SILIP: a novel stable isotope labeling method for in planta quantitative proteomic analysis

Due to ease of manipulation, metabolic isotope coding of samples for proteomic analysis is typically performed in cell culture, thus preventing an accurate in vivo quantitative analysis, which is only achievable in intact organisms. To address this issue in plant biology, we developed SILIP (stable isotope labeling in planta) using tomato plants (Solanum lycopersicum cv. Rutgers) as a method that allows soil-grown plants to be efficiently labeled using a 14N/15N isotope coding strategy. After 2 months of growth on 14N- and 15N-enriched nitrogen sources, proteins were extracted from four distinct tomato tissues (roots, stems, leaves and flowers), digested, and analyzed by LC/MS/MS (data-dependent acquisition, DDA) and alternating low- and elevated-energy MS scans (data-independent acquisition, MS(E)). Using a derived relationship to generate a theoretical standard curve, the measured ratio of the M (monoisotopic) and M-1 isotopologues of 70 identified 15N-labeled peptides from 16 different proteins indicated that 15N incorporation was almost 99%, which is in excellent agreement with the 99.3% 15N-enriched nitrate used in the soil-based medium. Values for the various tissues ranged from 98.2 +/- 0.3% 15N incorporation in leaves to 98.8 2 +/- 0.2% in stems, demonstrating uniform labeling throughout the plant. In addition, SILIP is compatible with root-knot nematode (Meloidogyne spp.) development, and thus provides a new quantitative proteomics tool to study both plant and plant-microorganism systems.     

Characterization of mature rat oligodendrocytes: a proteomic approach

Oligodendrocytes are glial cells responsible for the synthesis and maintenance of myelin in the central nervous system (CNS). Oligodendrocytes are vulnerable to damage occurring in a variety of neurological diseases. Understanding oligodendrocyte biology is crucial for the dissemination of de- and remyelination mechanisms. The goal of the present study is the construction of a protein database of mature rat oligodendrocytes. Post-mitotic oligodendrocytes were isolated from mature Wistar rats and subjected to immunocytochemistry. Proteins were extracted and analyzed by means of two-dimensional gel electrophoresis and two-dimensional liquid chromatography, both coupled to mass spectrometry. The combination of the gel-based and gel-free approach resulted in confident identification of a total of 200 proteins. A minority of proteins were identified in both proteomic strategies. The identified proteins represent a variety of functional groups, including novel oligodendrocyte proteins. The results of this study emphasize the power of the applied proteomic strategy to study known or to reveal new proteins and to investigate their regulation in oligodendrocytes in different disease models.     

磷蛋白组的研究技术及其进展

真核细胞中蛋白质磷酸化是一个重要事件。真核细胞利用可逆的蛋白磷酸化来控制许多细胞过程包括信号转换、基因表达、细胞周期等。磷蛋白组的研究涉及磷蛋白的分离和鉴定 ,磷酸化残基定位和定量分析。由于蛋白质磷酸化是一个动态过程 ,在细胞中磷蛋白含量低 ,磷酸化位点可变 ,且磷酸肽的质谱信号常常会受到抑制 ,所以磷蛋白的分析存在更多的困难。本文介绍了国内外在磷酸蛋白的分离鉴定及定量分析方面的研究技术以及进展情况。目前 ,质谱仍然是核心的鉴定技术 ,寻找更好富集方法是最大的挑战。定量蛋白组学是对蛋白质的差异表达进行精确的定量分析。目前还不存在一种独立的方法可以完成磷蛋白的分离、鉴定 ,以及磷酸位点的定位和定量分析。随着样品分离技术和相关仪器的发展 ,磷酸蛋白快速、准确、全面分析鉴定将能够实现。     

Mass spectrometry-based proteomic approaches to study pathogenic bacteria-host interactions

Elucidation of molecular mechanisms underlying hostpathogen interactions is important for control and treatment of infectious diseases worldwide. Within the last decade, mass spectrometry (MS)-based proteomics has become a powerful and effective approach to better understand complex and dynamic host-pathogen interactions at the protein level. Herein we will review the recent progress in proteomic analyses towards bacterial infection of their mammalian host with a particular focus on enteric pathogens. Large-scale studies of dynamic proteomic alterations during infection will be discussed from the perspective of both pathogenic bacteria and host cells.     

