4-巯基苯硼酸修饰二维二硫化钼纳米复合材料的制备及其用于N-糖肽特异性富集

蛋白质的N-糖基化修饰在多种生物过程中发挥着至关重要的作用，近年来的许多研究证实异常的蛋白质糖基化与多种疾病的发生发展密切相关，表明糖基化蛋白质具有较大的潜力成为新的生物标志物或者药物靶标。在样本的处理过程中，对N-糖基化肽段进行富集分离后再进行质谱分析已经成为糖蛋白质组学分析前的必要步骤。但是，由于复杂生物样本中N-糖基化肽段的丰度低和离子化效率差等问题，通过质谱鉴定N-糖肽仍然是一项艰巨的任务。研究通过将纳米金线（Au）、4-巯基苯硼酸（4-MPB）与超薄二维二硫化钼（2D-MoS₂）进行反应，成功制备了一种用于富集蛋白质N-糖基化肽段的新型功能纳米复合材料（MoS₂/Au/4-MPB）。二硫化钼纳米材料的层状结构可以为反应提供大量的可修饰位点，便于修饰纳米金线；功能基团4-巯基苯基硼酸对N-糖肽具有高度的选择性，可以对生物样品中N-糖基化肽段进行特异性富集。使用标准蛋白人免疫球蛋白G（IgG）和牛血清白蛋白（BSA）胰蛋白酶酶切产物对新型功能纳米材料的N-糖基化肽段的富集性能进行评估，其灵敏度达到5 fmol，选择性达到1：1000。将其用于生物样品中N-糖基化肽段的富集，从50 μg尿液外泌体蛋白胰蛋白酶酶切产物中共富集鉴定出768个N-糖肽，归属于377个蛋白质。这些结果表明该新型功能纳米复合材料对复杂生物样品中N-糖肽的选择富集有着巨大的应用潜力，为糖蛋白质组的研究提供了一种新方法。     

N-糖基化蛋白质组样品富集策略的研究进展

蛋白质的糖基化是生物体内重要的蛋白质翻译后修饰之一,但其丰度通常较低,糖基化蛋白质酶解肽段中仅有2%~5%为糖基化肽段,因此,为实现糖基化蛋白质组的深度覆盖分析,对糖基化蛋白质/肽段进行富集是非常必要的。该文对糖基化蛋白质组样品不同富集方法的原理、特点以及最新研究进展进行了综述,同时也对N-糖基化蛋白质组学富集策略的发展前景进行了展望。     

基于三甲基苯磺酰羟胺消除反应的氧连接氮乙酰葡萄糖胺修饰肽段的精准鉴定

氧连接氮乙酰葡萄糖胺(O-GlcNAc)是一种重要的蛋白质翻译后修饰,它在维持机体正常的生命活动中发挥着重要作用。许多研究证实,O-GlcNAc糖基化修饰稳态的破坏与人类多种疾病的发生相关,大规模富集鉴定O-GlcNAc糖基化修饰蛋白有助于发现新的临床疾病诊断标志物。由于O-GlcNAc糖基化修饰丰度较低,形成的糖苷键不稳定,O-GlcNAc糖基化修饰蛋白/肽段的富集鉴定面临一定挑战。近年来,全乙酰化的非天然糖代谢标记技术被广泛应用于O-GlcNAc糖基化修饰蛋白/肽段的富集鉴定。然而,最新的研究发现,在细胞代谢标记过程中,全乙酰化的非天然单糖会同时标记半胱氨酸的巯基而引入半胱氨酸巯基-叠氮糖人为修饰物。该副反应在一定程度上干扰了O-GlcNAc糖基化修饰蛋白/肽段的富集鉴定。鉴于此,研究发展了一种通过三甲基苯磺酰羟胺(MSH)特异性氧化消除半胱氨酸巯基-叠氮糖人为修饰物的方法,进而显著提高O-GlcNAc糖基化修饰肽段的精准鉴定。该方法建立于温和的磷酸钠缓冲液(50 mmol/L, pH=8)体系,利用过量的MSH,于95 ℃避光振荡反应30 min,可完全消除半胱氨酸巯基-叠氮糖人为修饰物。该方法应用于Hela细胞中,可有效消除叠氮全乙酰化半乳糖胺(Ac₄GalNAz)代谢产生的半胱氨酸巯基-叠氮糖人为修饰物,从而成功富集鉴定到157条O-GlcNAc糖基化修饰肽段,归属于130个蛋白质。该方法有效去除了半胱氨酸巯基-叠氮糖人为修饰物对代谢标记结果的干扰,为非天然糖代谢标记技术在糖蛋白组学分析中的应用提供了新的研究策略。     

