High throughput protein production for functional proteomics

A major impact of genome projects on human health will be their contribution to the understanding of protein function. Proteins are the engines of biological systems, nearly all pharmaceuticals act on proteins and increasingly proteins themselves are used therapeutically. As biology enters the post-genomic era, researchers have begun to embrace the exciting opportunity of investigating proteins in high throughput (HT) experiments. The study of proteins includes a vast array of techniques ranging from enzyme catalysis assays to interaction and structural studies. Many of these methods depend on purified proteins. The discovery of thousands of novel protein-coding sequences and the increased availability of large cDNA collections provide the opportunity to investigate protein function in a systematic manner and at an unprecedented scale. This opportunity highlights the need for development of HT methods for protein isolation. This article describes the challenges faced and the approaches taken to develop proteome-scale protein expression systems.     

Clinical application of quantitative glycomics

ABSTRACT

Introduction: Aberrant glycosylation has been associated with many diseases. Decades of research activities have reported many reliable glycan biomarkers of different diseases which enable effective disease diagnostics and prognostics. However, none of the glycan markers have been approved for clinical diagnosis. Thus, a review of these studies is needed to guide the successful clinical translation.

Area covered: In this review, we describe and discuss advances in analytical methods enabling clinical glycan biomarker discovery, focusing only on studies of released glycans. This review also summarizes the different glycobiomarkers identified for cancers, Alzheimer’s disease, diabetes, hepatitis B and C, and other diseases.

Expert commentary: Along with the development of techniques in quantitative glycomics, more glycans or glycan patterns have been reported as better potential biomarkers of different diseases and proved to have greater diagnostic/diagnostic sensitivity and specificity than existing markers. However, to successfully apply glycan markers in clinical diagnosis, more studies and verifications on large biological cohorts need to be performed. In addition, faster and more efficient glycomic strategies need to be developed to shorten the turnaround time. Thus, glycan biomarkers have an immense chance to be used in clinical prognosis and diagnosis of many diseases in the near future.     

High throughput and quantitative enzymology in the genomic era

Accurate predictions from models based on physical principles are the ultimate metric of our biophysical understanding. Although there has been stunning progress toward structure prediction, quantitative prediction of enzyme function has remained challenging. Realizing this goal will require large numbers of quantitative measurements of rate and binding constants and the use of these ground-truth data sets to guide the development and testing of these quantitative models. Ground truth data more closely linked to the underlying physical forces are also desired. Here, we describe technological advances that enable both types of ground truth measurements. These advances allow classic models to be tested, provide novel mechanistic insights, and place us on the path toward a predictive understanding of enzyme structure and function.     

Measurement of protein metabolism in epidermis and dermis

We found that, in the rabbit ear, the dermal protein contains 75.5% of cutaneous phenylalanine and 97.9% of cutaneous proline; the remaining 24.5% of phenylalanine and 2.1% of proline are in the epidermal protein. This finding led us to develop two novel models that use phenylalanine and proline tracers and the rabbit ear to quantify protein kinetics in the epidermis and dermis. The four-pool model calculates the absolute rates of protein kinetics and amino acid transport, and the two-pool model calculates the apparent rates of protein kinetics that are reflected in the blood. The results showed that both epidermis and dermis maintained their protein mass in the postabsorptive state. The rate of epidermal protein synthesis was 93.4 +/- 37.6 mg x 100 g(-1) x h(-1), which was 10-fold greater than that of the dermal protein (9.3 +/- 5.8 mg x 100 g(-1) x h(-1)). These synthetic rates were in agreement with those measured simultaneously by the tracer incorporation method. Comparison of the four-pool and two-pool models indicated that intracellular cycling of amino acids accounted for 75 and 90% of protein kinetics in the dermis and epidermis, respectively. We conclude that, in the skin, efficient reutilization of amino acids from proteolysis for synthesis enables the maintenance of protein mass in the postabsorptive state.     

