Flow field-flow fractionation: a pre-analytical method for proteomics

Proteome analysis requires a comprehensive approach including high-performance separation methods, mass spectrometric analysis, and bioinformatics. While recent advances in mass spectrometry (MS) have led to remarkable improvements in the ability to characterize complex mixtures of biomolecules in proteomics, a proper pre-MS separation step of proteins/peptides is still required. The need of high-performance separation and/or isolation/purification techniques of proteins is increasing, due to the importance of proteins expressed at extremely low levels in proteome samples. In this review, flow field-flow fractionation (F4) is introduced as a complementary pre-analytical separation method for protein separation/isolation, which can be effectively utilized for proteomic research. F4 is a set of elution-based techniques that are capable of separating macromolecules by differences in diffusion coefficient and, therefore, in hydrodynamic size. F4 provides protein separation without surface interaction of the analyte with packing or gel media. Separation is carried out in an open channel structure by a flow stream of a mobile phase of any composition, and it is solely based on the interaction of the analytes with a perpendicularly-applied, secondary flow of the fluid. Therefore, biological analytes such as proteins can be kept under a bio-friendly environment without losing their original structural configuration. Moreover, proteins fractionated on a size/shape basis can be readily collected for further characterization or proteomic analysis by MS using, for instance, either on-line or off-line methods based on electrospray ionization (ESI) or matrix-assisted laser desorption-ionization (MALDI). This review focuses on the advantages of F4 compared to most-assessed separation/isolation techniques for proteomics, and on selected applications based on size-dependent proteome separation. New method developments based on the hyphenation of F4 with on-line or off-line MS, and with other separation methods such as capillary isoelectric focusing (CIEF) are also described.     

Fourier transform infrared spectroscopic analysis of protein secondary structures

Infrared spectroscopy is one of the oldest and well established experimental techniques for the analysis of secondary structure of polypeptides and proteins. It is convenient, non-destructive, requires less sample preparation, and can be used under a wide variety of conditions. This review introduces the recent developments in Fourier transform infrared （FTIR） spectroscopy technique and its applications to protein structural studies. The experimental skills, data analysis, and correlations between the FTIR spectroscopic bands and protein secondary structure components are discussed. The applications of FTIR to the second- ary structure analysis, conformational changes, structural dynamics and stability studies of proteins are also discussed.     

Engineered affinity proteins—Generation and applications

The use of combinatorial protein engineering to design proteins with novel binding specificities and desired properties has evolved into a powerful technology, resulting in the recent advances in protein library selection strategies and the emerge of a variety of new engineered affinity proteins. The need for different protein library selection methods is due to that each target protein pose different challenges in terms of its availability and inherent properties. At present, alternative engineered affinity proteins are starting to complement and even challenge the classical immunoglobulins in different applications in biotechnology and potentially also for in vivo use as imaging agents or as biotherapeutics. This review article covers the generation and use of affinity proteins generated through combinatorial protein engineering. The most commonly used selection techniques for isolation of desired variants from large protein libraries are described. Different antibody derivatives, as well as a variety of the most validated engineered protein scaffolds, are discussed. In addition, we provide an overview of some of the major present and future applications for these engineered affinity proteins in biotechnology and medicine.     

Nuclear Magnetic Resonance Spectroscopy for the Study of B-Cell Epitopes

High-resolution nuclear magnetic resonance (NMR) spectroscopy is a structural technique that is finding increasing use in the study of antibody–antigen interactions. In this review we describe how the dynamic structural parameters obtained from NMR spectroscopy can further our understanding of B-cell epitopes and their function. Specific applications of NMR spectroscopy to examine the residues on peptides and proteins that contact the antibody combining site are also described. These include “footprinting” techniques using H–D exchange–COSY NMR spectroscopy, which are particularly useful for epitope mapping of protein antigens. For smaller systems, such as Fab–or Fv–peptide complexes, nuclear magnetization transfer difference NMR spectroscopy, transferred nuclear Overhauser effect spectroscopy, double-quantum-filtered NOE spectroscopy, and isotope editing techniques have been applied. The interpretation and limitations of the data obtained from these procedures and anticipated improvements in these applications in the future are discussed.     

Development of yeast molecular display systems focused on therapeutic proteins, enzymes, and foods: functional analysis of proteins and its application to bioconversion

Molecular display systems using yeast have been developed for industrial, medical, pharmaceutical, and biological studies. Although several host cells are available to construct a molecular display system, the yeast Saccharomyces cerevisiae is a well-established and convenient organism in eukaryotes. A wide variety of prokaryotic and eukaryotic proteins have been displayed on yeast cell surfaces. In addition, functional analyses and applications to bioconversion have been performed on the cell surface, and cells are conveniently engineered by molecular display systems. In this review, we focus on the yeast molecular display system with regard to therapeutic proteins, several enzymes, and food ingredients. In addition, recent patents on molecular display using yeast cell for production of those compounds, screening technology and related techniques are introduced. Development of devices for functional analysis of created and modified proteins in the yeast display system is also described.     

