ICP‐MS‐Based strategies for protein quantification

In the post‐genomics era, proteomics has become a central branch in life sciences. An understanding of biological functions will not only rely on protein identification, but also on protein quantification in a living organism. Most of the existing methods for quantitative proteomics are based on isotope labeling combined with molecular mass spectrometry. Recently, a remarkable progress that utilizes inductively coupled plasma‐mass spectrometry (ICP‐MS) as an attractive complement to electrospray MS and MALDI MS for protein quantification, especially for absolute quantification, has been achieved. This review will selectively discuss the recent advances of ICP‐MS‐based technique, which will be expected to further mature and to become one of the key methods in quantitative proteomics. © 2009 Wiley Periodicals, Inc., Mass Spec Rev 29:326–348, 2010     

Mass spectrometry-based clinical proteomics: head-and-neck cancer biomarkers and drug-targets discovery

Mass spectrometry (MS)-based proteomics is a rapidly developing technology for both qualitative and quantitative analyses of proteins, and investigations into protein posttranslational modifications, subcellular localization, and interactions. Recent advancements in MS have made tremendous impact on the throughput and comprehensiveness of cancer proteomics, paving the way to unraveling deregulated cellular pathway networks in human malignancies. In turn, this knowledge is rapidly being translated into the discovery of novel potential cancer markers (PCMs) and targets for molecular therapeutics. Head-and-neck cancer is one of the most morbid human malignancies with an overall poor prognosis and severely compromised quality of life. Early detection and novel therapeutic strategies are urgently needed for more effective disease management. The characterizations of protein profiles of head-and-neck cancers and non-malignant tissues, with unprecedented sensitivity and precision, are providing technology platforms for identification of novel PCMs and drug targets. Importantly, low-abundance proteins are being identified and characterized, not only from the tumor tissues, but also from bodily fluids (plasma, saliva, and urine) in a high-throughput and unbiased manner. This review is a critical appraisal of recent advances in MS-based proteomic technologies and platforms for facilitating the discovery of biomarkers and novel drug targets in head-and-neck cancer. A major challenge in the discovery and verification of these cancer biomarkers is the typically limited availability of well-characterized and adequately stored clinical samples in tumor and sera banks, collected using recommended procedures, and with detailed information on clinical, pathological parameters, and follow-up. Most biomarker discovery studies use limited number of clinical samples and verification of cancer markers in large number of samples is beyond the scope of a single laboratory. The validation of these potential markers in large sample cohorts in multicentric studies is needed for their translation from the bench to the bedside.     

Cancer proteomics

Cancer presents high mortality and morbidity globally, largely due to its complex and heterogenous nature, and lack of biomarkers for early diagnosis. A proteomics study of cancer aims to identify and characterize functional proteins that drive the transformation of malignancy, and to discover biomarkers to detect early‐stage cancer, predict prognosis, determine therapy efficacy, identify novel drug targets, and ultimately develop personalized medicine. The various sources of human samples such as cell lines, tissues, and plasma/serum are probed by a plethora of proteomics tools to discover novel biomarkers and elucidate mechanisms of tumorigenesis. Innovative proteomics technologies and strategies have been designed for protein identification, quantitation, fractionation, and enrichment to delve deeper into the oncoproteome. In addition, there is the need for high‐throughput methods for biomarker validation, and integration of the various platforms of oncoproteome data to fully comprehend cancer biology. © 2012 Wiley Periodicals, Inc. Mass Spec Rev 31:583–605, 2012     

基于质谱的蛋白质组学方法新进展

蛋白质组学是后基因组时代的科研热点,其研究方向主要涵盖蛋白质表达谱、蛋白质翻译后修饰谱、蛋白质相互作用谱和单细胞蛋白质组等定性分析以及相对和绝对定量分析,现已广泛应用于生物、医药及病理研究中。由于质谱具有灵敏、准确、高通量等特点,是蛋白质组学研究必不可少的工具。本文综述了近年来基于质谱技术的蛋白质组鉴定及定量方法,并展望未来蛋白质组学研究的发展方向。     