Differential growth of sensory neurons in vitro in presence of dermis and epidermis. A quantitative time-lapse analysis

The influence of dermal and epidermal cells on the growth of nerve fibres from chick embryo sensory neurons was investigated in vitro. A previous quantitative analysis showed that the growth of nerve fibres is profoundly modified in the close vicinity of epidermis. This change is mainly characterized by erratic trajectories of nerve fibres resulting from numerous lateral displacements of the growth cones. In contrast, no such behaviour is observed far away from the epidermis or in the presence of dermis. In this latter case, neurites exhibit a straighter direction of extension. These observations suggest that the epidermis exerts some kind of control on the establishment of nerve fibre pattern in the dermis.     

Mass spectrometry for high throughput quantitative proteomics in plant research: lessons from thylakoid membranes

Proteomics seeks to monitor the flux of protein through cells under variable developmental and environmental influences as programmed by the genome. Consequently, it is necessary to measure changes in protein abundance and turnover rate as faithfully as possible. In the absence of non-invasive technologies, the majority of proteomics approaches involve destructive sampling at various time points to obtain 'snapshots' that periodically report the genomes's product. The work has fallen to separations technologies coupled to mass spectrometry, for high throughput protein identification. Quantitation has become the major challenge facing proteomics as the field matures. Because of the variability of day-to-day measurements of protein quantities by mass spectrometry, a common feature of quantitative proteomics is the use of stable isotope coding to distinguish control and experimental samples in a mixture that can be profiled in a single experiment. To address limitations with separation technologies such as 2D-gel electrophoresis, alternative systems are being introduced including multi-dimensional chromatography. Strategies that accelerate throughput for mass spectrometry are also emerging and the benefits of these 'shotgun' protocols will be considered in the context of the thylakoid membrane and photosynthesis. High resolution Fourier-transform mass spectrometry is bringing increasingly accurate mass measurements to peptides and a variety of gas-phase dissociation mechanisms are permitting 'top-down' sequencing of intact proteins. Finally, a versatile workflow for sub-cellular compartments including membranes is presented that allows for intact protein mass measurements, localization of post-translational modifications and relative quantitation or turnover measurement.     

Steady-state heat distribution in epidermis,dermis and subdermal tissues

Steady-state temperature distribution is investigated in human skin and subdermal tissue exposed to a dry and cool environment with negligible insensible perspiration. The mathematical model incorporates the effect of blood mass flow and metabolic heat generation. The rates of the two and the tissue thermal conductivity are assumed to have different values in all the three parts, namely epidermis, dermis and subdermal tissues. A simple variational finite element approach is used to find numerical values of the interface temperatures for a wide range of the values of skin surface temperature and for different thicknesses of the above parts. These values are used to obtain approximate temperature profiles in the whole region. The biological and physical implications of the results are also discussed.     

High throughput two-dimensional blue-native electrophoresis: a tool for functional proteomics of mitochondria and signaling complexes

The recent upsurge in proteomics research has been facilitated largely by streamlining of two-dimensional (2-D) gel technology and the parallel development of facile mass spectrometry for analysis of peptides and proteins. However, application of these technologies to the mitochondrial proteome has been limited due to the considerable complement of hydrophobic membrane proteins in mitochondria, which precipitate during first dimension isoelectric focusing of standard 2-D gels. In addition, functional information regarding protein:protein interactions is lost during 2-D gel separation due to denaturing conditions in both gel dimensions. To resolve these issues, 2-D blue-native gel electrophoresis was applied to the mitochondrial proteome. In this technique, membrane protein complexes such as those of the respiratory chain are solubilized and resolved in native form in the first dimension. A second dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel then denatures the complexes and resolves them into their component subunits. Refinements to this technique have yielded the levels of throughput and reproducibility required for proteomics. By coupling to tryptic peptide fingerprinting using matrix-assisted laser desorption/ionization-time of flight mass spectrometry, a partial mitochondrial proteome map has been assembled. Applications of this functional mitochondrial proteomics method are discussed.     

Discovery of novel differentiation markers in the early stage of chondrogenesis by glycoform-focused reverse proteomics and genomics

Background

Osteoarthritis (OA) is one of the most common chronic diseases among adults, especially the elderly, which is characterized by destruction of the articular cartilage. Despite affecting more than 100 million individuals all over the world, therapy is currently limited to treating pain, which is a principal symptom of OA. New approaches to the treatment of OA that induce regeneration and repair of cartilage are strongly needed.

Methods

To discover potent markers for chondrogenic differentiation, glycoform-focused reverse proteomics and genomics were performed on the basis of glycoblotting-based comprehensive approach.

