What is targeted proteomics? A concise revision of targeted acquisition and targeted data analysis in mass spectrometry

Targeted proteomics has gained significant popularity in mass spectrometry‐based protein quantification as a method to detect proteins of interest with high sensitivity, quantitative accuracy and reproducibility. However, with the emergence of a wide variety of targeted proteomics methods, some of them with high‐throughput capabilities, it is easy to overlook the essence of each method and to determine what makes each of them a targeted proteomics method. In this viewpoint, we revisit the main targeted proteomics methods and classify them in four categories differentiating those methods that perform targeted data acquisition from targeted data analysis, and those methods that are based on peptide ion data (MS1 targeted methods) from those that rely on the peptide fragments (MS2 targeted methods).     

Targeted enrichment of genomic DNA regions for next-generation sequencing

In this review, we discuss the latest targeted enrichment methods and aspects of their utilization along with second-generation sequencing for complex genome analysis. In doing so, we provide an overview of issues involved in detecting genetic variation, for which targeted enrichment has become a powerful tool. We explain how targeted enrichment for next-generation sequencing has made great progress in terms of methodology, ease of use and applicability, but emphasize the remaining challenges such as the lack of even coverage across targeted regions. Costs are also considered versus the alternative of whole-genome sequencing which is becoming ever more affordable. We conclude that targeted enrichment is likely to be the most economical option for many years to come in a range of settings.     

Targeted proteomics on its way to discovery

For a long time, targeted and discovery proteomics covered different corners of the detection spectrum, with targeted proteomics focused on small target sets. This changed with the recent advances in highly multiplexed analysis. While discovery proteomics still pushes higher numbers of identified and quantified proteins, the advances in targeted proteomics rose to cover large parts of less complex proteomes or proteomes with low protein detection counts due to dynamic range restrictions, like the blood proteome. These new developments will impact, especially on the field of biomarker discovery and the possibility of using targeted proteomics for diagnostic purposes.     

Index-ion triggered MS2 ion quantification: a novel proteomics approach for reproducible detection and quantification of targeted proteins in complex mixtures

Biomedical research requires protein detection technology that is not only sensitive and quantitative, but that can reproducibly measure any set of proteins in a biological system in a high throughput manner. Here we report the development and application of a targeted proteomics platform termed index-ion triggered MS2 ion quantification (iMSTIQ) that allows reproducible and accurate peptide quantification in complex mixtures. The key feature of iMSTIQ is an approach called index-ion triggered analysis (ITA) that permits the reproducible acquisition of full MS2 spectra of targeted peptides independent of their ion intensities. Accurate quantification is achieved by comparing the relative intensities of multiple pairs of fragment ions derived from isobaric targeted peptides during MS2 analysis. Importantly, the method takes advantage of the favorable performance characteristics of the LTQ-Orbitrap, which include high mass accuracy, resolution, and throughput. As such it provides an attractive targeted proteomics tool to meet the demands of systems biology research and biomarker studies.     

Targeted proteomics by selected reaction monitoring mass spectrometry: applications to systems biology and biomarker discovery

Mass Spectrometry-based proteomics is now considered a relatively established strategy for protein analysis, ranging from global expression profiling to the identification of protein complexes and specific post-translational modifications. Recently, Selected Reaction Monitoring Mass Spectrometry (SRM-MS) has become increasingly popular in proteome research for the targeted quantification of proteins and post-translational modifications. Using triple quadrupole instrumentation (QqQ), specific analyte molecules are targeted in a data-directed mode. Used routinely for the quantitative analysis of small molecular compounds for at least three decades, the technology is now experiencing broadened application in the proteomics community. In the current review, we will provide a detailed summary of current developments in targeted proteomics, including some of the recent applications to biological research and biomarker discovery.     

利用人工锌指蛋白核酸酶进行植物基因定点突变和置换

基因定点突变技术在基因组原位改变基因特定序列,避免常规转基因过程中位置效应和插入失活。定点突变生物体不含转基因或标记基因,降低风险性。高等植物基因定点突变研究初见端倪,将可能为基因原位功能研究、作物遗传改良和分子设计提供有效策略。利用锌指蛋白核酸酶（Zinc Finger Nucleases, ZFN）引入DNA定点断裂（Double-Strand Breaks, DSBs）可以高效介导基因定点突变,使得ZFN在基因定点突变中倍受关注。文章综述了植物基因定点突变的一般策略,重点介绍了锌指蛋白的结构、原理、应用,特别是ZFN介导的植物基因定点突变与置换研究进展,并对ZFN介导的植物基因定点突变与置换应用前景进行了讨论。     

