Combining Phytate/Ca^(2+) Fractionation with Trichloroacetic Acid/Acetone Precipitation Improved Separation of Low-Abundant Proteins of Wheat (Triticum aestivum L.) Leaf for Proteomic Analysis

Proteomic assessment of low-abundance leaf proteins is hindered by the large quantity of ribulose-1,5-bisphosphate carboxylase/oxygenase （Rubisco） present within plant leaf tissues. In the present study, total proteins were extracted from wheat （Triticum aestivum L.） leaves by a conventional trichloroacetic acid （TCA）/acetone method and a protocol first developed in this work. Phytate/Ca2＋ fractionation and TCA/acetone precipitation were combined to design an improved TCA/acetone method. The extracted proteins were analysed by two-dimensional gel electrophoresis （2-DE）. The resulting 2-DE images were compared to reveal major differences. The results showed that large quantities of Rubisco were deleted from wheat leaf proteins prepared by the improved method. As many as （758±4） protein spots were detected from 2-DE images of protein extracts obtained by the improved method, 130 more than those detected by the TCA/acetone method. Further analysis indicated that more protein spots could be detected at regions of pI 4.00-4.99 and 6.50-7.00 in the improved method-based 2-DE images. Our findings indicated that the improved method is an efficient protein preparation protocol for separating low-abundance proteins in wheat leaf tissues by 2-DE analysis. The proposed protocol is simple, fast, inexpensive and also applicable to protein preparations of other plants.

Detection of Low-abundance Point Mutations by Competitive Strand Assisted Endonuclease Ⅳ Signal Amplification System

Genetic mutations are important molecular biomarkers for cancer diagnosis and surveillance. Therefore, the development of methods for mutation detection characterized with straightforward, highly specific and sensitive to low-level mutations within various sequence contexts is extremely needed. Although some of the currently available methods have shown very encouraging results, their discrimination efficiency is still very low. Herein, we demonstrate a fluorescent probe coupled with blocker and property of melting temperature discrimination, which is able to identify the presence of known or unknown single-base variations at abundances down to 0.1% within 20 min. The discrimination factors between the perfect-match target and single-base mismatched target are determined to be 10.15–38.48. The method is sequence independent, which assures a wide range of application. The new method would be an ideal choice for high-throughput in vitro diagnosis and precise clinical treatment.

Comprehensive and deep profiling of the plasma proteome with protein corona on zeolite NaY

Proteomic characterization of plasma is critical for the development of novel pharmacodynamic biomarkers.However,the vast dynamic range renders the profiling of proteomes extremely challenging.Here,we synthesized zeolite NaY and developed a simple and rapid method to achieve comprehensive and deep profiling of the plasma proteome using the plasma protein corona formed on zeolite NaY.Specifically,zeolite NaY and plasma were co-incubated to form plasma protein corona on zeolite NaY(NaY-PPC),followed by conventional protein identification using liquid chromatography-tandem mass spectrometry.NaY was able to significantly enhance the detection of low-abundance plasma proteins,minimizing the“masking”effect caused by high-abundance proteins.The relative abundance of middleand low-abundance proteins increased substantially from 2.54%to 54.41%,and the top 20 highabundance proteins decreased from 83.63%to 25.77%.Notably,our method can quantify approximately 4000 plasma proteins with sensitivity up to pg/mL,compared to only about 600 proteins identified from untreated plasma samples.A pilot study based on plasma samples from 30 lung adenocarcinoma patients and 15 healthy subjects demonstrated that our method could successfully distinguish between healthy and disease states.In summary,this work provides an advantageous tool for the exploration of plasma proteomics and its translational applications.

Diversity of oligotrich ciliates(Ciliophora, Spirotrichea) in the northern coast of South China Sea as revealed in LSU rDNA sequences

Although the taxonomy of oligotrich ciliates has been widely investigated,yet the species diversity remains poorly known.We newly designed a pair of oligotrich-specific LSU rDNA primers covering the 600-bp D1/D2 region,and it was effective for detecting oligotrich species.Using the primers,we constructed the cloning libraries to investigate the species diversity of oligotrichs in the northern coastal waters of the South China Sea.In total,165 oligotrich sequences were obtained from five widely separated sampling sites.Sixty operational taxonomic units(OTUs)were obtained at 99%similarity threshold,and low-abundance OTUs with no more than two sequences contributed most of these(about 78%).Our findings are consistent with previous morphological studies,Strombidium was found the most abundant and widely distributed genus in this area.In addition,the BLAST search in the NCBI database resulted in 95%OTUs matching with named oligotrich species in similarity below 99%.Therefore,oligotrich morphospecies diversity has been underestimated as low-abundance species,and the LSU rDNA oligotrich sequence database needs to be better promoted.