Results

Expression levels of high-mannose type N-glycans were up-regulated significantly at the late stage of differentiation of the mouse chondroprogenitor cells. Among 246 glycoproteins carrying this glycotype identified by ConA affinity chromatography and LC/MS, it was demonstrated that 52% are classified as cell surface glycoproteins. Gene expression levels indicated that mRNAs for 15 glycoproteins increased distinctly in the earlier stages during differentiation compared with Type II collagen. The feasibility of mouse chondrocyte markers in human chondrogenesis model was demonstrated by testing gene expression levels of these 15 glycoproteins during differentiation in human mesenchymal stem cells.

Conclusion

The results showed clearly an evidence of up-regulation of 5 genes, ectonucleotide pyrophosphatase/phosphodiesterase family member 1, collagen alpha-1(III) chain, collagen alpha-1(XI) chain, aquaporin-1, and netrin receptor UNC5B, in the early stages of differentiation.

General significance

These cell surface 5 glycoproteins become highly sensitive differentiation markers of human chondrocytes that contribute to regenerative therapies, and development of novel therapeutic reagents.     

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.     

Mechanobiological dysregulation of the epidermis and dermis in skin disorders and in degeneration

During growth and development, the skin expands to cover the growing skeleton and soft tissues by constantly responding to the intrinsic forces of underlying skeletal growth as well as to the extrinsic mechanical forces from body movements and external supports. Mechanical forces can be perceived by two types of skin receptors: (1) cellular mechanoreceptors/mechanosensors, such as the cytoskeleton, cell adhesion molecules and mechanosensitive (MS) ion channels, and (2) sensory nerve fibres that produce the somatic sensation of mechanical force. Skin disorders in which there is an abnormality of collagen [e.g. Ehlers–Danlos syndrome (EDS)] or elastic (e.g. cutis laxa) fibres or a malfunction of cutaneous nerve fibres (e.g. neurofibroma, leprosy and diabetes mellitus) are also characterized to some extent by deficiencies in mechanobiological processes. Recent studies have shown that mechanotransduction is crucial for skin development, especially hemidesmosome maturation, which implies that the pathogenesis of skin disorders such as bullous pemphigoid is related to skin mechanobiology. Similarly, autoimmune diseases, including scleroderma and mixed connective tissue disease, and pathological scarring in the form of keloids and hypertrophic scars would seem to be clearly associated with the mechanobiological dysfunction of the skin. Finally, skin ageing can also be considered as a degenerative process associated with mechanobiological dysfunction. Clinically, a therapeutic strategy involving mechanoreceptors or MS nociceptor inhibition or acceleration together with a reduction or augmentation in the relevant mechanical forces is likely to be successful. The development of novel approaches such as these will allow the treatment of a broad range of cutaneous diseases.     

Functional and quantitative proteomics using SILAC

Researchers in many biological areas now routinely characterize proteins by mass spectrometry. Among the many formats for quantitative proteomics, stable-isotope labelling by amino acids in cell culture (SILAC) has emerged as a simple and powerful one. SILAC removes false positives in protein-interaction studies, reveals large-scale kinetics of proteomes and - as a quantitative phosphoproteomics technology - directly uncovers important points in the signalling pathways that control cellular decisions.     

Lamellar bodies of human epidermis: proteomics characterization by high throughput mass spectrometry and possible involvement of CLIP-170 in their trafficking/secretion

