Proteomics strategies for protein identification

The information from genome sequencing provides new approaches for systems-wide understanding of protein networks and cellular function. DNA microarray technologies have advanced to the point where nearly complete monitoring of gene expression is feasible in several organisms. An equally important goal is to comprehensive survey cellular proteomes and profile protein changes under different cellular states. This presents a complex analytical problem, due to the chemical variability between proteins and peptides. Here, we discuss strategies to improve accuracy and sensitivity of peptide identification, distinguish represented protein isoforms, and quantify relative changes in protein abundance.     

Plastid proteomics

Plastids are essential organelles present in virtually all cells in plants and in green algae. The proteomes of plastids, and in particular of chloroplasts, have received significant amounts of attention in recent years. Various fractionation and mass spectrometry (MS) techniques have been applied to catalogue the chloroplast proteome and its membrane compartments. Neural network and hidden Markov models, in combination with experimentally derived filters, were used to try to predict the chloroplast subproteomes. Some of the many protein-protein interaction, as well as post-translational modifications have been characterized. Nevertheless, our understanding of the chloroplast proteome and its dynamics is very incomplete. Rapid improvements and wide-scale implementation of MS and new tools for comparative proteomics will undoubtedly accelerate this understanding in the near future. Proteomics studies often generate a large amount of data and these data are only meaningful if they can be easily accessed via the 'world-wide-web' and connected to other types of biological information. The plastid proteome data base (PPDB at http://www.ppdb.tc.cornell.edu/) and other web resources are discussed. This review will briefly summarize recent experimental and theoretical efforts, attempt to translate these data into the functions of the chloroplast and outline expectations and possibilities for (comparative) chloroplast proteomics.     

Web Resources for Mass Spectrometry-based Proteomics

With the development of high-resolution and high-throughput mass spectrometry(MS)technology, a large quantum of proteomic data is continually being generated. Collecting and sharing these data are a challenge that requires immense and sustained human effort. In this report, we provide a classification of important web resources for MS-based proteomics and present rating of these web resources, based on whether raw data are stored, whether data submission is supported,and whether data analysis pipelines are provided. These web resources are important for biologists involved in proteomics research.     

Glucotoxicity and pancreatic proteomics

Chronic hyperglycaemia is one of the main characteristics of a diabetic state. This is also the first cause of diabetic complications. However, it is now generally accepted that glucotoxicity also participates in the worsening of type 2 diabetes, by affecting the secretion of β-cells. So far, different mechanisms have been proposed to explain the adverse effects of chronic hyperglycaemia. One of them suggests that the modulation of expression of several key proteins during a hyperglycaemia state, may explain the toxic effect of glucotoxicity. Therefore, proteomic analysis of biological samples represents an interesting method to study the effect of chronic hyperglycaemia on protein expression. The discovery of new proteins for which the expression could be modulated by chronic hyperglycaemia may probably help to better understand the mechanisms underlying glucotoxicity. In this review, we will first present an introduction of the different mechanisms known to be involved in the control of glucose homeostasis and in the development of glucotoxicity. In a second part, some proteomic data linked with the effect of glucotoxicity in pancreas, pancreatic islets and β-cells will be presented and discussed.     

Miniaturized proteomics and peptidomics using capillary liquid separation and high resolution mass spectrometry

Knowledge of the protein and peptide content in a tissue or a body fluid is vital in many areas of medical and biomedical sciences. Information from proteomic and peptidomic studies may reveal alterations in expression due to, e.g., a disease and facilitate the understanding of the pathophysiology and the identification of biological markers. In this minireview, we discuss miniaturized proteomic and peptidomic approaches that have been applied in our laboratory in order to investigate the protein and peptide contents of body fluids (such as plasma, cerebrospinal and amniotic fluid), as well as extracted tissues. The methods involve miniaturized liquid separation, i.e., capillary liquid chromatography and capillary electrophoresis, combined with high resolution mass spectrometry (MS), i.e., Fourier transform ion cyclotron resonance MS. These approaches provide the opportunity to analyze samples of small volumes with high throughput, high sensitivity, good dynamic range and minimal sample handling. Also, the experiments are relatively easy to automate.     

Discovery of biomarkers for gastric cancer: a proteomics approach

Gastric cancer is the second leading cause of cancer-related deaths worldwide. Although many treatment options exist for patients with gastric tumors, the incidence and mortality rate of gastric cancer are on the rise. The early stages of gastric cancer are non-symptomatic, and the treatment response is unpredictable. This situation is further aggravated by a lack of diagnostic biomarkers that can aid in the early detection and prognosis of gastric cancer and in the prediction of chemoresistance. Moreover, clinical surgical specimens are rarely obtained, and traditional biomarkers of gastric cancer are not very effective. Many studies in the field of proteomics have contributed to the discovery and establishment of powerful diagnostic tools (e.g., ProteinChip array) in the management of cancer. The evolution in proteomic technologies has not only enabled the screening of a large number of samples but also enabled the identification of pathologically significant proteins, such as phosphoproteins, and the quantitation of difference in protein expression under different conditions. Multiplexed assays are used widely to accurately fractionate various complex samples such as blood, tissue, cells, and Helicobacter pylori-infected specimens to identify differentially expressed proteins. Biomarker detection studies have substantially contributed to the areas of secretome, metabolome, and phosphoproteome. Here, we review the development of potential biomarkers in the natural history of gastric cancer, with specific emphasis on the characteristics of target protein convergence.     