Optimization of low-abundance protein extraction and abundant protein removal from defatted soybean meal

The aim of this study was to optimize the conditions for the extraction of low-abundance proteins（LAPs） and the removal of abundant proteins（APs; β-conglycinin and glycinin） from soybean meal.Single factor and orthogonal experiments were designed to determine the effects of four factors（isopropanol concentration, total extraction time, ultrasonic power, and ultrasonic time） on protein concentration in isopropanol extracts.Proteins in the isopropanol supernatant and the cold acetone precipitate of isopropanol were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS-PAGE） and matrix-assisted laser desorption/ionization-time of flight mass spectrometry（MALDI-TOF-MS）.The results showed that the optimal conditions were 50% isopropanol, ultrasonic pretreatment for 15 min at 350 W, and a total extraction time of 1 h.Under these conditions, the protein concentration in the isopropanol extracts reached 0.8081 g/L.Many LAPs were detected, including β-amylase, soybean agglutinin, soybean trypsin inhibitor, fumarylacetoacetase-like, phospholipase D alpha 1-like, oleosin, and even some unknown soybean proteins.The soybean APs（β-conglycinin and glycinin） were not found.The method may be useful for discovering new soybean proteins and extracting enough LAPs of soybean to allow further studies of their physiological effects on animals without the influence of APs.

DNA origami-based nano-hunter enriches low-abundance point mutations by targeting wild-type gene segments

Point mutations can be used as biomarkers to perform diagnosis for diseases. In this study, a nanorobot for low-abundance point mutation enrichment was constructed using DNA origami. The novel design achieved limits of detection of 0.1% and 1% for synthesized DNA samples and clinical gene samples, respectively. Resettability was a key property of this method, which also involved a simpler process, lower cost and shorter detection duration than traditional enrichment methods. This novel DNA nanorobot may enable the detection of tumor markers, potentially facilitating early cancer diagnosis.

Staged-probability strategy of processing shotgun proteomic data to discover more functionally important proteins

Biologically important proteins related to membrane receptors,signal transduction,regulation,transcription,and translation are usually low in abundance and identified with low probability in mass spectroscopy(MS)-based analyses.Most valuable proteomics information on them were hitherto discarded due to the application of excessively strict data filtering for accurate identification.In this study,we present a stagedprobability strategy for assessing proteomic data for potential functionally important protein clues.MS-based protein identifications from the second(L2)and third(L3)layers of the cascade affinity fractionation using the Trans-Proteomic Pipeline software were classified into three probability stages as 1.00–0.95,0.95–0.50,and 0.50–0.20 according to their distinctive identification correctness rates(i.e.100%–95%,95%–50%,and 50%–20%,respectively).We found large data volumes and more functionally important proteins located at the previously unacceptable lower probability stages of 0.95–0.50 and 0.50–0.20 with acceptable correctness rate.More importantly,low probability proteins in L2 were verified to exist in L3.Together with some MS spectrogram examples,comparisons of protein identifications of L2 and L3 demonstrated that the stagedprobability strategy could more adequately present both quantity and quality of proteomic information,especially for researches involving biomarker discovery and novel therapeutic target screening.

Thermodynamics-guided two-way interlocking DNA cascade system for universal multiplexed mutation detection

Detection of point mutations in driver genes is of great significance for the early diagnosis,treatment,and prognostic evaluation of cancer.However,current detection methods do not offer versatility,specificity,and rapid performance simultaneously.Thus,multiple mutation detection processes are necessary,which results in long processing times and high costs.In this study,we developed a thermodynamics-guided two-way interlocking DNA cascade system for universal multiplexed mutation detection(TTI-CS).This strategy is based on the DNA probe,which changes the thermodynamic balance of the DNA cascade by the designed bubble structure,thereby achieving a good distinction between mutant and wild-type DNA.The designed method greatly shortens the detection time through two-way intrusion.In addition,this method only changes two inexpensive trigger and bridge sequences,which replace the specific and expensive nucleic acid probes used in analyses based on traditional DNA probe methods,thereby enabling multiple detections.We performed the detection of synthetic single-stranded DNA for the five mutation points and successfully detected in endometrial cancer specimens.The detection limit of this method is0.1%,which better meets the needs of clinical low-abundance multiple mutation detection.Overall,TTI-CS is currently one of the best methods for detecting multiple mutation detections.