Lamellar bodies (LBs) are tubulovesicular secretory organelles of epithelial cells related to lysosomes. In the epidermis, they play a crucial role in permeability barrier homeostasis, secreting their contents, lipids, a variety of hydrolases, protease inhibitors, and antimicrobial peptides, in the upper keratinocyte layers. The identification of proteins transported in epidermal LBs is still far from complete, and the way their secretion is controlled unknown. In this study, we describe the first proteomics characterization by nano-LC-MS/MS of a fraction enriched in epidermal LBs. We identified 984 proteins, including proteins known or thought to be secreted by LBs. Moreover 31 proteins corresponded to lysosomal components further suggesting that LBs are a new class of secretory lysosomes. Many of the newly found proteins could play a role in the epidermal barrier and desquamation (one acid ceramidase-like protein, apolipoproteins, glycosidases, protease inhibitors, and peptidases) and in LB trafficking (e.g. Rab, Arf, and motor complex proteins). We focus here on CLIP-170/restin, a protein that mediates interactions between organelles and microtubules. Western blotting confirmed the presence of CLIP-170 and its known effectors IQGAP1 and Cdc42 in the LB-enriched fraction. We showed, by confocal microscopy analysis of skin cryosections, that CLIP-170 was expressed in differentiated keratinocytes, first at the periphery of the nucleus then with a granular cytoplasmic labeling evocative of LBs. It was preferentially co-localized with Cdc42 and with the known LB protein cathepsin D. CLIP-170 was also largely co-localized with Rab7. This study strongly suggests a new function for CLIP-170, its involvement together with Cdc42 and/or Rab7 in the intracellular trafficking of LBs, and provides evidence that nano-LC-MS/MS combined with monodimensional electrophoresis separation constitutes a powerful method for identifying proteins in a complex mixture such as subcellular structures.     

High throughput quantitative analysis of serum proteins using glycopeptide capture and liquid chromatography mass spectrometry

It is expected that the composition of the serum proteome can provide valuable information about the state of the human body in health and disease and that this information can be extracted via quantitative proteomic measurements. Suitable proteomic techniques need to be sensitive, reproducible, and robust to detect potential biomarkers below the level of highly expressed proteins, generate data sets that are comparable between experiments and laboratories, and have high throughput to support statistical studies. Here we report a method for high throughput quantitative analysis of serum proteins. It consists of the selective isolation of peptides that are N-linked glycosylated in the intact protein, the analysis of these now deglycosylated peptides by liquid chromatography electrospray ionization mass spectrometry, and the comparative analysis of the resulting patterns. By focusing selectively on a few formerly N-linked glycopeptides per serum protein, the complexity of the analyte sample is significantly reduced and the sensitivity and throughput of serum proteome analysis are increased compared with the analysis of total tryptic peptides from unfractionated samples. We provide data that document the performance of the method and show that sera from untreated normal mice and genetically identical mice with carcinogen-induced skin cancer can be unambiguously discriminated using unsupervised clustering of the resulting peptide patterns. We further identify, by tandem mass spectrometry, some of the peptides that were consistently elevated in cancer mice compared with their control littermates.     

定量蛋白质组学研究的技术体系

从蛋白质组水平上对基因表达进行准确的定量分析,是比较蛋白质组学的重要内容,是研究重大疾病致病机制以及药理控制机制的必要手段。定量分析蛋白质组的方法主要包括:2DE结合串联质谱技术,稳定同位素标记技术等。     

Precise mapping of increased sialylation pattern and the expression of acute phase proteins accompanying murine tumor progression in BALB/c mouse by integrated sera proteomics and glycomics

Inbred BALB/c mouse implanted with murine tumors serves as an attractive model system for the studies of cancer biology in immuno-competent individuals. It is anticipated that tumor progression would induce notable pathophysiological consequences, some of which manifested as alteration in serum proteomic and glycomic profiles. Similar to sera derived from human cancer patients and immuno-compromised mice bearing human tumors, we show in this work that BALB/c mice of the same genetic background but bearing two distinct tumor origins both exhibited elevated expression levels of acute phase proteins including haptoglobin and serum amyloid P protein, in response to tumor progression. Such common traits are generally not informative nor qualifying as biomarkers. Additional mass spectrometry (MS)-based glycomic mapping nevertheless detected distinctive changes of sialylation pattern on the complex type N-glycans. MALDI MS/MS sequencing afforded a facile but definitive identification of an increase in internal Neu5Gcalpha2-6 sialylation on the GlcNAc of the Neu5Gc2-3Gal1-3GlcNAc terminal sequence as a common feature whereas a substitution of Neu5Gc by Neu5Ac was found to be induced by colonic but not breast tumor. A more pronounced change was similarly detected on N-glycans derived from ascitic fluids representing late tumor progression stages. We next demonstrated that such distinct change in glycotope expression can be localized to a particular protein carrier by LC-MS/MS analysis of glycopeptides. Serotransferrin was identified as one such abundant serum glycoprotein, which changed significantly not in protein expression level but in terminal glycosylation pattern.