The importance of the digest: proteolysis and absolute quantification in proteomics

Virtually all mass spectrometric-based methods for quantitative proteomics are at the peptide level, whether label-mediated or label-free. Absolute quantification in particular is based on the measurement of limit peptides, defined as those peptides that cannot be further fragmented by the protease in use. Complete release of analyte and (stable isotope labelled) standard ensures that the most reliable quantification data are recovered, especially when the standard peptides are in a different primary sequence context, such as sometimes occurs in the QconCAT methodology. Moreover, in label-free methods, incomplete digestion would diminish the ion current attributable to limit peptides and lead to artifactually low quantification data. It follows that an essential requirement for peptide-based absolute quantification in proteomics is complete and consistent proteolysis to limit peptides. In this paper we describe strategies to assess completeness of proteolysis and discuss the potential for variance in digestion efficiency to compromise the ensuing quantification data. We examine the potential for kinetically favoured routes of proteolysis, particularly at the last stages of the digestion, to direct products into ‘dead-end’ mis-cleaved products.     

Label-free quantitative proteomics reveals differentially regulated proteins in the latex of sticky diseased Carica papaya L. plants

Papaya meleira virus (PMeV) is so far the only described laticifer-infecting virus, the causal agent of papaya (Carica papaya L.) sticky disease. The effects of PMeV on the laticifers' regulatory network were addressed here through the proteomic analysis of papaya latex. Using both 1-DE- and 1D-LC-ESI-MS/MS, 160 unique papaya latex proteins were identified, representing 122 new proteins in the latex of this plant. Quantitative analysis by normalized spectral counting revealed 10 down-regulated proteins in the latex of diseased plants, 9 cysteine proteases (chymopapain) and 1 latex serine proteinase inhibitor. A repression of papaya latex proteolytic activity during PMeV infection was hypothesized. This was further confirmed by enzymatic assays that showed a reduction of cysteine-protease-associated proteolytic activity in the diseased papaya latex. These findings are discussed in the context of plant responses against pathogens and may greatly contribute to understand the roles of laticifers in plant stress responses.     

Translating clinical proteomics: the importance of study design

Mass spectrometry-based clinical proteomics approaches were introduced into the biomedical field more than two decades ago. Despite recent developments both in the field of mass spectrometry and bioinformatics, the gap between proteomics results and their translation into clinical practice still needs to be closed, as implementation of proteomics results in the clinic appears to be scarce. An extra focus on the importance of the experimental design is therefore of crucial importance.     

Combining bioinformatics and MS-based proteomics: clinical implications

Clinical proteomics research aims at i) discovery of protein biomarkers for screening, diagnosis and prognosis of disease, ii) discovery of protein therapeutic targets for improvement of disease prevention, treatment and follow-up, and iii) development of mass spectrometry (MS)-based assays that could be implemented in clinical chemistry, microbiology or hematology laboratories. MS has been increasingly applied in clinical proteomics studies for the identification and quantification of proteins. Bioinformatics plays a key role in the exploitation of MS data in several aspects such as the generation and curation of protein sequence databases, the development of appropriate software for MS data treatment and integration with other omics data and the establishment of adequate standard files for data sharing. In this article, we discuss the main MS approaches and bioinformatics solutions that are currently applied to accomplish the objectives of clinical proteomic research.     

Optimization of reversed-phase microcapillary liquid chromatography for quantitative proteomics

A specific capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS) method for the determination of serotonin (5HT) and its precursors tryptophan (Trp) and 5-hydroxytryptophan (5HTP) in human platelet rich plasma is described. The analytes were removed from the plasma samples and preconcentrated by solid phase extraction (SPE) on mixed mode cation-exchange sorbents. The SPE recoveries were 71.6 +/- 3.1 for 5HT, 91.0 +/- 2.8 for Trp, and 95.3 +/- 5.9% for 5HTP. Deuterated analogues of 5HT and Trp were used as internal standards for quantitation purposes. Submicromolar detection limits were obtained for standard mixtures of all compounds and their deuterated isotopes, except 5HTP, which had detection limits in the low micromolar range. The potential usefulness of this method in the clinical setting was demonstrated by analyzing plasma extracts from healthy volunteers as well as from pathological samples. While 5HTP was not present in any of the analyzed samples, the levels of 5HT and Trp in both normal and pathological plasma were determined.     