麦胚凝集素亲和色谱富集糖肽过程中的肽键裂解

蛋白质的糖基化是最重要的翻译后修饰之一,与蛋白质结构和功能的关系密切。凝集素亲和色谱是蛋白质糖基化研究中很常用的工具,不同的凝集素可以对不同的单糖或寡糖有特异的富集作用。麦胚凝集素（WGA）由于其特异作用的糖型广泛存在而成为使用最多的凝集素之一。在本研究中,发现将WGA用于糖肽亲和富集会导致部分肽段的降解,从而导致后续的肽段序列分析的失败。本文用4种标准蛋白质对这种现象进行了验证,结果表明肽段的降解可以发生在多个位点,其中较多地发生在酪氨酸、苯丙氨酸及亮氨酸的羧基端。这一结果提示：在糖蛋白质组研究中,如果应用WGA富集糖肽并采用质谱进行鉴定,则采用半酶切或非特异性酶切的检索策略更为合适。     

硼酸功能化的介孔氧化硅用于糖肽的高特异富集

复旦大学的赵东元院士和陆豪杰教授合作研究开发的将介孔材料成功运用于糖基化肽段富集的新技术（Anal．Chem．,2009,81（1）：503～508）。与传统策略相比,此方法不仅大大缩短了孵育时间,并且将灵敏度提高两个数量级,有望为大规模的蛋白质组后修饰鉴定提供了强有力的技术保障。     

基于亲和色谱的肺癌细胞磷酸化蛋白质组研究及其应用

磷酸化是蛋白质翻译后修饰的重要形式之一,其异常往往会导致细胞内信号通路的紊乱和疾病的发生。固定化金属离子亲和色谱(IMAC)是磷酸化肽段的高效富集技术,在磷酸化蛋白质组研究方面应用广泛。该研究以金属钛离子(Ti⁴⁺)螯合IMAC材料(Ti⁴⁺-IMAC)为载体,进行磷酸化肽段富集。比较了10 μm Ti⁴⁺-IMAC通过振荡法和固相萃取法(SPE)富集磷酸肽的效果,发现振荡法可以富集到更多的磷酸肽;对比了两种尺寸(10 μm和30 μm)Ti⁴⁺-IMAC在磷酸化肽段富集中的差异,发现小尺寸材料富集效果更佳。进一步采用优化的策略比较了不同转移能力肺癌细胞的磷酸化蛋白质组,免标记定量蛋白质组学结果表明,优化的Ti⁴⁺-IMAC方法可以从正常的肺成纤维细胞MRC5、低转移肺癌细胞95C和高转移肺癌细胞95D中分别鉴定到510、863和1108种磷酸化蛋白质,其中317种为3组所共有。该研究共鉴定到1268种磷酸化蛋白质上的7560个磷酸化位点,其中1130个为差异磷酸化位点,文献报道显示部分异常表达的激酶与癌症转移密切相关。通过生信对比分析发现,异常表达的磷酸化蛋白质主要与细胞侵袭、迁移和死亡等细胞迁移方面的功能有关。通过优化磷酸化肽富集策略,初步阐明了磷酸化蛋白质网络的异常与肺癌转移之间的相关性,该方法有望用于肺癌进展相关的磷酸化位点、磷酸化蛋白质及其信号通路研究。     