急性髓系白血病mRNA与非编码RNA差异表达谱及CeRNA调控网络分析

利用GEO数据库(gene expression omnibus database)通过生物信息学分析方法探讨急性髓系白血病(acute myelogenous leukemia,AML)的发病机制。检索GEO数据库中AML相关芯片数据集GSE142698、GSE142699和GSE96535。利用GEO2R分析得到差异mRNAs、miRNAs以及差异lncRNAs。利用在线生物信息学分析工具DAVID对差异mRNAs进行GO富集分析和KEGG通路分析。利用miRWalk数据库预测AML相关miRNAs的靶向mRNAs,利用Spongescan数据库预测AML相关miRNAs的靶向lncRNAs,构建lncRNA-miRNA-mRNA竞争性内源RNA （competing endogenous RNA,ceRNA）调控网络。共筛选出29个显著差异mRNAs、70个显著差异miRNAs和20 005个显著差异lncRNAs。GO富集分析和KEGG通路分析显示,差异表达基因主要涉及蛋白磷酸化、细胞分裂、细胞增殖的负调控、基因表达的正向调节、周期蛋白依赖的丝氨酸/苏氨酸激酶活性的调节等生物过程以及细胞周期、细胞衰老、癌症通路、PI3K-Akt通路等信号通路。将miRWalk数据库预测的靶向mRNAs与差异mRNAs取交集,Spongescan数据库预测的靶向lncRNAs与差异lncRNAs取交集,分别确定了25个mRNAs、6个lncRNAs参与AML相关ceRNA调控网络的构建。结果表明,lncRNAs可能作为关键的ceRNA,通过调控miRNA和相关靶基因参与AML的发生与发展,研究结果为AML诊断和治疗的分子生物学研究提供了新的依据。     

中国人群中16个已知肺癌基因突变的综合性分析

目的:对过去已知的肺癌基因在中国人群中的突变分布进行综合性的分析,指导肺癌的临床治疗。方法:通过文献查阅,挑选出16个已知的肺癌基因。在112例肺癌样本中,对这16个基因进行大样本的靶基因测序并用Sanger测序来验证。同时,对突变在不同亚组中的分布差异进行分析。结果:16个已知肺癌基因突变可评价60.4%肺癌样本。同时,这些基因的在不同的样本亚组中表现出不同的突变特点;通过功能域的分析及蛋白空间结构的模拟,发现9个可能的突变热点既位于蛋白的功能域内又能导致蛋白空间结构或者表面电荷分布的异常。结论:通过靶基因深度测序,全面分析了16个已知肺癌基因在肺癌不同亚组中的突变分布差异,发现并初步验证了9个可能的肺癌突变热点。     

Multiplexed array-based and in-solution genomic enrichment for flexible and cost-effective targeted next-generation sequencing

The unprecedented increase in the throughput of DNA sequencing driven by next-generation technologies now allows efficient analysis of the complete protein-coding regions of genomes (exomes) for multiple samples in a single sequencing run. However, sample preparation and targeted enrichment of multiple samples has become a rate-limiting and costly step in high-throughput genetic analysis. Here we present an efficient protocol for parallel library preparation and targeted enrichment of pooled multiplexed bar-coded samples. The procedure is compatible with microarray-based and solution-based capture approaches. The high flexibility of this method allows multiplexing of 3-5 samples for whole-exome experiments, 20 samples for targeted footprints of 5 Mb and 96 samples for targeted footprints of 0.4 Mb. From library preparation to post-enrichment amplification, including hybridization time, the protocol takes 5-6 d for array-based enrichment and 3-4 d for solution-based enrichment. Our method provides a cost-effective approach for a broad range of applications, including targeted resequencing of large sample collections (e.g., follow-up genome-wide association studies), and whole-exome or custom mini-genome sequencing projects. This protocol gives details for a single-tube procedure, but scaling to a manual or automated 96-well plate format is possible and discussed.     

Analytical strategies for LC-MS-based targeted metabolomics

Recent advances in mass spectrometry are enabling improved analysis of endogenous metabolites. Here we discuss several issues relevant to developing liquid chromatography-electrospray ionization-mass spectrometry methods for targeted metabolomics (i.e., quantitative analysis of dozens to hundreds of specific metabolites). Sample preparation and liquid chromatography approaches are discussed, with an eye towards the challenge of dealing with a diversity of metabolite classes in parallel. Evidence is presented that heated electrospray ionization (ESI) generally gives improved signal compared to the more traditional unheated ESI. Applicability to targeted metabolomics of triple quadrupole mass spectrometry operating in multiple reaction monitoring (MRM) mode and high mass resolution full scan mass spectrometry (e.g., time-of-flight, Orbitrap) are described. We suggest that both are viable solutions, with MRM preferred when targeting a more limited number of analytes, and full scan preferred for its potential ability to bridge targeted and untargeted metabolomics.     