Mass spectrometry analysis of nitrotyrosine‐containing proteins

Oxidative stress plays important roles in a wide range of diseases such as cancer, inflammatory disease, neurodegenerative disorders, etc. Tyrosine nitration in a protein is a chemically stable oxidative modification, and a marker of oxidative injuries. Mass spectrometry (MS) is a key technique to identify nitrotyrosine‐containing proteins and nitrotyrosine sites in endogenous and synthetic nitroproteins and nitropeptides. However, in vivo nitrotyrosine‐containing proteins occur with extreme low‐abundance to severely challenge the use of MS to identify in vivo nitroproteins and nitrotyrosine sites. A preferential enrichment of nitroproteins and/or nitropeptides is necessary before MS analysis. Current enrichment methods include immuno‐affinity techniques, chemical derivation of the nitro group plus target isolations, followed with tandem mass spectrometry analysis. This article reviews the MS techniques and pertinent before‐MS enrichment techniques for the identification of nitrotyrosine‐containing proteins. This article reviews future trends in the field of nitroproteomics, including quantitative nitroproteomics, systems biological networks of nitroproteins, and structural biology study of tyrosine nitration to completely clarify the biological functions of tyrosine nitration. © 2013 Wiley Periodicals, Inc. Mass Spec Rev 34: 423–448, 2015.     

Quantitative mass spectrometry to investigate epidermal growth factor receptor phosphorylation dynamics

Identifying proteins of signaling networks has received much attention, because an array of biological processes are entirely dependent on protein cross-talk and protein-protein interactions. Protein posttranslational modifications (PTM) add an additional layer of complexity, resulting in complex signaling networks. Of particular interest to our working group are the signaling networks of epidermal growth factor (EGF) receptor, a transmembrane receptor tyrosine kinase involved in various cellular processes, including cell proliferation, differentiation, and survival. Ligand binding to the N-terminal residue of the extracellular domain of EGF receptor induces conformational changes, dimerization, and (auto)-phosphorylation of intracellular tyrosine residues. In addition, activated EGF receptor may positively affect survival pathways, and thus determines the pathways for tumor growth and progression. Notably, in many human malignancies exaggerated EGF receptor activities are commonly observed. An understanding of the mechanism that results in aberrant phosphorylation of EGF receptor tyrosine residues and derived signaling cascades is crucial for an understanding of molecular mechanisms in cancer development. Here, we summarize recent labeling methods and discuss the difficulties in quantitative MS-based phosphorylation assays to probe for receptor tyrosine kinase (RTK) activity. We also review recent advances in sample preparation to investigate membrane-bound RTKs, MS-based detection of phosphopeptides, and the diligent use of different quantitative methods for protein labeling.     

Plant organelle proteomics: Collaborating for optimal cell function

Organelle proteomics describes the study of proteins present in organelle at a particular instance during the whole period of their life cycle in a cell. Organelles are specialized membrane bound structures within a cell that function by interacting with cytosolic and luminal soluble proteins making the protein composition of each organelle dynamic. Depending on organism, the total number of organelles within a cell varies, indicating their evolution with respect to protein number and function. For example, one of the striking differences between plant and animal cells is the plastids in plants. Organelles have their own proteins, and few organelles like mitochondria and chloroplast have their own genome to synthesize proteins for specific function and also require nuclear‐encoded proteins. Enormous work has been performed on animal organelle proteomics. However, plant organelle proteomics has seen limited work mainly due to: (i) inter‐plant and inter‐tissue complexity, (ii) difficulties in isolation of subcellular compartments, and (iii) their enrichment and purity. Despite these concerns, the field of organelle proteomics is growing in plants, such as Arabidopsis, rice and maize. The available data are beginning to help better understand organelles and their distinct and/or overlapping functions in different plant tissues, organs or cell types, and more importantly, how protein components of organelles behave during development and with surrounding environments. Studies on organelles have provided a few good reviews, but none of them are comprehensive. Here, we present a comprehensive review on plant organelle proteomics starting from the significance of organelle in cells, to organelle isolation, to protein identification and to biology and beyond. To put together such a systematic, in‐depth review and to translate acquired knowledge in a proper and adequate form, we join minds to provide discussion and viewpoints on the collaborative nature of organelles in cell, their proper function and evolution. © 2010 Wiley Periodicals, Inc., Mass Spec Rev 30:772–853, 2011     

Three-dimensional quantitative mass spectrometry imaging in complex system: From subcellular to whole organism