基于精氨酸酶切的蛋白质C端肽段富集方法的优化及评估北大核心CSCD

基于聚合物的蛋白质C端反向富集策略是用于研究蛋白质C端最为广泛的策略之一。目前,基于胰蛋白酶(trypsin)切割精氨酸残基C端(ArgC型酶切)的蛋白C端组学方法对蛋白质C端的鉴定深度仍有待提高。为解决这一问题,该研究对此方法进行了优化和评估:建立了基于“V型”过滤装置的“一锅法”富集流程,避免了副反应的干扰,缩短了样本的制备时间;优化了蛋白水平乙酰化反应条件,最大限度地降低了丝氨酸、苏氨酸、酪氨酸残基上的副反应,提高了肽段鉴定的可信性;优化了基于固相萃取枪头膜片过滤柱(StageTip柱)的样品分离过程,使C端肽段的鉴定深度增加至原来的4倍。通过以上优化,按照肽段水平错误发现率(FDR)<0.01、离子分数(ion score)≥20,且C端带有乙醇胺修饰的数据筛选标准,从人HEK 293T细胞中共鉴定出696个蛋白质C端。若仅要求肽段水平FDR<0.01,鉴定数目进一步增加到933个,这是基于聚合物富集策略的蛋白质C端组学方法所得的最大数据集之一。探索了胰蛋白酶镜像酶(LysargiNase)切割精氨酸残基N端(ArgN型酶切)与不同肽段N端衍生化修饰组合对蛋白质C端鉴定数目和种类的影响,“LysargiNase酶切+肽段N端乙酰化”新策略在原有“胰蛋白酶酶切+肽段N端二甲基化”策略的基础上将鉴定蛋白质C端的种类提升了47%。综上,该研究通过对基于Arg型酶切的蛋白C端组学方法的优化,提升了C端肽段的鉴定深度,扩大了C端肽段鉴定的覆盖范围。该方法将有望成为系统性表征蛋白质C端的有力工具。     

纳米材料在蛋白磷酸化分析中的应用研究

蛋白质磷酸化修饰是一种重要的蛋白质翻译后修饰,在细胞代谢过程中发挥着重要作用。当蛋白质的正常磷酸化调节发生异常时,会导致癌症、糖尿病、心脏病等各种疾病的发生。因此,蛋白磷酸化分析对于疾病的早期快速诊断、药物筛选和治疗等方面具有重大的意义。由于蛋白质磷酸化过程是动态的,并且磷酸化肽段或蛋白在生物样品中的含量较低,因此高灵敏的蛋白磷酸化分析面临着巨大的挑战。该文依据在检测过程中,选择性识别或捕获磷酸化的肽段或蛋白的主要机理,综述了近几年纳米材料对磷酸化肽段的富集和信号放大作用在蛋白磷酸化分析中的研究进展,并对其未来研究方向进行了展望。     

完整糖基化肽段的富集与质谱解析新技术研究进展

蛋白质糖基化是生物体内最重要的翻译后修饰之一,在蛋白质稳定性、细胞内和细胞间信号转导、激素活化或失活和免疫调节等生理过程和病理进程中发挥重要作用。而异常的蛋白质糖基化往往和多种疾病的发生发展密切相关,目前应用于临床检测的多种肿瘤生物标志物大多属于糖蛋白或者糖抗原。因此在组学层次系统分析蛋白质糖基化的变化对阐明生物体内糖基化修饰的调控机理和发现新型疾病标志物都非常重要。基于质谱的蛋白质组学技术为全面分析蛋白质及其修饰提供了有效的分析手段。在自下而上的蛋白质组学研究中,由于完整糖基化肽段同时存在性质各异的肽段骨架和糖链结构、糖肽的相对丰度和离子化效率较低以及糖基化修饰有高度异质性等特点,完整糖肽的分析比其他翻译后修饰更加困难。近年来,为了更全面、系统地分析蛋白质糖基化,研究人员发展了一些新技术,包括完整糖肽的富集技术、质谱的碎裂模式和数据采集模式、质谱数据的解析方法和定量策略等等,大力推进了该领域的研究水平,也为研究蛋白质糖基化相关的生物标志物提供了技术支持。该篇综述主要关注近年来基于质谱的糖蛋白质组学研究中的新进展,重点介绍针对完整N-和O-糖基化肽段的富集新技术和谱图解析新方法,并讨论其在肿瘤早期诊断方面的应用潜力。     