The effects of plant extracts on microbial community structure in a rumen-simulating continuous-culture system as revealed by molecular profiling

An in vitro study in dual-flow continuous-culture fermentors was conducted with two different concentrations of monensin, cinnamaldehyde or garlic extract added to 1:1 forage-to-concentrate diet in order to determine their effects on selected rumen bacterial populations. Samples were subjected to total DNA extraction, restriction analysis of PCR amplified parts of 16S rRNA genes (ARDRA) and subsequent analysis of the restriction profiles by lab-on-chip technology with the Agilent's Bioanalyser 2100. Eub338-BacPre primer pair was used to select for the bacteria from the genera Bacteroides, Porphyromonas and Prevotella, especially the latter representing the dominant Gram-negative bacterial population in the rumen. Preliminary results of HaeIII restriction analysis show that the effects of monensin, cinnamaldehyde and garlic extract on the BacPre targeted ruminal bacteria are somewhat different in regard to targeted populations and to the nature of the effect. Garlic extract was found to trigger the most intensive changes in the structure of the BacPre targeted population. Comparison of the in silico restriction analysis of BacPre sequences deposited in different DNA databanks and of the results of performed amplified ribosomal DNA restriction analysis showed differences between the predicted and obtained HaeIII restriction profiles, and suggested the presence of novel, still unknown Prevotella populations in studied samples.     

Unraveling the Distribution and Evolution of miR156 targeted SPLs in Plants by Phylogenetic Analysis

Squamosa promoter binding protein like genes (SPLs) are critical during plant development and mostly regulated by miR156. However, little is known about phylogenetic distribution and evolutionary patterns of miR156 targeted SPLs. In this study, 183 SPLs from nine genome sequenced species representing algae, bryophytes, lycophyte, monocots, and eudicots were computationally analyzed. Our results showed that miR156 responsive elements (MREs) on SPLs were present in land plants but absent from unicellular green algae. Phylogenetic analysis revealed that miR156 targeted SPLs only distributed in group II not group I of land plants, suggesting they originated from a common ancestor. In addition, group II were further divided into seven subgroups (IIa IIg) and miR156 targeted SPLs distributed in some specific members of SPLs from six subgroups except subgroup IId. Such distribution pattern was well elucidated by gene structure evolution of miR156 targeted SPLs based on the correlation of phylogenetic classification and gene structure. They could suffer from the exon loss events combined with MREs loss during evolution. Moreover, gene duplication contributed to the abundance of miR156 targeted SPLs, which had significantly increased after angiosperms and lower plants split. With Arabidopsis as the model species, we found segmental and tandem gene duplications predominated during miR156 targeted SPLs expansion. Taken together, these results provide better insights in understanding the function diversity and evolution of miR156 targeted SPLs in plants.     

Application of targeted quantitative proteomics analysis in human cerebrospinal fluid using a liquid chromatography matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometer (LC MALDI TOF/TOF) platform

Targeted quantitative proteomics by mass spectrometry aims to selectively detect one or a panel of peptides/proteins in a complex sample and is particularly appealing for novel biomarker verification/validation because it does not require specific antibodies. Here, we demonstrated the application of targeted quantitative proteomics in searching, identifying, and quantifying selected peptides in human cerebrospinal spinal fluid (CSF) using a matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometer (MALDI TOF/TOF)-based platform. The approach involved two major components: the use of isotopic-labeled synthetic peptides as references for targeted identification and quantification and a highly selective mass spectrometric analysis based on the unique characteristics of the MALDI instrument. The platform provides high confidence for targeted peptide detection in a complex system and can potentially be developed into a high-throughput system. Using the liquid chromatography (LC) MALDI TOF/TOF platform and the complementary identification strategy, we were able to selectively identify and quantify a panel of targeted peptides in the whole proteome of CSF without prior depletion of abundant proteins. The effectiveness and robustness of the approach associated with different sample complexity, sample preparation strategies, as well as mass spectrometric quantification were evaluated. Other issues related to chromatography separation and the feasibility for high-throughput analysis were also discussed. Finally, we applied targeted quantitative proteomics to analyze a subset of previously identified candidate markers in CSF samples of patients with Parkinson's disease (PD) at different stages and Alzheimer's disease (AD) along with normal controls.     

