Tb^(3+)-nucleic acid probe-based label-free and rapid detection of mercury pollution in food

Mercury is a threatening pollutant in food,herein,we developed a Tb^(3+)-nucleic acid probe-based label-free assay for mix-and-read,rapid detection of mercury pollution.The assay utilized the feature of light-up fluorescence of terbium ions(Tb^(3+))via binding with single-strand DNA.Mercury ion,Hg^(2+)induced thymine(T)-rich DNA strand to form a double-strand structure(T-Hg^(2+)-T),thus leading to fluorescence reduction.Based on the principle,Hg^(2+)can be quantified based on the fluorescence of Tb^(3+),the limit of detection was 0.0689μmol/L and the linear range was 0.1-6.0μmol/L.Due to the specificity of T-Hg^(2+)-T artificial base pair,the assay could distinguish Hg^(2+)from other metal ions.The recovery rate was ranged in 98.71%-101.34%for detecting mercury pollution in three food samples.The assay is low-cost,separation-free and mix-to-read,thus was a competitive tool for detection of mercury pollution to ensure food safety.

Proteomic study of vitreous in proliferative diabetic retinopathy patients after treatment with aflibercept:a quantitative analysis based on 4D label-free technique

AIM:To identify different metabolites,proteins and related pathways to elucidate the causes of proliferative diabetic retinopathy(PDR)and resistance to anti-vascular endothelial growth factor(VEGF)drugs,and to provide biomarkers for the diagnosis and treatment of PDR.METHODS:Vitreous specimens from patients with diabetic retinopathy were collected and analyzed by Liquid Chromatography-Mass Spectrometry(LC-MS/MS)analyses based on 4D label-free technology.Statistically differentially expressed proteins(DEPs),Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway representation and protein interactions were analyzed.RESULTS:A total of 12 samples were analyzed.The proteomics results showed that a total of 58 proteins were identified as DEPs,of which 47 proteins were up-regulated and 11 proteins were down-regulated.We found that C1q and tumor necrosis factor related protein 5(C1QTNF5),Clusterin(CLU),tissue inhibitor of metal protease 1(TIMP1)and signal regulatory protein alpha(SIRPα)can all be specifically regulated after aflibercept treatment.GO functional analysis showed that some DEPs are related to changes in inflammatory regulatory pathways caused by PDR.In addition,protein-protein interaction(PPI)network evaluation revealed that TIMP1 plays a central role in neural regulation.In addition,CD47/SIRPαmay become a key target to resolve anti-VEGF drug resistance in PDR.CONCLUSION:Proteomic analysis is an approach of choice to explore the molecular mechanisms of PDR.Our data show that multiple proteins are differentially changed in PDR patients after intravitreal injection of aflibercept,among which C1QTNF5,CLU,TIMP1 and SIRPαmay become targets for future treatment of PDR and resolution of anti-VEGF resistance.