Mass spectrometry-based quantitative proteomics

A major aim of present-day proteomics is to study changes in protein expression levels at a global level, ideally monitoring all proteins present in cells or tissue. Mass spectrometry is a well-respected technology in proteomics that is widely used for the identification of proteins. More recently, methodologies have been introduced showing that mass spectrometry can also be used for protein quantification. This article reviews various mass spectrometry-based technologies in quantitative proteomics, highlighting several interesting applications in areas ranging from cell biology to clinical applications.     

蛋白质N末端组学研究进展

蛋白质的N末端作为合成的起始,其氨基酸序列组成及翻译后修饰直接影响着蛋白质的活性、稳定性和细胞内定位,调控着细胞内的信号转导,甚至决定了这些蛋白质的命运。对蛋白质N末端组学的系统研究不仅可以揭示N末端区域对整个蛋白质的重要作用,有助于我们深入地了解蛋白质在各种生命活动中所扮演的角色,同时在实现蛋白质组高覆盖、基因组重注释等方面也有着重要的价值。本文结合我们的现有工作,综述了近年来蛋白质N末端组学的研究进展,尤其是一些重要的基于质谱的N末端富集技术和方法。     

基于质谱技术的蛋白质组定量方法

对不同状态下的蛋白质在表达和修饰水平上进行精确定量,对于探索蛋白质的生物功能、发现疾病的生物标志物都具有重要意义,也是当前蛋白质组学的一个重要研究前沿。近年来,各种蛋白质组定量的新技术和新方法不断涌现,但仍面临着巨大挑战。本文就基于质谱技术的多种蛋白质组定量方法的基本原理、近几年的研究进展和应用进行评述。     

The methods of quantitative proteomics

In modern science proteomic analysis is inseparable from other fields of systemic biology. Possessing huge resources quantitative proteomics operates colossal information on molecular mechanisms of life. Advances in proteomics help researchers to solve complex problems of cell signaling, posttranslational modification, structure and funciotnal homology of proteins, molecular diagnostics etc. More than 40 various methods have been developed in proteomics for quantitative analysis of proteins. Although each method is unique and has certain advantages and disadvantages all these use various isotope labels (tags). In this review we will consider the most popular and effective methods employing both chemical modifications of proteins and also metabolic and enzymatic methods of isotope labeling.     

Tissue and serum proteomic profiling for diagnostic and prognostic bladder cancer biomarkers

A panel of biomarkers for the early detection of bladder cancer has not yet been identified. Many different molecules, including DNA, RNA or proteins have been reported but none have provided adequate sensitivity for a single-tier screening test or a test to replace cystoscopy. Therefore, multimarker panels are discussed at present to give a more-precise answer to the biomarker quest. Mass spectrometry or 2D gel-electrophoresis have evolved greatly within recent years and are capable of analyzing multiple proteins or peptides in parallel with high sensitivity and specificity. However, transmission of screening results from one laboratory to another is still the main pitfall of those methods; a fact that emphasizes the need for consistent and standardized procedures as suggested by the Human Proteome Organization (HUPO). In this article, recent results in screening approaches and other proteomic techniques used for biomarker evaluation in bladder cancer are discussed with a focus on serum and tissue biomarkers.     

Advances in quantitative proteomics using stable isotope tags

A great deal of current biological and clinical research is directed at the interpretation of the information contained in the human genome sequence in terms of the structure, function and control of biological systems and processes. Proteomics, the systematic analysis of proteins, is becoming a critical component in this endeavor because proteomic measurements are carried out directly on proteins – the catalysts and effectors of essentially all biological functions. To detect changes in protein profiles that might provide important diagnostic or functional insights, proteomic analyses necessarily have to be quantitative. This article summarizes recent technological advances in quantitative proteomics.     