Proteome analysis of mouse model systems: A tool to model human disease and for the investigation of tissue-specific biology

The molecular dissections of the mechanistic pathways involved in human disease have always relied on the use of model organisms. Among the higher mammalian organisms, the laboratory mouse (Mus musculus) is the most widely used model. A large number of commercially-available, inbred strains are available to the community, including an ever growing collection of transgenic, knock-out, and disease models. Coupled to availability is the fact that animal colonies can be kept under standardized housing condition at most major universities and research institutes, with relative ease and cost efficiency (compared to larger vertebrates). As such, mouse models to study human biology and disease remains extremely attractive. In the current review we will provide an historic overview of the use of mouse models in proteome research with a focus on general tissue and organelle biology, comparative proteomics of human and mouse and the use of mouse models to study cardiac disease.     

蛋白质组学研究技术及其在寄生虫学中的应用

介绍了蛋白质组学研究的核心技术 ,即蛋白质组分分离、蛋白质组分鉴定、利用蛋白质组信息学进行结构和功能预测 ,以及蛋白质组学技术在寄生虫学中的应用和研究进展。     

Building and searching tandem mass (MS/MS) spectral libraries for peptide identification in proteomics

Spectral library searching is an emerging approach in peptide identifications from tandem mass spectra, a critical step in proteomic data analysis. In spectral library searching, a spectral library is first meticulously compiled from a large collection of previously observed peptide MS/MS spectra that are conclusively assigned to their corresponding amino acid sequence. An unknown spectrum is then identified by comparing it to all the candidates in the spectral library for the most similar match. This review discusses the basic principles of spectral library building and searching, describes its advantages and limitations, and provides a primer for researchers interested in adopting this new approach in their data analysis. It will also discuss the future outlook on the evolution and utility of spectral libraries in the field of proteomics.     

A feasibility study to identify proteins in the residual Pap test fluid of women with normal cytology by mass spectrometry-based proteomics

Background

The proteomic analysis of body fluids is a growing technology for the identification of protein biomarkers of disease. Given that Papanicolaou tests (Pap tests) are routinely performed on over 30 million women annually in the U.S. to screen for cervical cancer, we examined the residual Pap test fluid as a source of protein for analysis by mass spectrometry (MS). In the liquid-based Pap test, cervical cells are collected from the ectocervix and placed into an alcohol-based fixative prior to staining and pathologic examination. We hypothesized that proteins shed by cells of the female genital tract can be detected in the Pap test fixative by MS-based proteomic techniques. We examined the feasibility of using residual fluid from discarded Pap tests with cytologically “normal” results to optimize sample preparation for MS analysis. The protein composition of the cell-free Pap test fluid was determined by silver staining of sodium dodecyl sulfate -polyacrylamide gels, and the abundance of serum proteins was examined by Western immunoblot using an antibody against human serum albumin. Both pooled and individual samples were trypsin digested and analyzed by two-dimensional MS/MS. Proteins were identified by searching against the Human Uniprot database, and characterized for localization, function and relative abundance.

Results

The average volume of the residual Pap test fluid was 1.5 ml and the average protein concentration was 0.14 mg/ml. By Western immunoblot we showed that the amount of albumin in each sample was significantly reduced compared to normal serum. By MS/MS, we identified 714 unique proteins in pooled Pap test samples and an average of 431 proteins in individual samples. About 40% of the proteins identified were extracellular or localized to the plasma membrane. Almost 20% of the proteins identified were involved in immunity and defense, characteristic of the healthy cervical-vaginal proteome. By merging the protein sets from the individual and pooled Pap test samples, we created a “Normal Pap test Core Proteome” consisting of 153 proteins.

Conclusions

Residual Pap test fluid contains a sufficient amount of protein for analysis by MS and represents a valuable biospecimen source for the identification of protein biomarkers for gynecological diseases.     

Electrospray ionization mass spectroscopic analysis of peptides modified with N-ethylmaleimide or iodoacetanilide

The cysteine-specific modifiers we reported previously, N-ethylmaleimide (NEM) and iodoacetanilide (IAA), have been applied to label cysteine residues of peptides in combination with electrospray ionization mass spectrometry (ESI-MS/MS), and their scope in proteomic studies was examined. Peptides modified with N-ethylmaleimide (NEM) or iodoacetanilide (IAA) showed significant enhancement in ionization efficiencies. These modifiers were also found to remain intact in tandem mass spectrometry. Both combinations of N-ethylmaleimide (NEM) and d₅-N-ethylmaleimide (d₅-NEM), and iodoacetanilide (IAA) and ¹³C₆-iodoacetanilide (¹³C₆-IAA) were also shown to be applicable to quantitative analysis of a peptide.     