基于label-free定量蛋白质组学方法筛选沉默CHAF1B基因后心肌细胞差异表达蛋白及调控网络分析

目的分析沉默染色质装配因子1亚基B(chromatin assembly factor 1 subunit B,CHAF1B)基因后心肌细胞中差异表达蛋白,预测CHAF1B基因调控网络,为寻找促进心肌细胞修复的潜在治疗靶点提供参考。方法采用转染和蛋白质印迹法筛选沉默CHAF1B基因的有效小干扰RNA(small interfering RNA,siRNA)。应用有效siRNA沉默人源心肌AC16细胞CHAF1B基因后,采用细胞活力检测方法检测细胞活力;提取总蛋白质进行定量、还原、烷基化和胰蛋白酶裂解成肽段,利用高效液相串联质谱法鉴定肽段;搜索UniProt蛋白库筛选差异表达的蛋白质进行基因本体(Gene Ontology,GO)富集分析、京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路富集和蛋白质互作网络(protein-protein interaction networks,PPI)分析。结果siRNA有效沉默CHAF1B基因后,心肌细胞存活明显受到抑制;label-free定量蛋白质组学方法鉴定结果显示,共有69个差异表达蛋白质,其中50个表达显著上调(差异倍数≥2,P<0.05),19个表达显著下调(差异倍数≤0.5,P<0.05)。GO分析显示,差异表达蛋白质主要参与大分子复合亚基体、细胞组分生物合成和组装等生物学过程,分布在细胞质和囊泡等区域,发挥蛋白质结合等分子功能。KEGG通路富集和PPI分析显示,差异表达蛋白质参与的信号通路包括蛋白酶体、氨酰tRNA生物合成、胞吞、嘧啶代谢和氨基酸生物合成等10条信号途径;表达显著上调的蛋白质如蛋白酶体亚单位A2和B7、26 S蛋白酶体调节亚单位6B和10B参与蛋白酶体途径,丝氨酸、甘氨酸、谷氨酰胺和赖氨酸tRNA合成酶介导氨酰tRNA生物合成;表达显著下调的蛋白质包括骨架相关蛋白2/3复合体亚单位3和热休克70蛋白1样参与胞吞作用,核糖核苷-二磷酸还原酶大亚基介导嘧啶代谢等通路。实时荧光定量聚合酶链式反应结果证实,转染CHAF1B siRNA后心肌细胞中合成骨架相关蛋白2/3复合体亚单位3的基因ARPC3和氨酰tRNA生物合成关键基因QARS1的mRNA水平均显著降低。结论CHAF1B为心肌细胞存活的关键蛋白质,参与调控心肌细胞的胞吞和氨基酸生物合成等多种生物学过程,参考其调控网络可帮助寻找促进心肌细胞修复的干预环节。

Characterization of natural peptides in Pheretima by integrating proteogenomics and label-free peptidomics

Pheretima,also called“earthworms”,is a well-known animal-derived traditional Chinese medicine that is extensively used in over 50 Chinese patent medicines(CPMs)in Chinese Pharmacopoeia(2020 edition).However,its zoological origin is unclear,both in the herbal market and CPMs.In this study,a strategy for integrating in-house annotated protein databases constructed from close evolutionary relationship-sourced RNA sequencing data from public archival resources and various sequencing algorithms(restricted search,open search,and de novo)was developed to characterize the phenotype of natural peptides of three major commercial species of Pheretima,including Pheretima aspergillum(PA),Pheretima vulgaris(PV),and Metaphire magna(MM).We identified 10,477 natural peptides in the PA,7,451 in PV,and 5,896 in MM samples.Five specific signature peptides were screened and then validated using synthetic peptides;these demonstrated robust specificity for the authentication of PA,PV,and MM.Finally,all marker peptides were successfully applied to identify the zoological origins of Brain Heart capsules and Xiaohuoluo pills,revealing the inconsistent Pheretima species used in these CPMs.In conclusion,our integrated strategy could be used for the in-depth characterization of natural peptides of other animal-derived traditional Chinese medicines,especially non-model species with poorly annotated protein databases.

Label-free breast cancer detection and classification by convolutional neural network-based on exosomes surface-enhanced raman scattering

Because the breast cancer is an important factor that threatens women's lives and health,early diagnosis is helpful for disease screening and a good prognosis.Exosomes are nanovesicles,secreted from cells and other body fluids,which can reflect the genetic and phenotypic status of parental cells.Compared with other methods for early diagnosis of cancer(such as circulating tumor cells(CTCs)and circulating tumor DNA),exosomes have a richer number and stronger biological stability,and have great potential in early diagnosis.Thus,it has been proposed as promising biomarkers for diagnosis of early-stage cancer.However,distinguishing different exosomes remain is a major biomedical challenge.In this paper,we used predictive Convolutional Neural model to detect and analyze exosomes of normal and cancer cells with surface-enhanced Raman scattering(SERS).As a result,it can be seen from the SERS spectra that the exosomes of MCF-7,MDA-MB-231 and MCF-10A cells have similar peaks(939,1145 and 1380 cm^(-1)).Based on this dataset,the predictive model can achieve 95%accuracy.Compared with principal component analysis(PCA),the trained CNN can classify exosomes from different breast cancer cells with a superior performance.The results indicate that using the sensitivity of Raman detection and exosomes stable presence in the incubation period of cancer cells,SERS detection combined with CNN screening may be used for the early diagnosis of breast cancer in the future.