Mass spectrometry imaging (MSI) has been applied for label-free three-dimensional (3D) imaging from position array across the whole organism, which provides high-dimensional quantitative data of inorganic or organic compounds that may play an important role in the regulation of cellular signaling, including metals, metabolites, lipids, drugs, peptides, and proteins. While MSI is suitable for investigation of the spatial distribution of molecules, it has a limitation with visualization and quantification of multiple molecules. 3D-MSI, however, can be applied toward exploring metabolic pathway as well as the interactions of lipid–protein, protein–protein, and metal–protein in complex systems from subcellular to the whole organism through an untargeted methodology. In this review, we highlight the methods and applications of MS-based 3D imaging to address the complexity of molecular interaction from nano- to micrometer lateral resolution, with particular focus on: (a) common and hybrid 3D-MSI techniques; (b) quantitative MSI methodology, including the methods using a stable isotope labeling internal standard (SILIS) and SILIS-free approaches with tissue extinction coefficient or virtual calibration; (c) reconstruction of the 3D organ; (d) application of 3D-MSI for biomarker screening and environmental toxicological research. 3D-MSI quantitative analysis provides accurate spatial information and quantitative variation of biomolecules, which may be valuable for the exploration of the molecular mechanism of the disease progresses and toxicological assessment of environmental pollutants in the whole organism. Additionally, we also discuss the challenges and perspectives on the future of 3D quantitative MSI.     

The application of quantification techniques in proteomics for biomedical research

The systematic analysis of biological processes requires an understanding of the quantitative expression patterns of proteins, their interacting partners and their subcellular localization. This information was formerly difficult to accrue as the relative quantification of proteins relied on antibody‐based methods and other approaches with low throughput. The advent of soft ionization techniques in mass spectrometry plus advances in separation technologies has aligned protein systems biology with messenger RNA, DNA, and microarray technologies to provide data on systems as opposed to singular protein entities. Another aspect of quantitative proteomics that increases its importance for the coming few years is the significant technical developments underway both for high pressure liquid chromatography and mass spectrum devices. Hence, robustness, reproducibility and mass accuracy are still improving with every new generation of instruments. Nonetheless, the methods employed require validation and comparison to design fit for purpose experiments in advanced protein analyses. This review considers the newly developed systematic protein investigation methods and their value from the standpoint that relative or absolute protein quantification is required de rigueur in biomedical research. © 2012 Wiley Periodicals, Inc., Mass Spec Rev 32:1–26, 2013     

Prospective highlights of functional skin proteomics

Although a wide variety of protein profiles have been extensively constructed via proteomic analysis, the comprehensive proteomic profiling of the skin, which is considered to be the largest organ of the human body, is still far from complete. Our efforts to establish the functional skin proteome, a protein database describing the protein networks that underlie biological processes, has set in motion the identification and characterization of proteins expressed in the epidermis and dermis of the BALB/c mice. In this review, we will highlight various cutaneous proteins we have characterized and discuss their biological functions associated with skin distress, immunity, and cancer. This type of research into functional skin proteomics will provide a critical step toward understanding disease and developing successful therapeutic strategies.     

Proteomics analysis of tumor microenvironment: Implications of metabolic and oxidative stresses in tumorigenesis

Tumorigenesis is always concomitant with microenvironmental alterations. The tumor microenvironment is a heterogeneous and complex milieu, which exerts a variety of stresses on tumor cells for proliferation, survival, or death. Recently, accumulated evidence revealed that metabolic and oxidative stresses both play significant roles in tumor development and progression that converge on a common autophagic pathway. Tumor cells display increased metabolic autonomy, and the hallmark is the exploitation of aerobic glycolysis (termed Warburg effect), which increased glucose consumption and decreased oxidative phosphorylation to support growth and proliferation. This characteristic renders cancer cells more aggressive; they devour tremendous amounts of nutrients from microenvironment to result in an ever‐growing appetite for new tumor vessel formation and the release of more “waste,” including key determinants of cell fate like lactate and reactive oxygen species (ROS). The intracellular ROS level of cancer cells can also be modulated by a variety of stimuli in the tumor microenvironment, such as pro‐growth and pro‐inflammatory factors. The intracellular redox state serves as a double‐edged sword in tumor development and progression: ROS overproduction results in cytotoxic effects and might lead to apoptotic cell death, whereas certain level of ROS can act as a second‐messenger for regulation of such cellular processes as cell survival, proliferation, and metastasis. The molecular mechanisms for cancer cell responses to metabolic and oxidative stresses are complex and are likely to involve multiple molecules or signaling pathways. In addition, the expression and modification of these proteins after metabolic or oxidative stress challenge are diverse in different cancer cells and endow them with different functions. Therefore, MS‐based high‐throughput platforms, such as proteomics, are indispensable in the global analysis of cancer cell responses to metabolic and oxidative stress. Herein, we highlight recent advances in the understanding of the metabolic and oxidative stresses associated with tumor progression with proteomics‐based systems biology approaches. © 2012 Wiley Periodicals, Inc., Mass Spec Rev 32:267–311, 2013     

