recognized that evolution may occur in ecological time. It is not clear, however, how fast evolution – or phenotypic change more generally – may be in comparison with the associated ecology, or whether systems with fast ecological dynamics generally have relatively fast rates of phenotypic change. We developed a new dataset on standardized rates of change in population size and phenotypic traits for a wide range of species and taxonomic groups. We show that rates of change in phenotypes are generally no more than 2/3, and on average about 1/4, the concurrent rates of change in population size. There was no relationship between rates of population change and rates of phenotypic change across systems. We also found that the variance of both phenotypic and ecological rates increased with the mean across studies following a power law with an exponent of two, while temporal variation in phenotypic rates was lower than in ecological rates. Our results are consistent with the view that ecology and evolution may occur at similar time scales, but clarify that only rarely do populations change as fast in traits as they do in abundance.     

Proteomic changes in response to crystal formation in Drosophila Malpighian tubules

Kidney stone disease is a major health burden with a complex and poorly understood pathophysiology. Drosophila Malpighian tubules have been shown to resemble human renal tubules in their physiological function. Herein, we have used Drosophila as a model to study the proteomic response to crystal formation induced by dietary manipulation in Malpighian tubules. Wild-type male flies were reared in parallel groups on standard medium supplemented with lithogenic agents: control, Sodium Oxalate (NaOx) and Ethylene Glycol (EG). Malpighian tubules were dissected after 2 weeks to visualize crystals with polarized light microscopy. The parallel group was dissected for protein extraction. A new method of Gel Assisted Sample Preparation (GASP) was used for protein extraction. Differentially abundant proteins (p<0.05) were identified by label-free quantitative proteomic analysis in flies fed with NaOx and EG diet compared with control. Their molecular functions were further screened for transmembrane ion transporter, calcium or zinc ion binder. Among these, 11 candidate proteins were shortlisted in NaOx diet and 16 proteins in EG diet. We concluded that GASP is a proteomic sample preparation method that can be applied to individual Drosophila Malpighian tubules. Our results may further increase the understanding of the pathophysiology of human kidney stone disease.     

Frequency‐dependent selection for rare genotypes promotes genetic diversity of a tropical palm

Negative frequency‐dependent selection among species is a key driver of community diversity in natural systems, but the degree to which negative frequency‐dependent selection shapes patterns of survival and genetic diversity within species is poorly understood. In a 5‐year field experiment, we show that seedlings of a tropical palm with rare genotypes had a pronounced survival advantage over seedlings with common genotypes, with effect sizes comparable to that of light availability. This ‘rare genotype advantage’ led to an increase in population‐wide genetic diversity among seedlings compared to null expectations, as predicted by negative frequency‐dependent selection, and increased reproductive success in adult trees with rare genotypes. These results suggest that within‐species negative frequency‐dependent selection of genotypes can shape genetic variation on ecologically relevant timescales in natural systems and may be a key, overlooked source of non‐random mortality for tropical plants.     

Phylogeography of Arabis serrata (Brassicaceae) in the Japanese Archipelago based on chloroplast DNA variations

Arabis serrata (Brassicaceae), a perennial plant widely distributed along the Japanese Archipelago, occurs in various habitats: for example, limestone zones, serpentine barrens, volcanic soils, and roadsides. It likely survived by adapting to its surrounding environment, resulting in great morphological and ecological variation. In this study, we performed a phylogeographic analysis to examine past changes in the distribution of A. serrata following climate oscillations during the Pleistocene. To cover most of A. serrata's range, leaves were collected from eight to ten individuals randomly selected from each of 37 populations in the Japanese Archipelago. Two chloroplast noncoding regions of the samples were amplified and sequenced: trnT(GGU)‐psbD and trnH(GUG)‐psbA spacers. Twenty‐five haplotypes were detected and distinguished by 31 substitutions. Four main haplotypes were observed in many populations distributed throughout the Japanese Archipelago. According to the genetic boundaries detected using the Monmonier algorithm, A. serrata is clustered into four groups, each including several populations: Hokkaido Island, northern mainland Honshu, central Japan, and western Japan. The boundaries, however, were not robust because all genetic parameters did not support the differentiation among groups. These results indicate the absence of an obvious geographic structure in the distribution of A. serrata, suggesting that this species has experienced a rapid range expansion in postglacial times.     