季铵化磁性壳聚糖复合材料对磷酸化肽的富集研究

蛋白质磷酸化是最重要和最普遍的翻译后修饰之一,蛋白质磷酸化的测定对于全面了解生物过程中的磷酸化途径至关重要。质谱技术是分析蛋白质磷酸化的重要手段,但磷酸化肽固有的低丰度、低电离效率以及与非磷酸化肽共存等特性严重影响质谱对其进行直接分析。为解决此问题,需在质谱分析前对磷酸化肽进行选择性富集。本研究制备了一种基于季铵化磁性壳聚糖的复合材料用于磷酸化肽的富集。此磁性材料具有快速的磁响应性、良好的生物相容性、正电性以及廉价易得等优点。采用β-酪蛋白作为模型蛋白质,结果表明此材料对磷酸化肽具有良好的富集选择性。经过富集后,结合基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）检测手段,方法的检出限为0.4 fmol。本方法被成功用于脱脂牛奶中磷酸化肽的检测,表明其对复杂样品中磷酸化肽的富集和检测具有良好的应用潜力。     

Discovery of Post-Translational Modifications in Emiliania huxleyi

Emiliania huxleyi is a cosmopolitan coccolithophore that plays an essential role in global carbon and sulfur cycling, and contributes to marine cloud formation and climate regulation. Previously, the proteomic profile of Emiliania huxleyi was investigated using a three-dimensional separation strategy combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The current study reuses the MS/MS spectra obtained, for the global discovery of post-translational modifications (PTMs) in this species without specific enrichment methods. Twenty-five different PTM types were examined using Trans-Proteomic Pipeline (Comet and PeptideProphet). Overall, 13,483 PTMs were identified in 7421 proteins. Methylation was the most frequent PTM with more than 2800 modified sites, and lysine was the most frequently modified amino acid with more than 4000 PTMs. The number of proteins identified increased by 22.5% to 18,780 after performing the PTM search. Compared to intact peptides, the intensities of some modified peptides were superior or equivalent. The intensities of some proteins increased dramatically after the PTM search. Gene ontology analysis revealed that protein persulfidation was related to photosynthesis in Emiliania huxleyi. Additionally, various membrane proteins were found to be phosphorylated. Thus, our global PTM discovery platform provides an overview of PTMs in the species and prompts further studies to uncover their biological functions. The combination of a three-dimensional separation method with global PTM search is a promising approach for the identification and discovery of PTMs in other species.     

Method for the synthesis of mono-ADP-ribose conjugated peptides

ADP-ribosylation is an important post-translational modification involved in processes including cellular replication, DNA repair, and cell death. Despite these roles, the functions of ADP-ribosylation, in particular mono-ADP-ribosylation, remain poorly understood. The development of a technique to generate large amounts of site-specific, ADP-ribosylated peptides would provide a useful tool for deconvoluting the biochemical roles of ADP-ribosylation. Here we demonstrate that synthetic histone H2B tail peptides, incorporating aminooxy or N-methyl aminooxy functionalized amino acids, can be site-specifically conjugated to ADP-ribose. These peptides are recognized as substrates by the ADP-ribosylation biochemical machinery (PARP1), can interact with the ADP-ribose binding proteins macroH2A1.1 and PARP9, and demonstrate superior enzymatic and chemical stability when compared to ester-linked ADP-ribose. In addition, the incorporation of benzophenone photo-cross-linkers into these peptides is demonstrated to provide a means to probe for and enrich ADP-ribose binding proteins.     