Proteolytic 18O-labeling strategies for quantitative proteomics

A number of proteomic techniques have been developed to quantify proteins in biological systems. This review focuses on the quantitative proteomic technique known as "proteolytic 18O-labeling." This technique utilizes a protease and H(2)18O to produce labeled peptides, with subsequent chromatographic and mass spectrometric analysis to identify and quantify (relative) the proteins from which the peptides originated. The technique determines the ratio of individual protein's expression level between two samples relative to each other, and can be used to quantitatively examine protein expression (comparative proteomics) and post-translational modifications, and to study protein-protein interactions. The present review discusses various aspects of the 18O-labeling technique, including: its history, the advantages and disadvantages of the proteolytic 18O-labeling technique compared to other techniques, enzymatic considerations, the problem of variable incorporation of 18O atoms into peptides with a discussion on recent advancements of the technique to overcome it, computational tools to interpret the data, and a review of the biological applications.     

A review of current applications of mass spectrometry for neuroproteomics in epilepsy

The brain is unquestionably the most fascinating organ, and the hippocampus is crucial in memory storage and retrieval and plays an important role in stress response. In temporal lobe epilepsy (TLE), the seizure origin typically involves the hippocampal formation. Despite tremendous progress, current knowledge falls short of being able to explain its function. An emerging approach toward an improved understanding of the complex molecular mechanisms that underlie functions of the brain and hippocampus is neuroproteomics. Mass spectrometry has been widely used to analyze biological samples, and has evolved into an indispensable tool for proteomics research. In this review, we present a general overview of the application of mass spectrometry in proteomics, summarize neuroproteomics and systems biology‐based discovery of protein biomarkers for epilepsy, discuss the methodology needed to explore the epileptic hippocampus proteome, and also focus on applications of ingenuity pathway analysis (IPA) in disease research. This neuroproteomics survey presents a framework for large‐scale protein research in epilepsy that can be applied for immediate epileptic biomarker discovery and the far‐reaching systems biology understanding of the protein regulatory networks. Ultimately, knowledge attained through neuroproteomics could lead to clinical diagnostics and therapeutics to lessen the burden of epilepsy on society. © 2009 Wiley Periodicals, Inc., Mass Spec Rev 29:197–246, 2010     

Silencing of long non-coding RNA CCHE1 inhibits the ovarian cancer SKOV3 cell invasion and migration and inactivates the p38-MAPK signaling pathway

Ovarian cancer (OC) is a major cause of cancer-related deaths among gynaecological malignancies. Emerging studies suggest that the long non-coding RNA (lncRNA) may be the potential biomarker for the diagnosis and prognosis of the cancer. The current study was carried out to investigate the role of lncRNA CCHE1 silencing in OC cell invasion and migration. Expression of lncRNA CCHE1 in normal ovarian cell Hose and OC cell lines HO 8910, A2780 and SKOV3 was detected. LncRNA were transfected with siRNA, and then the proliferation of cells was detected by using MTT assay. Cell invasion and migration was measured by using Transwell assay and scratch test, respectively. The protein levels of E-cadherin, N-cadherin, ERK, p38-MAPK and the phosphorylation of ERK and p38-MAPK in cells after siRNA transfection were detected by using Western blot analysis. Consequently, lncRNA CCHE1 expression was highly expressed in OC cell lines, especially in SKOV3 cells. siRNA1, siRNA2 and siRNA3 all decreased. lncRNA CCHE1 expression in SKOV3 cells and siRNA2 showed the best silencing efficacy. Silencing of lncRNA CCHE1 decreased proliferation, invasion and migration, and reduced the protein levels of N-cadherin, ERK, p38-MAPK and the phosphorylation of ERK and p38-MAPK, while reducing the protein level of E-cadherin in SKOV3 cells. Collectively, our study proved that the silencing of lncRNA CCHE1 could inhibit SKOV3 cell invasion and migration via inactivating the p38-MAPK signaling pathway.     