Diffprot - software for non-parametric statistical analysis of differential proteomics data

Mass spectrometry-based global proteomics experiments generate large sets of data that can be converted into useful information only with an appropriate statistical approach. We present Diffprot - a software tool for statistical analysis of MS-derived quantitative data. With implemented resampling-based statistical test and local variance estimate, Diffprot allows to draw significant results from small scale experiments and effectively eliminates false positive results. To demonstrate the advantages of this software, we performed two spike-in tests with complex biological matrices, one label-free and one based on iTRAQ quantification; in addition, we performed an iTRAQ experiment on bacterial samples. In the spike-in tests, protein ratios were estimated and were in good agreement with theoretical values; statistical significance was assigned to spiked proteins and single or no false positive results were obtained with Diffprot. We compared the performance of Diffprot with other statistical tests - widely used t-test and non-parametric Wilcoxon test. In contrast to Diffprot, both generated many false positive hits in the spike-in experiment. This proved the superiority of the resampling-based method in terms of specificity, making Diffprot a rational choice for small scale high-throughput experiments, when the need to control the false positive rate is particularly pressing.     

Chlamydomonas reinhardtii proteomics

Proteomics, based on the expanding genomic resources, has begun to reveal new details of Chlamydomonas reinhardtii biology. In particular, analyses focusing on subproteomes have already provided new insight into the dynamics and composition of the photosynthetic apparatus, the chloroplast ribosome, the oxidative phosphorylation machinery of the mitochondria, and the flagellum. It assisted to discovered putative new components of the circadian clockwork as well as shed a light on thioredoxin protein-protein interactions. In the future, quantitative techniques may allow large scale comparison of protein expression levels. Advances in software algorithms will likely improve the use of genomic databases for mass spectrometry (MS) based protein identification and validation of gene models that have been predicted from the genomic DNA sequences. Although proteomics has only been recently applied for exploring C. reinhardtii biology, it will likely be utilized extensively in the near future due to the already existing genetic, genomic, and biochemical tools.     

Reciprocal Changes in Phosphorylation and Methylation of Mammalian Brain Sodium Channels in Response to Seizures

Voltage-gated sodium (Nav) channels initiate action potentials in brain neurons and are primary therapeutic targets for anti-epileptic drugs controlling neuronal hyperexcitability in epilepsy. The molecular mechanisms underlying abnormal Nav channel expression, localization, and function during development of epilepsy are poorly understood but can potentially result from altered posttranslational modifications (PTMs). For example, phosphorylation regulates Nav channel gating, and has been proposed to contribute to acquired insensitivity to anti-epileptic drugs exhibited by Nav channels in epileptic neurons. However, whether changes in specific brain Nav channel PTMs occur acutely in response to seizures has not been established. Here, we show changes in PTMs of the major brain Nav channel, Nav1.2, after acute kainate-induced seizures. Mass spectrometry-based proteomic analyses of Nav1.2 purified from the brains of control and seizure animals revealed a significant down-regulation of phosphorylation at nine sites, primarily located in the interdomain I-II linker, the region of Nav1.2 crucial for phosphorylation-dependent regulation of activity. Interestingly, Nav1.2 in the seizure samples contained methylated arginine (MeArg) at three sites. These MeArgs were adjacent to down-regulated sites of phosphorylation, and Nav1.2 methylation increased after seizure. Phosphorylation and MeArg were not found together on the same tryptic peptide, suggesting reciprocal regulation of these two PTMs. Coexpression of Nav1.2 with the primary brain arginine methyltransferase PRMT8 led to a surprising 3-fold increase in Nav1.2 current. Reciprocal regulation of phosphorylation and MeArg of Nav1.2 may underlie changes in neuronal Nav channel function in response to seizures and also contribute to physiological modulation of neuronal excitability.     

Integration of an on-line protein digestion microreactor to a nanoelectrospray emitter for peptide mapping

A method for integrating nanoelectrospray mass spectrometry with a microreactor for on-line digestion and fast peptide mass mapping from dilute protein samples is presented. Fused silica capillaries (i.d. 50 microm, o.d. 360 microm) are employed as the digestion microreactor and the nanoelectrospray emitter by immobilizing trypsin onto the surface of the inner wall of the fused silica capillary tubing. The procedure is demonstrated using solutions of 1pmol/mul angiotensin II, cytochrome c, hemoglobin, and beta-casein. Because the inner walls of the capillaries are modified by covalent chemical bonds, the adsorption of peptides and proteins to the inner walls of the capillaries is suppressed. This procedure was performed with solutions as dilute as 1fmol/mul (1nM) cytochrome c. This method shows generation of tryptic peptides with sequence coverage up to 90% within minutes; trypsin autolysis products are not detected. In addition, the immobilized enzyme can be cleaned easily, enabling the microreactor to be reused for nanoelectrospray.