Design of peptide–dendrimer conjugates with tumor homing and antitumor effects

Development of drug delivery systems for cancer therapy is a crucial issue. Previously, some peptides were designed as tumor homing cell-penetrating peptides with antitumor activities. In this study, dual function dendrimers with tumor targeting activities and antitumor effects were designed using the tumor targeting CPP44 peptide for acute myelogenous leukemia (AML) and the antitumor p16^INK4a peptide. Two types of peptide–dendrimer conjugates were synthesized. One was a CPP44-linked p16^INK4a peptide-conjugated dendrimer (tandem linked dendrimer) and the other was a dendrimer conjugated with separate CPP44 and p16^INK4a peptides (parallel linked dendrimer). In addition, a peptide cathepsin B substrate was linked to the antitumor p16^INK4a peptide to release it from the carriers. These peptide–dendrimer conjugates produced more effective antitumor effects than a CPP44-linked p16^INK4a peptide. The parallel linked dendrimer showed less association with AML cells than the tandem linked dendrimer, but had greater antitumor effects. This suggested that both cellular uptake and antitumor peptide cleavage affected the antitumor activities of dual functional peptide-conjugated dendrimers.     

Gel electrophoresis/electroelution sorting fractionator combined with filter-aided sample preparation for deep proteomic analysis

Sample preparation and protein fractionation are important issues for proteomic studies. Protein extraction procedures strongly affect the performance of fractionation methods by provoking protein dispersion in several fractions. The most notable exception is the gel-based electrophoretic protein fractionation due to its resolution and effectiveness of sodium dodecyl sulfate as a solubilizing agent, while its main limitation lies in the poor recovery of the gel-trapped proteins. We created a fractionator device to separate complex mixture of proteins and peptides that is based on the continuous gel electrophoresis/electroelution sorting of these molecules. In an unsupervised process, complex mixtures of proteins or peptides are fractionated into the gel while separated fractions are simultaneously and sequentially electroeluted to the solution containing wells. The performance of the device was studied for protein fractionation in terms of reproducibility, protein recovery, and loading capacity. In a setup free of sodium dodecyl sulfate, complex peptide mixtures can also be fractionated. More than 11,700 proteins were identified in the whole-cell lysate of the CaSki cell line by using the fractionator combined with the filter-aided sample preparation method and mass spectrometry analysis. Fractionator-based proteome characterization increased 1.7-fold the number of identified proteins compared to the unfractionated sample analysis.     

基于高分辨质谱技术的婴幼儿食品中过敏原蛋白质的高灵敏检测

基于高效液相色谱-串联质谱(LC-MS/MS)技术,选择稳定性好、灵敏度高的特征肽段,利用平行反应监测(PRM)技术,实现了多类过敏原蛋白质的高灵敏度同时检测,并成功应用于婴幼儿食品中过敏原成分的分析。对于婴幼儿食品中蛋白质的提取,与传统的丙酮沉淀法比,采用膜上原位样品预处理方法(i-FASP)可实现更高的蛋白质提取效率和抗干扰能力。所检测的过敏原蛋白质的定量限(LOQ)最小可达到0.028 mg/L,其线性范围最宽可跨越4个数量级,且线性关系良好(相关系数R~2≥0.99)。该方法为食品中过敏原蛋白质组学快速分析提供了一种可靠的分析方法。     

Fabrication of metal nanoparticle monolayers on amphiphilic poly(amido amine) dendrimer Langmuir films