Proteome analysis of apoptotic cells

Apoptosis, a genetically determined form of cell death, is a central and complex process involved in the development of multicellular organisms in the maintenance of cell homeostasis. During apoptosis, a large number of proteins involved in transducing signals are posttranslationally modified. Classical proteomics, the combination of protein separation by two-dimensional gel electrophoresis (2DGE) and protein identification by mass spectrometry (MS), enabled the discovery of more than 100 proteins altered during apoptosis. Functional data about protein degradation, modification, translocation, and synthesis were obtained. In addition to classical proteomics, some specifically designed proteome studies were carried out to analyze specific apoptotic components such as the mitochondrial releasing factors, death-inducing signaling complex (DISC), inhibitor of apoptosis (IAP) interacting proteins, and caspases. The identification of main regulators significantly influenced the elucidation of the concept underlying apoptosis signaling. Thus, the application of detailed protein analytical methods in the young field of apoptosis research was particularly fruitful.     

CRIP1生物学功能的定量蛋白质组学分析

CRIP1（cysteine-rich intestinal protein 1）是含有双锌指结构域的蛋白,在很多肿瘤细胞中高表达,但其在肺癌细胞中的生理学功能尚不明确。本研究应用定量蛋白质组学探究CRIP1过表达对肺癌顺铂耐药细胞（A549/DDP）的影响及作用机制。运用慢病毒载体系统构建了CRIP1过表达的A549/DDP稳转细胞系,利用Western Blotting证实了单克隆细胞系中CRIP1的过表达。发现CRIP1过表达能够加快细胞增殖,增加细胞的耐药性。通过定量蛋白质组学分析,鉴定了CRIP1过表达引起的蛋白质组的变化,并且对上调和下调的蛋白进行聚类分析。结果表明,CRIP1过表达上调了烟酰胺磷酸核糖转移酶（NAMPT）和NAD依赖型氧化还原酶的表达,从而促进细胞的增殖,并且提高了细胞的耐药性。     

Techniques for phosphopeptide enrichment prior to analysis by mass spectrometry

Mass spectrometry is the tool of choice to investigate protein phosphorylation, which plays a vital role in cell regulation and diseases such as cancer. However, low abundances of phosphopeptides and low degrees of phosphorylation typically necessitate isolation and concentration of phosphopeptides prior to MS analysis. This review discusses the enrichment of phosphopeptides with immobilized metal affinity chromatography, reversible covalent binding, and metal oxide affinity chromatography. Capture of phosphopeptides on TiO₂ seems especially promising in terms of selectivity and recovery, but the success of all methods depends on careful selection of binding, washing, and elution solutions. Enrichment techniques are complementary, such that a combination of methods greatly enhances the number of phosphopeptides isolated from complex samples. Development of a standard series of phosphopeptides in a highly complex mixture of digested proteins would greatly aid the comparison of different enrichment methods. Phosphopeptide binding to magnetic beads and on‐plate isolation prior to MALDI‐MS are emerging as convenient methods for purification of small (µL) samples. On‐plate enrichment can yield >70% recoveries of phosphopeptides in mixtures of a few digested proteins and can avoid sample‐handling steps, but this technique is likely limited to relatively simple samples such as immunoprecipitates. With recent advances in enrichment techniques in hand, MS analysis should provide important insights into phosphorylation pathways. © 2009 Wiley Periodicals, Inc., Mass Spec Rev 29:29–54, 2010     

Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research

Progress in proteomics research has led to a demand for powerful analytical tools with high separation efficiency and sensitivity for confident identification and quantification of proteins, posttranslational modifications, and protein complexes expressed in cells and tissues. This demand has significantly increased interest in capillary electrophoresis‐mass spectrometry (CE‐MS) in the past few years. This review provides highlights of recent advances in CE‐MS for proteomics research, including a short introduction to top‐down mass spectrometry and native mass spectrometry (native MS), as well as a detailed overview of CE methods. Both the potential and limitations of these methods for the analysis of proteins and peptides in synthetic and biological samples and the challenges of CE methods are discussed, along with perspectives about the future direction of CE‐MS. @ 2019 Wiley Periodicals, Inc. Mass Spec Rev 00:1–16, 2019.