4D label-free proteomic analysis of vitreous from patients with rhegmatogenous retinal detachment

AIM:To identify metabolites,proteins,and related pathways involved in the etiology of rhegmatogenous retinal detachment(RRD)for use as biomarkers in diagnosing and treating RRD.METHODS:Vitreous specimens were collected and liquid chromatography-tandem mass spectrometry analysis was per formed using the four-dimensional label-free technique.Statistically significant differentially expressed proteins,gene ontology(GO)terms,Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway representations,and protein interactions were analyzed.RESULTS:Nine specimens were subjected to proteomic analysis.In total,161 proteins were identified as differentially expressed proteins(DEPs),including 53 upregulated proteins and 108 downregulated proteins.GO functional analysis revealed that some DEPs were enriched in neuron-related terms and membrane protein terms.Moreover,KEGG analysis indicated that the cell adhesion molecule metabolic pathway was associated with the greatest number of DEPs.Finally,the evaluation of protein-protein interaction network revealed that DEPs were clustered in neuronal adhesion,apoptosis,inflammation and immune responses,correct protein folding,and glycolysis.CONCLUSION:Proteomic profiling is useful for the exploration of molecular mechanisms that underlie RRD.This study reveals increased expression levels of proteins related to heat shock protein content,glycolysis,and inflammatory responses in RRD.Knowledge regarding biomarkers of RRD pathogenesis may help to prevent the occurrence of RRD in the future.

基于Label-free技术的高州油茶铝胁迫蛋白质组学研究

【目的】南方地区拥有大面积的酸性红壤,导致土壤中铝元素多以离子的形式溶出,严重抑制油茶的生长及高产。研究采用Label-free技术探究高州油茶在铝胁迫下的蛋白质组学响应,为揭示高州油茶应答铝胁迫的分子响应机制奠定了理论基础。【方法】以高州油茶两年生实生苗为试验材料,测定不同浓度铝处理条件下高州油茶叶片部分代谢指标及抗氧化生理相关指标,同时比较对照组(CK)和4 mmol/L铝处理组(GZ4)差异蛋白质的表达。【结果】生理试验结果表明,不同浓度铝胁迫下油茶幼苗都会发生氧化胁迫导致细胞活性氧(ROS)积累,2 mmol/L铝浓度下油茶叶片通过增加渗透调节物质含量和增强多种抗氧化酶活性以清除细胞活性氧,使细胞免受氧化胁迫的损害;而4 mmol/L铝浓度下叶片丙二醛(MDA)含量和脯氨酸(PRO)含量明显升高,积累的活性氧超出了自身所能清除的范围,叶片可溶性糖含量和可溶性蛋白含量显著减少,抗氧化酶活性均有不同程度减弱,油茶正常的生理功能受到抑制。蛋白质组学数据表明,CK组和GZ4组共鉴定到4282个蛋白质,与CK组相比,GZ4处理组有207个蛋白质的表达丰度显著增加,129个蛋白质的表达丰度显著降低。差异表达蛋白质的通路富集和蛋白质互作网络分析结果表明,4 mmol/L铝胁迫下高州油茶主要通过提高谷胱甘肽代谢,增强细胞清除活性氧和自由基负离子的能力缓解铝胁迫造成的氧化损伤;4 mmol/L铝浓度下高州油茶蛋白质合成和光合作用受到明显抑制,抗氧化酶活性减弱可能与细胞供能能力不足有关。【结论】铝胁迫下高州油茶主要通过能量及碳水化合物代谢、氨基酸合成及代谢、生物碱合成、蛋白质加工、卟啉和叶绿素代谢等代谢途径抵抗铝毒。

基于label-free技术的梅花鹿(Cervus nippon)茸角蛋白组分比较

旨在探究不同生长时期梅花鹿鹿茸的蛋白质组分信息,为鹿茸有效活性物质的挖掘提供理论依据。本研究利用label-free蛋白质组学技术及生物信息学方法对不同生长时期(10、20、40、60、130与360天)梅花鹿茸角蛋白质组分进行比较研究。结果显示,梅花鹿茸角含有丰富的蛋白质,10、20、40、60、130与360天蛋白质含量依次为65.35、70.90、74.00、82.25、56.00、28.02mg·g^-1。应用label-free蛋白质组学技术共鉴定出636种梅花鹿茸角蛋白质,其中218种蛋白质为显著差异表达,主要参与了蛋白质合成、发育、细胞骨架、转运等生物学过程。不同生长阶段茸角的蛋白质表达有各自的特点,为梅花鹿茸角药理活性成分的筛选及茸角相关产品的开发奠定理论基础。