Mouse thymus targeted peptide isolated by in vivo phage display can inhibit bioactivity of thymus output in vivo

Heterogeneity of the vasculature in different organs has been well documented by the method of in vivo phage display. Using this technology, several peptide ligands that home to tissue-specific vascular endothelial cell have been isolated. Such peptide ligands directed against specific vascular surface molecules can be used as targeted therapeutic compounds or imaging agents to the vasculature of the specific organ in vivo. In this study, the authors perform in vivo selection in mice using a phage display random peptide library and separated phage peptides homing to mouse thymus by 3 rounds of in vivo panning. Sequence analysis showed that CHAQGSAEC is the dominant peptide sequence. Immunohistochemistry confirmed that the phage peptide CHAQGSAEC can bind specifically to thymus blood vessels in mice. Furthermore, phage peptide CHAQGSAEC and free peptide CHAQGSAEC can inhibit the bioactivity of thymus output in vivo. These results indicate the feasibility of the targeted peptide for possible function as a kind of tool to inhibit thymus bioactivity or as a targeted compound for targeted medicine.     

Proteomic analysis of phosphorylation in cancer

Constitutive activity of kinases is known to be crucial for a tumor to maintain its malignant phenotype, a phenomenon which is often referred to as oncogene addiction. The in-depth analysis of aberrant signaling pathways by the analysis of protein phosphorylation has become feasible through recent advances in proteomics technology. In this article we will review developments in the field of phosphoproteomics and its application in cancer research. The most widely used technologies for the generic enrichment of phosphopeptides are discussed as well as targeted approaches for the analysis of a specific subset of phosphopeptides. Validation experiments of phosphorylation sites using targeted mass spectrometry are also explained. Finally, we will highlight applications of phosphoproteomic technology in cancer research using cell lines and tissue.     

Optimum combination of targeted 131I therapy and total-body irradiation for treatment of disseminated tumors of differing radiosensitivity.

131I is the radionuclide most commonly used in biologically targeted radiotherapy at the present time. Microdosimetric analysis has shown that microtumors whose diameters are less than the beta-particle maximum range absorb radiation energy inefficiently from targeted radionuclides. Micrometastases of diameters < 1 mm are likely to be spared if targeted 131I is used as a single modality. Because of this, combined modality therapy incorporating targeted 131I, external beam total-body irradiation (TBI), and bone marrow rescue has been proposed. In this study, the minimum necessary TBI component is shown to depend on the radiosensitivity of the tumor cells. The analysis shows that the TBI component, to achieve radiocurability, increases directly with tumor radioresistance. For the most radiosensitive tumors, a whole-body TBI treatment dose 2 x 2 Gy is calculated to be obligatory, whereas practical arguments exist in favor of higher doses. For more radioresistant tumors, the analysis implies that a TBI treatment delivery of 5 x 2 Gy is obligatory. In all situations, external beam TBI appears to be an essential factor in providing reasonable probability of cure of disseminated malignant disease. Reasonable prospects of tumor cure by combination strategies incorporating 131I exist for the more radiosensitive tumor types (e.g., neuroblastoma, lymphoma, leukemia, myeloma, seminoma), but more resistant tumors are unlikely to be curable at present. Superior targeting agents, and the possible use of panels of different radionuclides, may be necessary to achieve high cure probabilities for less radiosensitive tumor types.     

miR156靶基因SPL在植物中的系统分布和进化模式分析

Squamosa promoterbinding proteinlike genes （SPLs）在植物发育过程中具有重要作用。很多SPLs被miR156调节,然而,对于它们在植物中的系统分布和进化模式还知之甚少。本文对9个测序物种（藻类,苔藓,石松,单子叶和双子叶植物）的183个SPLs进行了生物信息学分析。结果表明miR156应答元件（MREs）仅在陆生植物SPLs中发现,藻类中不存在。系统进化分析显示陆生植物SPLs分为两大分支：group I和group II。 MiR156靶基因仅分布于group II,表明它们有着共同的祖先。Group II进一步分为7个亚支（IIaIIg）,miR156靶基因分布在除IId外的其余6个亚支的特定SPLs。系统分类与基因结构的相关性反映了SPL靶基因结构上的变化。在进化过程中,它们可能发生外显子的丢失且伴随MRE的丢失。另外,基因重复对SPL靶基因的丰度变化影响很大,尤其是被子植物与低等植物分歧后它们数量明显增加。以拟南芥为模式植物分析发现串联重复和片段重复是SPL靶基因扩张的主要机制。     

NOP56在乳腺癌组织中的表达情况及对临床预后的意义

NOP56是一种与癌基因表达密切相关的核仁蛋白。本文通过对在线数据进行差异表达基因分析,发现NOP56在乳腺癌组织中高表达。再以NOP56的表达高低为表型,分析不同表型与临床预后的差异,结果表明NOP56高表达与乳腺癌不良临床病理参数和预后密切相关。通过富集分析获得NOP56的蛋白互作网络、计算共表达基因语义相似性。最后通过在线数据库获得NOP56及其共表达基因的的临床靶向药物放线菌素D(更生霉素)。这些结果为乳腺癌防治提供了潜在的新的预测指标,完善了临床靶向药物使用的分子机制,为靶向药物的临床使用提供依据和线索。