Bioinformatics approaches in clinical proteomics

Protein expression profiling is increasingly being used to discover, validate and characterize biomarkers that can potentially be used for diagnostic purposes and to aid in pharmaceutical development. Correct analysis of data obtained from these experiments requires an understanding of the underlying analytic procedures used to obtain the data, statistical principles underlying high-dimensional data and clinical statistical tools used to determine the utility of the interpreted data. This review summarizes each of these steps, with the goal of providing the nonstatistician proteomics researcher with a working understanding of the various approaches that may be used by statisticians. Emphasis is placed on the process of mining high-dimensional data to identify a specific set of biomarkers that may be used in a diagnostic or other assay setting.     

Label-free quantitative proteomic analysis of the inhibitory activities of juglone against translation and energy metabolism in Escherichia coli

Plant-derived antimicrobial agents have received increasing attention owing to their potential to control pathogens and excellent efficacy despite the growing prevalence of antibiotic resistance. However, the antibacterial mechanism of juglone, a traditional medicine used to cure skin infections, is still unclear. Therefore, in this study, in order to elucidate the mechanisms underlying the antibacterial activity of juglone, label-free quantitative proteomic technology was applied for analysis of the 417 proteins that were differentially expressed in Escherichia coli after treatment with juglone at one-half of the minimum inhibitory concentration. Gene ontology enrichment analysis of differentially expressed proteins suggested that juglone effectively repressed the expression of dehydrogenase and cytochrome oxidase, indicating that energy generation was blocked. Additionally, juglone induced RNA formation and ribosome assembly, resulting in inhibition of translation. This is the first study to adopt a proteomic approach to investigate the antibacterial mechanism of action of juglone against E. coli.     

Application of proteomics to graft-versus-host disease: from biomarker discovery to potential clinical applications

Introduction: Graft-versus-host disease (GVHD) is a frequent and potentially life-threatening complication that occurs in many patients who undergo hematopoietic stem cell transplantation. In an effort to develop blood and tissue-based biochemical assays for GVHD diagnosis, high throughput proteomic platforms have been widely utilized for the identification and validation of disease biomarkers for both acute and chronic GVHD.

Areas covered: This article reviews biomarker research findings on acute and chronic GVHD ascertained by studying peripheral blood, urine and saliva that gives biological information on systemic or localized disease. While the primary focus of GVHD biomarker discovery has been on identification and validation of prognostic and predictive biomarkers that might allow stratification of disease risk, molecular biomarkers that might aid patient diagnosis and/or response to treatment have also been reported.

Expert commentary: Unbiased as well as targeted proteomic studies of acute and chronic GVHD have identified some distinguishing features of the two diseases especially the role of certain immune cell populations. A combination of patient risk stratification using panels of biomarkers and the application of novel targeted therapeutics should help to reduce the burden of GVHD, and hence improve the quality of life for patients following hematopoietic stem cell transplantation.     

High-coverage quantitative proteomics using amine-specific isotopic labeling

Peptide dimethylation with isotopically coded formaldehydes was evaluated as a potential alternative to techniques such as the iTRAQ method for comparative proteomics. The isotopic labeling strategy and custom-designed protein quantitation software were tested using protein standards and then applied to measure proteins levels associated with Alzheimer's disease (AD). The method provided high accuracy (10% error), precision (14% RSD) and coverage (70%) when applied to the analysis of a standard solution of BSA by LC-MS/MS. The technique was then applied to measure protein abundance levels in brain tissue afflicted with AD relative to normal brain tissue. 2-D LC-MS analysis identified 548 unique proteins (p<0.05). Of these, 349 were quantified with two or more peptides that met the statistical criteria used in this study. Several classes of proteins exhibited significant changes in abundance. For example, elevated levels of antioxidant proteins and decreased levels of mitochondrial electron transport proteins were observed. The results demonstrate the utility of the labeling method for high-throughput quantitative analysis.     