Label-free与iTRAQ蛋白质组学方法在IgA肾病中的应用

目的:探讨蛋白质非标记(label-free)定量技术与同位素标记相对和绝对定量(iTRAQ)技术两种尿液蛋白组学技术在IgA肾病(IgA-N)中的应用价值。方法:选取正常人(健康对照组)和IgA-N患者(IgA-N组)各12例,均留取新鲜晨尿,组内混合,预实验判断样品质量,初步确定蛋白质鉴定数量,FASP酶解后分两部分进行:一部分iTRAQ标记分级后与另一部分进行label-free的LC-MS/MS质谱、数据分析。比较两种实验方法蛋白覆盖情况、实验重复性及差异蛋白数量。结果:经过质谱鉴定,label-free经质谱鉴定到的肽段长度7~26,高峰10~18,各肽段长度范围内的数目最多达800种。iTRAQ实验经质谱鉴定到的肽段长度6~26,高峰分布6~16,各肽段长度范围内的数目最多达1400种。iTRAQ计算组内变异系数(CV)为5.7%~11.2%,组间CV为7.9%~22.0%(CV<0.3)。Label-free实验计算组内CV为3.7%~13.2%,组间CV为5.9%~19.0%(CV<0.3)。Label-free鉴定到624种蛋白,iTRAQ鉴定到621种蛋白。通过火山图分析两种实验方法均可以很好筛选出差异蛋白,且聚类效果好;经GO分析两种实验方案均显示出相似度较高的生物分类功能。结论:iTRAQ标记可检测到更多的蛋白,且在低分子量检测中占优势,而label-free实验得到的CV相对较小,需要依据实验目的优化实验方案。

基于Label-free技术分析牛卵泡蛋白质组分及关键调控蛋白

旨在研究牛卵泡发育过程中蛋白质组表达变化并筛选卵泡发育关键调控蛋白,利用非标记(label-free)定量蛋白质组学技术对牛卵泡颗粒细胞(granulesa cells, GCs)蛋白质组分进行比较分析。采集牛发情周期优势卵泡(dominant follicles, DF)和从属卵泡(subordinate follicles, SF),分别分离GCs,并提取总蛋白,胰蛋白酶酶解,液相色谱-串联质谱(LC-MS/MS)进行蛋白质组分分析,数据库检索分析DF和SF中蛋白质表达情况,并应用生物信息学方法筛选牛卵泡发育关键调控蛋白。结果表明:本试验从30 321个肽段中共成功鉴定出3 409种蛋白质(FDR≤0.01),其中,DF中表达2 895种蛋白质,SF中表达3 102种蛋白质,获得差异表达蛋白质(差异倍数>2,P<0.05) 259种,17种差异表达蛋白质可能与牛卵泡优势化过程相关,SERPINB2可能调控牛卵泡闭锁。该研究筛选获得的牛卵泡差异表达蛋白质和特异表达蛋白质,丰富了牛卵泡发育调控理论,为进一步研究卵泡闭锁及优势化奠定基础。

基于Label-free组学研究南极磷虾油对脂代谢的影响

为了研究南极磷虾油对脂代谢的调节作用,利用非标记(Label-free)蛋白质定量技术,比较分析灌胃南极磷虾油对高脂模型小鼠肝脏蛋白的表达影响。结果表明,与模型组相比,灌胃磷虾油后,试验组和正常组中差异蛋白表达上调的分别有125个和109个,下调表达的分别有99个和95个。进一步分析脂代谢差异蛋白发现,在试验组和正常组中表达上调的分别是棕榈酰蛋白硫酯酶1 (PPT1)、载脂蛋白B100(APOB100)、短支链酰基辅酶A脱氢酶(ACADSB)、3-羟基酰基-CoA脱水酶3(HACD3)和磺基转移酶1A1(SULT1A1);表达下调的分别为酰基辅酶A合成酶中链家族成员3(ACSM3)和酰基辅酶A合成酶家族成员2(线粒体)(ACSF2)。结合脂代谢通路分析和蛋白质相互作用网络图进一步推测,ACADSB、ACSM3和ACSF2等蛋白质在南极磷虾油调节脂代谢中起着重要的调控作用。本研究结果为深入解析南极磷虾油的作用机理和调节脂代谢的分子机制提供了依据。