评价新型稳定同位素赖氨酸标记在定量蛋白质组学中的应用

为了评价基于2-甲氧基-4,5-二氢-1氢-咪唑稳定同位素试剂在定量蛋白质组学中的应用价值,合成了轻型(D0)和重型(D4)的2-甲氧基-4,5-二氢-1氢-咪唑,通过对标准蛋白BSA酶解后产物的标记确认标记反应的特异性,并观察了标记物在MALDI-TOF-MS和LC-ESI-MS中定量的准确性,标记肽在串联质谱中的离子特点,以及对反相液相色谱行为的影响。结果表明,2-甲氧基-4,5-二氢-1氢-咪唑只与酶解后的肽段赖氨酸侧链氨基反应,具有良好的标记特异性;差异表达蛋白的定量可以通过MALDI和ESI电离模式实现;标记肽的串联质谱主要产生y离子,测序更为简便;反相液相色谱可以保持较好的分离效果,氘原子的引入不会影响保留时间,侧链修饰可以用于涉及液相色谱分离的蛋白质组学技术。2-甲氧基-4,5-二氢-1氢-咪唑稳定同位素试剂可以用于定量蛋白质组学。     

Evaluation of four phosphopeptide enrichment strategies for mass spectrometry-based proteomic analysis

Information about phosphorylation status can be used to prioritize and characterize biological processes in the cell. Various analytical strategies have been proposed to address the complexity of phosphorylation status and comprehensively identify phosphopeptides. In this study, we evaluated four strategies for phosphopeptide enrichment, using titanium dioxide (TiO₂) and Phos-tag ligand particles from in-gel or in-solution digests prior to mass spectrometry-based analysis. Using TiO₂ and Phos-tag magnetic beads, it was possible to enrich phosphopeptides from in-gel digests of phosphorylated ovalbumin separated by Phos-tag SDS-PAGE or in-solution serum digests, while minimizing non-specific adsorption. The tip-column strategy with TiO₂ particles enabled enrichment of phosphopeptides from in-solution digests of whole-cell lysates with high efficiency and selectivity. However, the tip-column strategy with Phos-tag agarose beads yielded the greatest number of identified phosphopeptides. The strategies using both types of tip columns had a high degree of overlap, although there were differences in selectivity between the identified phosphopeptides. Together, our results indicate that multi-enrichment strategies using TiO₂ particles and Phos-tag agarose beads are useful for comprehensive phosphoproteomic analysis.     

The dynamics of the proteome: strategies for measuring protein turnover on a proteome-wide scale.

Quantitative proteomics captures the steady-state amount of a protein in a cell but does not explain how a change in protein amount is manifest -- whether through a change in synthesis or a change in degradation. If we are to understand the changes in the proteome, we will need to define such processes. In this brief review, strategies for the determination of intracellular protein dynamics on a proteome-wide scale are discussed.     

Molecular constituents of the postsynaptic density fraction revealed by proteomic analysis using multidimensional liquid chromatography-tandem mass spectrometry

Protein constituents of the postsynaptic density (PSD) fraction were analysed using an integrated liquid chromatography (LC)-based protein identification system, which was constructed by coupling microscale two-dimensional liquid chromatography (2DLC) with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) and an automated data analysis system. The PSD fraction prepared from rat forebrain was solubilized in 6 m guanidium hydrochloride, and the proteins were digested with trypsin after S-carbamoylmethylation under reducing conditions. The tryptic peptide mixture was then analysed with the 2DLC-MS/MS system in a data-dependent mode, and the resultant spectral data were automatically processed to search a genome sequence database for protein identification. In triplicate analyses, the system allowed assignments of 5264 peptides, which could finally be attributed to 492 proteins. The PSD contained various proteins involved in signalling transduction, including receptors, ion channel proteins, protein kinases and phosphatases, G-protein and related proteins, scaffold proteins, and adaptor proteins. Structural proteins, including membrane proteins involved in cell adhesion and cell-cell interaction, proteins involved in endocytosis, motor proteins, and cytoskeletal proteins were also abundant. These results provide basic data on a major protein set associated with the PSD and a basis for future functional studies of this important neural machinery.     