A newly designed 1.5th generation poly(amido amine) dendrimer with an azacrown core, hexylene spacers, and octyl terminals was spread on gold nanoparticle (Au-NP) suspension. The surface pressure-area isothermal curves indicated that the molecular area of dendrimer on Au-NP suspension was significantly smaller than that on water, indicating the formation of dendrimer/Au-NP composites. The dendrimer Langmuir films on the Au-NP suspension were transferred to copper grids at various surface pressures and observed by transmission electron microscopy. The transferred films consisted of a fractal-like network of nanoparticles at low surface pressure and of a defect-rich monolayer of nanoparticles at high surface pressure. From these results, it was suggested that the dendrimers bind Au-NPs, and dendrimer/Au-NP composites formed networks or monolayers at the interface. From the intensity decrease of the Au plasmon band of Au-NP suspension after the formation of composite, it was estimated that some (approximately 14) dendrimer molecules bind to one Au-NP. Furthermore, neutron reflectivity at the air/suspension interface and X-ray reflectivity of the film transferred on a silicon substrate revealed that the dendrimer molecules are localized on the upper-half surface of Au-NP. Metal affinity of azacrown, flexibility of hexylene spacer, and amphiphilicity of dendrimer with octyl terminals played important roles for the formation of dendrimer/Au-NP hybrid films. The present investigation proposed a new method to fabricate the self-assembled functional polymer/nanoparticle hybrid film.     

A Method for Microalgae Proteomics Analysis Based on Modified Filter-Aided Sample Preparation

With the fast development of microalgal biofuel researches, the proteomics studies of microalgae increased quickly. A filter-aided sample preparation (FASP) method is widely used proteomics sample preparation method since 2009. Here, a method of microalgae proteomics analysis based on modified filter-aided sample preparation (mFASP) was described to meet the characteristics of microalgae cells and eliminate the error caused by over-alkylation. Using Chlamydomonas reinhardtii as the model, the prepared sample was tested by standard LC-MS/MS and compared with the previous reports. The results showed mFASP is suitable for most of occasions of microalgae proteomics studies.     

Evaluation of Sample Preparation Strategies for Human Milk and Plasma Proteomics

Sample preparation is the most critical step in proteomics as it directly affects the subset of proteins and peptides that can be reliably identified and quantified. Although a variety of efficient and reproducible sample preparation strategies have been developed, their applicability and efficacy depends much on the biological sample. Here, three approaches were evaluated for the human milk and plasma proteomes. Protein extracts were digested either in an ultrafiltration unit (filter-aided sample preparation, FASP) or in-solution (ISD). ISD samples were desalted by solid-phase extraction prior to nRPC-ESI-MS/MS. Additionally, milk and plasma samples were directly digested by FASP without prior protein precipitation. Each strategy provided inherent advantages and disadvantages for milk and plasma. FASP appeared to be the most time efficient procedure with a low miscleavage rate when used for a biological sample aliquot, but quantitation was less reproducible. A prior protein precipitation step improved the quantitation by FASP due to significantly higher peak areas for plasma and a much better reproducibility for milk. Moreover, the miscleavage rate for milk, the identification rate for plasma, and the carbamidomethylation efficiency were improved. In contrast, ISD of both milk and plasma resulted in higher miscleavage rates and is therefore less suitable for targeted proteomics.     

Selective enrichment of glycopeptides for mass spectrometry analysis using C18 fractionation and titanium dioxide chromatography

Comprehensive glycoprotein characterization based on mass spectrometry (MS) is challenging because of low concentration of glycopeptides and suppression effect of abundant non-glycosylated peptides in MS. Therefore, it is vital to enrich glycopeptides before MS analysis. A new method was developed to selectively enrich glycopeptides from complex sample by coupling C18 fractionation with titanium dioxide (TiO(2)) enrichment. The new method allows to selectively enrich N-linked glycopeptides with various glycan forms and different sequence lengths. Compared with single TiO(2) method, the established method demonstrated higher glycopeptide selectivity and higher glycosylation heterogeneity coverage. Further application of this method to mixture of non-glycosylated protein and glycoprotein digests at different levels reveals the feasibility of enrichment of tryptic glycopeptides from simple proteomics samples.