应用Label-free技术研究人牙囊细胞和牙周膜成纤维细胞的差异蛋白质

目的:应用Label-free蛋白定量技术研究牙囊细胞(h DFCs)和牙周膜成纤维细胞(h PDLFs)差异表达蛋白质,为发现h DFCs向h PDLFs转化过程中的重要蛋白质提供参考。方法:应用Label-free蛋白定量技术,结合ultramate 3000 nano-UPLC软件,对h DFCs和h PDLFs总蛋白提取液进行蛋白定性定量检测。然后分别对蛋白质的鉴定结果行重复性、关联性、蛋白相对含量和差异表达蛋白等进行分析;对总体蛋白质和差异表达蛋白质行生物信息学GO或KEGG分析。结果:h DFCs和h PDLFs蛋白相对含量及总体蛋白GO分析结果相似。定量分析显示,当h DFCs分化形成h PDLFs后,有22个差异表达蛋白质,其中下调蛋白15个(P<0.05,FC>2),上调蛋白7个(P<0.05,FC>2)。结论:h DFCs和h PDLFs蛋白质表达谱相似;22个差异表达蛋白质为研究h DFCs向h PDLFs的转化机制提供了方向。

活动性肺结核患者外周血CD4+T淋巴细胞的label-free蛋白组学初步分析

目的应用非标记label-free蛋白质组学方法筛选出活动性肺结核患者外周血CD4+ T淋巴细胞差异表达蛋白质,为阐明结核病的发病机制和早期诊断提供理论依据.方法收集分离9例活动性肺结核患者和9例健康人外周血CD4 T淋巴细胞,应用label-free非标记蛋白组学方法检测外周血CD4+ T淋巴细胞蛋白质谱,筛选出差异表达蛋白质。并用基因本体论(GO)及京都基因与基因组百科全书(KEGG)生物信息学分析软件对差异表达蛋白质进行分析.结果活动性肺结核与健康人外周血CD4+T淋巴细胞中的蛋白质组分布的基本框架很相似,但可发现二者之间有38个明显差异表达的蛋白点,其中有26种蛋白质在肺结核组中表达上调(差异倍数>1.5, P<0.05), 12种蛋白质在肺结核组中表达下调(差异倍数<0.67, P<0.05 ).GO分析结果表明,大部分的差异蛋白质主要定位于胞内区域,结核感染相关差异蛋白质功能体现在结合、调控以及代谢过程中.KEGG分析表明,磷酸戊糖途径、cGMP/PKG信号通路、磷脂酰肌醇信号系统等与结核分枝杆菌感染密切相关.结论对活动性肺结核患者外周血CD4+T淋巴的比较蛋白质组学研究一共鉴定岀38种差异蛋白质,他们可能在肺结核发病过程中起着重要作用,也可能可做为诊断结核病的潜在生物学标志物。

基于label-free技术的青杨3个叶位叶片比较蛋白质组学分析

为了研究青杨不同叶位叶片蛋白质组的表达变化规律和青杨叶片衰老的蛋白质组学机制,采用非标记蛋白质组学(label-free)方法对杂交青杨子代3个叶位叶片蛋白质组变化进行了分析。结果表明:3个不同叶位叶片共检测到111个差异表达蛋白质,其中55%的差异蛋白质仅在25叶位与5叶位的比较中差异显著;差异表达蛋白质功能分析表明,青杨叶片成熟到衰老过程中与光合作用、生长发育相关蛋白在10和25叶位多呈现下调表达,逆境反应、营养分解转运相关蛋白在10和25叶位多上调表达,而一些转录翻译与修饰调控相关蛋白质则主要呈现波动趋势;青杨3个叶位中,10叶位光合作用和物质合成比5叶位活跃,25叶位叶片由于衰老营养物质分解转运加强,物质合成减弱。蛋白质是细胞功能执行者,可见,青杨叶片成熟到衰老是受蛋白质表达变化有序调控的过程。