Introduction to proteomics: tools for the new biology

Towards revolutionary biomarkers, a considerable amount of research funds and time have been dedicated to proteomics. Although the discovery of novel biomarkers at the dawn of proteomics was a promising development, only a few identified biomarkers seemed to be beneficial for cancer patients. We may need to approach this issue differently, instead of only extending the conventional approaches that have been used historically. The study of biomarkers is essentially a study of diseases and the biochemistry relating to peptide, protein and post-translational modifications is only a tool. A problem-oriented approach should be needed in biomarker development. Clinician participation in the study of biomarkers will lead to realistic, practical and interesting biomarker candidates, which justify the time and expense involved in validation studies. Although discussion in this article is focused on cancer biomarkers, it can generally be applied to biomarker studies for other diseases.     

A label-free quantification method by MS/MS TIC compared to SILAC and spectral counting in a proteomics screen

In order to assess the biological function of proteins and their modifications for understanding signaling mechanisms within cells as well as specific biomarkers to disease, it is important that quantitative information be obtained under different experimental conditions. Stable isotope labeling is a powerful method for accurately determining changes in the levels of proteins and PTMs; however, isotope labeling experiments suffer from limited dynamic range resulting in signal change ratios of less than approximately 20:1 using most commercial mass spectrometers. Label-free approaches to relative quantification in proteomics such as spectral counting have gained popularity since no additional chemistries are needed. Here, we show a label-free method for relative quantification based on the TIC from peptide MS/MS spectra collected from data-dependent runs can be used effectively as a quantitative measure and expands the dynamic range over isotope labeling experiments allowing for abundance differences up to approximately 60:1 in a screen for proteins that bind to phosphotyrosine residues.     

Simultaneous Improvement in the Precision,Accuracy, and Robustness of Label-free Proteome Quantification by Optimizing Data Manipulation Chains,

1. Download : Download high-res image (158KB)
2. Download : Download full-size image

Highlights

• •High-quality LFQ is valuable technique yet remains extremely challenging.
• •Fluctuating precision, limited robustness, and compromised accuracy are known issues.
• •We proposed a strategy collectively improving LFQ precision, robustness, and accuracy.
• •An online tool incorporating this novel strategy was also developed.

     

Alternative profiling platform based on MELDI and its applicability in clinical proteomics

The presence of numerous proteomics data and their results in literature reveal the importance and influence of proteins and peptides on human cell cycle. For instance, the proteomic profiling of biological samples, such as serum, plasma or cells, and their organelles, carried out by surface-enhanced laser desorption/ionization mass spectrometry, has led to the discovery of numerous key proteins involved in many biological disease processes. However, questions still remain regarding the reproducibility, bioinformatic artifacts and cross-validations of such experimental set-ups. The authors have developed a material-based approach, termed material-enhanced laser desorption/ionization mass spectrometry (MELDI-MS), to facilitate and improve the robustness of large-scale proteomic experiments. MELDI-MS includes a fully automated protein-profiling platform, from sample preparation and analysis to data processing involving state-of-the-art methods, which can be further improved. Multiplexed protein pattern analysis, based on material morphology, physical characteristics and chemical functionalities provides a multitude of protein patterns and allows prostate cancer samples to be distinguished from non-prostate cancer samples. Furthermore, MELDI-MS enables not only the analysis of protein signatures, but also the identification of potential discriminating peaks via capillary liquid chromatography mass spectrometry. The optimized MELDI approach offers a complete proteomics platform with improved sensitivity, selectivity and short sample preparation times.     

Advances in shotgun proteomics and the analysis of membrane proteomes

The emergence of shotgun proteomics has facilitated the numerous biological discoveries made by proteomic studies. However, comprehensive proteomic analysis remains challenging and shotgun proteomics is a continually changing field. This review details the recent developments in shotgun proteomics and describes emerging technologies that will influence shotgun proteomics going forward. In addition, proteomic studies of integral membrane proteins remain challenging due to the hydrophobic nature in integral membrane proteins and their general low abundance levels. However, there have been many strategies developed for enriching, isolating and separating membrane proteins for proteomic analysis that have moved this field forward. In summary, while shotgun proteomics is a widely used and mature technology, the continued pace of improvements in mass spectrometry and proteomic technology and methods indicate that future studies will have an even greater impact on biological discovery.     