小蓬NeMT2基因的克隆及其植物表达载体的构建

该研究利用RACE ( Rapid amplification of cDNA ends)技术从小蓬中成功分离编码金属硫蛋白( Metal-lothionein,MT)的cDNA序列,命名为NeMT2,在GenBank中登录号为KT835290。该基因全长590 bp,开放阅读框为237 bp,编码78个氨基酸,编码的氨基酸序列中含有14个半胱氨酸残基( Cys,C),呈C-C,C-X-C,C-X-X-C排列,集中分布在肽链的N端和C端,基因编码蛋白的分子量为7.6036 kD,等电点为4.71。系统发育分析表明,小蓬金属硫蛋白NeMT2与藜科的海蓬子( AEF01492)和盐穗木( AHI62953)同源性最高,其次是甜菜( XP 010667708.1)。生物信息学分析表明,金属硫蛋白NeMT2无信号肽结构,属于非跨膜亲水性蛋白;疏水性分析表明,NeMT2蛋白的35~45个氨基酸之间有较强的疏水性,其中第41位Asp具最强的疏水性(1.444);结构预测分析该蛋白质二级结构的主要元件是无规则卷曲。通过RT-PCR对NeMT2基因的表达分析发现, NeMT2基因在铜矿区和非铜矿区的小蓬叶片中均有表达,但该基因在铜矿区小蓬叶片的表达量明显高于非铜矿区。将小蓬NeMT2基因定向克隆到植物表达载体pCAMBIA1300的35S 启动子下游,构建该基因的植物超表达载体pCAMBIA1300＋NeMT2。该研究结果为进一步研究该基因的功能和小蓬响应重金属胁迫的分子机制提供了一定基础。     

不同全血过滤方法用于去白细胞血液制备的临床对照研究

目的:观察不同全血过滤方法用于去白细胞血液制备的效果。方法:采用两种全血过滤方法进行对比研究,对照组采用常规法,将采集后全血混匀后直接与白细胞滤器连接直接过滤;实验组采用湿润滤盘法,血液采集完成混匀后静置,先用上层血清10～20 m L湿润滤盘,再混匀与白细胞滤器连接后进行过滤。比较两组制备方法所用的过滤时间、血液回收率、过滤前后血液指标情况及24小时内溶血的发生情况。结果:两组全血过滤方法过滤前后白细胞、红细胞、血红蛋白、血小板及血浆游离血红蛋白水平比较差异均无明显统计学意义(P0.05)。而实验组过滤时间短于对照组,血液回收率高于对照组,且24小时内溶血比例明显低于对照组(P0.05)。结论:常规法与湿润滤盘法均能达到去白细胞血液标准,但湿润滤盘法较常规法能有效的降低过滤时间、增加血液回收率,减少去白细胞悬浮红细胞因溶血造成的血液不合格率,值得临床推广应用。     

A Fast and Economical Sample Preparation Protocol for Interaction Proteomics Analysis

A simple and fast immunoprecipitation (IP) protocol is designed with the sample preparation incorporated, applicable to both low and high throughput. This new protocol combines two procedures based on magnetic beads in 96‐well plate format. Protein complexes are captured by antibodies and magnetic beads conjugated with protein A. Proteins are washed and on‐bead digested by using Single‐Pot solid‐phase sample preparation (SP3). The whole IP‐SP3 approach can be completed in one day, which is considerably faster compared to the classical approach. No major quantitative differences are found between SP3 and FASP (filter‐aided sample preparation) or a longer incubation protocol. Taken together, the IP‐SP3 protocol is a fast and economical approach easily applicable for large‐scale protein interactome analysis.