Label-free visualization of cholesteatoma in the mastoid and tympanic membrane using CARS microscopy

Objective:The present study aimed to evaluate the possibility of using coherent anti-Stokes Raman spectroscopy(CARS) microscopy to determine the specific molecular morphology of cholesteatoma by detecting the natural vibrational contrast of the chemical bonds without any staining.Materials and methods:Specimens from the mastoid and tympanic membrane with and without cholesteatoma were analyzed using CARS microscopy,two-photon excited fluorescence(TPEF) microscopy,and the second harmonic generation(SHG) microscopy.Results:In cholesteatoma tissues from the mastoid,a strong resonant signal at 2845 cm^(-1) was observed by CARS,which indicated the detection of the CH_2 hydro-carbon lipid bonds that do not generate visible signals at 2940 cm^(-1) suggestive of CH_3 bonds in amino acids.A strong resonant signal at 2940 cm^(-1) appeared in an area of the same specimen,which also generated abundant signals by TPEF and SHG microscopy at 817 nm,which was suggestive of collagen.In the tympanic membrane specimen with cholesteatoma,a strong resonant signal with corrugated morphology was detected,which indicated the presence of lipids.A strong signal was detected in the tympanic membrane with chronic otitis media using TPEF/SHG at 817 nm,which indicated collagen enrichment.The CARS and TPEF/SHG images were in accordance with the histology results.Conclusion:These results suggest the need to develop a novel CARS microendoscope that can be used in combination with TPEF/SHG to distinguish cholesteatoma from inflammatory tissues.

The application of label-free imaging technologies in transdermal research for deeper mechanism revealing

The penetration behavior of topical substances in the skin not only relates to the transdermal delivery efficiency but also involves the safety and therapeutic effect of topical products,such as sunscreen and hair growth products.Researchers have tried to illustrate the transdermal process with diversified theories and technologies.Directly observing the distribution of topical substances on skin by characteristic imaging is the most convincing approach.Unfortunately,fluorescence labeling imaging,which is commonly used in biochemical research,is limited for transdermal research for most topical substances with a molecular mass less than 500 Da.Label-free imaging technologies possess the advantages of not requiring any macromolecular dyes,no tissue destruction and an extensive substance detection capability,which has enabled rapid development of such technologies in recent years and their introduction to biological tissue analysis,such as skin samples.Through the specific identification of topical substances and endogenous tissue components,label-free imaging technologies can provide abundant tissue distribution information,enrich theoretical and practical guidance for transdermal drug delivery systems.In this review,we expound the mechanisms and applications of the most popular label-free imaging technologies in transdermal research at present,compare their advantages and disadvantages,and forecast development prospects.

基于Label-free技术探讨肌少-骨质疏松症与骨质疏松症患者骨组织差异蛋白的研究与分析

目的对“骨质疏松症(OP)”与“肌少-骨质疏松症(SO)”研究对象的骨组织样品进行蛋白定量检测、蛋白差异分析及差异蛋白功能富集分析,旨在筛选与鉴定出调控SO发生发展的差异蛋白。方法共收集6例SO与OP患者骨组织样品,采用Label-free定量蛋白质组技术进行鉴定与筛选;在基因本体论(GO)生物学资源库、蛋白互作网络(PPI)系统及京都基因与基因组百科全书(KEGG)对具有显著差异的蛋白开展生物信息研究。结果在SO组与OP组中共筛选出1395个差异蛋白,经差异显著性筛选其中上调表达有21个,表达下调有9个,上下调蛋白有统计学意义;两组间差异显著的蛋白分别是过氧化物酶-1、转化生长因子-β1、线粒体转录因子A和细胞色素C氧化酶等;差异蛋白主要涉及细胞氧化还原稳态、氧化磷酸化和代谢过程。结论过氧化物酶-1、转化生长因子-β1、线粒体转录因子A和细胞色素C氧化酶等差异蛋白可能参与了SO发生发展过程。