免标记荧光适体传感器的研究进展

核酸适体在治疗、诊断和生物传感等领域都引起了强烈的关注和广泛的应用。与传统的识别元素-- 抗体相比较,适体展现 出很多的优点：尺寸小,化学性质稳定,容易制备和修饰。更重要的是适体在生物传感的设计上更为灵活,因此,产生了很多高选 择性、高灵敏度的新型适体传感器。目前,很多的检测手段都被应用到适体传感器中,其中荧光的检测手段占有重要的地位。虽然 荧光适体传感器已经取得了重大的进展,但是荧光标记给传感器的设计带来很多的不便,因此,免标记的荧光适体传感器备受关 注。在本文中,我们将对免标记的荧光适体传感器的研究进展进行综述,为分析工作者发展更加灵敏、更加简单、更加应用广泛的 免标记荧光适体传感器提供依据。     

Cancer biomarker discovery and translation: proteomics and beyond

Introduction: Cancer is often diagnosed at late stages when the chance of cure is relatively low and although research initiatives in oncology discover many potential cancer biomarkers, few transition to clinical applications. This review addresses the current landscape of cancer biomarker discovery and translation with a focus on proteomics and beyond.

Areas covered: The review examines proteomic and genomic techniques for cancer biomarker detection and outlines advantages and challenges of integrating multiple omics approaches to achieve optimal sensitivity and address tumor heterogeneity. This discussion is based on a systematic literature review and direct participation in translational studies.

Expert commentary: Identifying aggressive cancers early on requires improved sensitivity and implementation of biomarkers representative of tumor heterogeneity. During the last decade of genomic and proteomic research, significant advancements have been made in next generation sequencing and mass spectrometry techniques. This in turn has led to a dramatic increase in identification of potential genomic and proteomic cancer biomarkers. However, limited successes have been shown with translation of these discoveries into clinical practice. We believe that the integration of these omics approaches is the most promising molecular tool for comprehensive cancer evaluation, early detection and transition to Precision Medicine in oncology.     

Lung cancer proteomics,clinical and technological considerations

The overall survival of lung cancer patients is disappointingly low. This is due to several factors, including the lack of an effective screening strategy to detect tumors at a potentially curable early stage, a marked resistance of lung cancer cells to drug treatment and a still superficial knowledge about the multifactorial cellular networks that are activated or suppressed during cancer progression. Furthermore, the armamentarium of clinicians and researchers in the field does not yet include reliable biomarkers to predict tumor response to treatment and foresee the natural history of the disease. In the present situation, a potential breakthrough is presented by proteomics technologies with the potential to discover relevant biomarkers which can be accurately quantified in multiplexed assays. Proteomics field can also contribute greatly in the understanding of mechanisms in tumor progression and treatment response.     

Advances in qualitative and quantitative plant membrane proteomics

The membrane proteome consists of integral and membrane-associated proteins that are involved in various physiological and biochemical functions critical for cellular function. It is also dynamic in nature, where many proteins are only expressed during certain developmental stages or in response to environmental stress. These proteins can undergo post-translational modifications in response to these different conditions, allowing them to transiently associate with the membrane or other membrane proteins. Along with their increased size, hydrophobicity, and the additional organelle and cellular features of plant cells relative to mammalian systems, the characterization of the plant membrane proteome presents unique challenges for effective qualitative and quantitative analysis using mass spectrometry (MS) analysis. Here, we present the latest advancements developed for the isolation and fractionation of plant organelles and their membrane components amenable to MS analysis. Separations of membrane proteins from these enriched preparations that have proven effective are discussed for both gel- and liquid chromatography-based MS analysis. In this context, quantitative membrane proteomic analyses using both isotope-coded and label-free approaches are presented and reveal the potential to establish a wider-biological interpretation of the function of plant membrane proteins that will ultimately lead to a more comprehensive understanding of plant physiology and their response mechanisms.