定量蛋白质组学分析方法及应用

蛋白质组学经历了近10年的发展,现在已经初具规模。但是由于它是动态地观察生物体不断变化的所有蛋白质,所以技术难度非常之大。为使研究简化并更具针对性,人们着重进行比较蛋白质组学的研究。为了具体量化这些蛋白质的变化产生了定量蛋白质组学,近几年各种标记技术的进步使得该学科得以迅猛发展。     

Absolute quantification strategies in proteomics based on mass spectrometry

The strong need for quantitative information in proteomics has fueled the development of mass spectrometry-based analytical methods that are able to determine protein abundances. This article reviews mass spectrometry experiments aimed at providing an absolute quantification of proteins. The experiments make use of the isotope-dilution concept by spiking a known amount of synthetic, isotope-labeled reference peptide into the analyte sample. Quantification is achieved by comparing the mass spectrometry signal intensities of the reference with an endogenous peptide that is generated upon proteolytic cleavage of the target protein. In an analogous manner, the level of post-translational modification at a distinct residue within a target protein can be determined. Among the strengths of absolute quantification are low detection limits reaching subfemtomole levels, a high dynamic range spanning approximately five orders of magnitude, low requirements for sample clean-up, and a fast and straightforward method development. Recent studies have demonstrated the compatibility of absolute quantification with various mass spectrometry readout techniques and sample purification steps such as 1D gel electrophoresis, size-exclusion chromatography, isoelectric peptide focusing, strong cation exchange and reversed phase or affinity chromatography. Under ideal conditions, quantification errors and coefficients of variation below 5% have been reported. However, the fact that at the start of the experiment the analyte is a protein and the internal standard is a peptide, severe quantification errors may result due to the selection of unsuitable reference peptides and/or imperfect protein proteolysis. Within the ensemble of mass spectrometry-based quantification methods, absolute quantification is the method of choice in cases where absolute numbers, many repetitive experiments or precise levels of post-translational modifications are required for a few, preselected species of interest. Consequently, prominent application areas include biomarker quantification, the study of post-translational modifications such as phosphorylation or ubiquitination and the comparison of concentrations of interacting proteins.     

Phospholipid capture combined with non-linear chromatographic correction for improved serum metabolite profiling

Serum analysis with LC/MS can yield thousands of potential metabolites. However, in metabolomics, biomarkers of interest will often be of low abundance, and ionization suppression from high abundance endogenous metabolites such as phospholipids may prevent the detection of these metabolites. Here a cerium-modified column and methyl-tert-butyl-ether (MTBE) liquid–liquid extraction were employed to remove phospholipids from serum in order to obtain a more comprehensive metabolite profile. XCMS, an in-house developed data analysis software platform, showed that the intensity of existing endogenous metabolites increased, and that new metabolites were observed. This application of phospholipid capture in combination with XCMS non-linear data processing has enormous potential in metabolite profiling, for biomarker detection and quantitation.     

Comparative proteomic analysis of the insulin‐induced L6 myotube secretome

Emerging evidence has revealed an endocrine function for skeletal muscle; in fact, certain anti‐inflammatory cytokines are secreted only from contractile skeletal muscle. However, the skeletal muscle secretome as a whole is poorly characterized, as is how it changes in response to extracellular stimuli. Herein, we sought to identify and characterize the members of the skeletal muscle secretome, and to determine which protein secretion levels were modulated in response to insulin stimulation. To conduct these studies, we treated differentiated L6 rat skeletal muscle cells with insulin or left them untreated, and we comparatively analyzed the proteins secreted into the media. We fractionated this conditioned media using offline RP HPLC, digested the fractionated proteins, and analyzed the resulting peptides with LC‐ESI‐MS/MS. We identified a total of 254 proteins, and by using three different filtering methods, we identified 153 of these as secretory proteins. Fourteen proteins were secreted at higher levels under insulin stimulation, including several proteins known to be highly secreted in metabolic diseases; 19 proteins were secreted at lower levels under insulin stimulation. These result not only pinpointed several previously unknown, insulin induced, secretory proteins of skeletal muscle, it also described a novel approach for conditioned secretome analysis.