基于Label-free技术比较马和驴血清蛋白质组分差异

旨在探究马和驴血清蛋白质生物学特征,为马属动物种间生理特征比较和健康保障提供理论依据。本研究选取辽宁省大连市某集约化养殖场的9匹蒙古马和9头辽西驴,雌性,年龄4~10岁,均处于正常发情周期的间情期,健康无病、精神状态及采食状况良好,分别提供相同的饲养条件。采集马和驴各9份血清样品分别随机分成3组,每组内的3份血清样品均匀混合成1个生物学重复,各获得3个生物学重复的血清样品。利用Label-free蛋白质组学技术及生物信息学方法对马和驴的血清蛋白质组分进行比较研究,提取血清蛋白质,再对蛋白质进行酶解,液相色谱-串联质谱(LC-MS/MS)进行蛋白质组分分析,数据库检索分析马和驴血清样品中蛋白质表达情况,并应用生物信息学方法筛选马和驴血清差异关键调控蛋白质。结果显示,本研究共鉴定出361种蛋白质,其中,马血清中表达288种蛋白质,分子量范围为1.52~511.24 ku;驴血清中表达244种蛋白质,分子量范围为1.53~611.47 ku;共获得231种显著差异表达蛋白质(差异倍数≥1.5,P≤0.05)。差异表达蛋白质主要参与蛋白质激活、补体激活、免疫应答、凝血和脂蛋白氧化调控等生物学过程。显著富集的KEGG通路为补体和凝血级联,吞噬体,内质网蛋白质加工,抗原加工递呈,甘氨酸、丝氨酸和苏氨酸的代谢,癌症蛋白多糖等。通过差异蛋白互作分析显示,差异表达蛋白质紧密相连富集最为显著的功能模块主要为代谢途径、补体和凝血级联、吞噬体,HSP90AA1、HSPA8、APOD、APOM、SERPING1、MASP1、CALR、TUBA1B、TUBB等处在关系互作网的重要节点。马和驴在生理条件下的血清蛋白质组成特征存在一定差异,该研究为进一步揭示马属动物种间生理特征差异和有效保障健康提供数据支撑。

LABEL-FREE DETECTION OF PROTEIN MICROARRAY WITH HIGH THROUGHPUT SURFACE PLASMON RESONANCE IMAGING(SPRI)

A surface plasmon resonance imaging(SPRI)system was developed for the discrimination of proteins on a gold surface.As a label-free and high-throughput technique,SPRI enables simultaneously monitoring of the biomolecular interactions at low concentrations.We used SPRI as a label-free and parallel method to detect different proteins based on protein microarray.Bovine Serum Albumin(BSA),Casein and Immunoglobulin G(IgG)were immobilized onto the Au surface of a gold-coated glass chip as spots forming a 6×6 matrix.These proteins can be discriminated directly by changing the incident angle of light.Excellent reproducibility for label-free detection of protein molecules was achieved.This SPRI platform represents a simple and robust method for performing high-sensitivity detection of protein microarray.

Proteomic Analysis of Chrysanthemum Lateral Buds after Removing Apical Dominance Based on Label-Free Technology

Studying the genetic basis and regulatory mechanism of chrysanthemum lateral bud outgrowth is of great significance for reduction the production cost of cut chrysanthemum.To clarify the molecular basis of lateral bud elongation after removal of apical dominance in chrysanthemum,label-free quantification analysis was used to analyze the proteome changes after apical bud removal.Quantitative real-time PCR(qPCR)was used to analyze the changes in the expression of three plant hormone-related genes.A total of 440 differentially expressed proteins were successfully identified at three time points during the lateral bud elongation.The number of differentially expressed proteins in the three stages(24 h/0 h,48 h/0 h,48 h/24 h)were 219,332,and 97,respectively.The difference in expressed proteins in the three comparison stages mainly involves RNA processing and modification;translation,ribosomal structure and biogenesis;Posttranslational modification,protein turnover,and chaperones.Path analysis showed that there was various physiological activities in the process of lateral bud dormancy breaking and elongation,which involved energy metabolism,biosynthesis,signal transduction and stress response in the growth process of lateral buds.qPCR indicated that the expression of cytokinin synthesis related gene was significantly increased after the removal of apical dominance,while the expression of strigolactones synthesis related gene experiences a dramatic fall to promote the development of the lateral buds.However,there was a drop before a slight increase in the expression of the auxin synthesis related gene,which was mainly due to the removal of apical dominance that led to the loss of indoleacetic acid in the main stem.However,with formation of the new apical source,indoleacetic acid can be released again.