Multidimensional separations for protein/peptide analysis in the post-genomic era

Proteomics is the study of all or part of the protein complement of genes in an organism, often involving the analysis of complex protein/peptide samples. Such complex samples are beyond the separation capacity of 1-D separation techniques. This review describes several multidimensional separations for proteins and peptides. First, several variants of 2-D liquid chromatography (2DLC) are reviewed, including coupled size exclusion-reversed phase, ion exchange-reversed phase, and reversed phase-reversed phase chromatography. Second, we describe coupled liquid chromatography and capillary electrophoresis methods. Finally, a multidimensional protein identification technique (MudPIT) is explained in detail. Each of the described techniques has a much higher separation capacity than 1-D methods and can potentially be automated for high-throughput experiments. In particular, MudPIT takes advantage of both the high separation capacity of 2DLC and the powerful peptide characterization ability of tandem mass spectrometry to analyze complex protein samples. Additional applications and developments of multidimensional liquid separations for proteomics are expected in the future.     

Glycoproteomics based on tandem mass spectrometry of glycopeptides

Next to the identification of proteins and the determination of their expression levels, the analysis of post-translational modifications (PTM) is becoming an increasingly important aspect in proteomics. Here, we review mass spectrometric (MS) techniques for the study of protein glycosylation at the glycopeptide level. Enrichment and separation techniques for glycoproteins and glycopeptides from complex (glyco-)protein mixtures and digests are summarized. Various tandem MS (MS/MS) techniques for the analysis of glycopeptides are described and compared with respect to the information they provide on peptide sequence, glycan attachment site and glycan structure. Approaches using electrospray ionization and matrix-assisted laser desorption/ionization (MALDI) of glycopeptides are presented and the following fragmentation techniques in glycopeptide analysis are compared: collision-induced fragmentation on different types of instruments, metastable fragmentation after MALDI ionization, infrared multi-photon dissociation, electron-capture dissociation and electron-transfer dissociation. This review discusses the potential and limitations of tandem mass spectrometry of glycopeptides as a tool in structural glycoproteomics.     

Membrane protein reconstitution for functional and structural studies

Membrane proteins are involved in various critical biological processes,and studying membrane proteins represents a major challenge in protein biochemistry.As shown by both structural and functional studies,the membrane environment plays an essential role for membrane proteins.In vitro studies are reliant on the successful reconstitution of membrane proteins.This review describes the interaction between detergents and lipids that aids the understanding of the reconstitution processes.Then the techniques of detergent removal and a few useful techniques to refine the formed proteoliposomes are reviewed.Finally the applications of reconstitution techniques to study membrane proteins involved in Ca2+ signaling are summarized.     

Turnover of oxidatively modified proteins: the usage of in vitro and metabolic labeling

Cellular reactions to oxidative stress always include a response in the protein turnover. Therefore, cellular handling of proteins is important to observe. In this method review, radioactive labeling of proteins in vitro and in intact cells is described. The use of techniques based on the radioactive quantification of amino acids is much more selective and reliable than other nonradioactive methods for studying the protein turnover of both long- and short-lived proteins. Variations of such measurements allow one to measure protein synthesis, protein degradation, formation of insoluble proteins, and, perhaps, the turnover of individual proteins.     

Applications of cell-based phage display panning to proteomic analysis

In the post-genomic era, proteomics together with genomic tools have led to powerful new strategies in basic and clinical research. These combined “omics” technologies are being integrated into the drug target discovery process. Unlike the genome, the proteome is a highly dynamic entity that requires techniques capable of analyzing on selected populations of proteins in specific biological conditions that reflect the proteins’ functional characteristics. Antibodies have become one of the most important reagents for the analysis of selected populations of proteins, and the application of phage-display antibody libraries to high-throughput antibody generation against large numbers of various antigens provides a tool for proteome-wide protein expression analysis. In this review, we will discuss the utility of phage-display antibodies in proteomics applications, specifically for the discovery of novel disease markers and therapeutic targets.     

Deep Profiling of Cellular Heterogeneity by Emerging Single‐Cell Proteomic Technologies

The ability to comprehensively profile cellular heterogeneity in functional proteome is crucial in advancing the understanding of cell behavior, organism development, and disease mechanisms. Conventional bulk measurement by averaging the biological responses across a population often loses the information of cellular variations. Single‐cell proteomic technologies are becoming increasingly important to understand and discern cellular heterogeneity. The well‐established methods for single‐cell protein analysis based on flow cytometry and fluorescence microscopy are limited by the low multiplexing ability owing to the spectra overlap of fluorophores for labeling antibodies. Recent advances in mass spectrometry (MS), microchip, and reiterative staining‐based techniques for single‐cell proteomics have enabled the evaluation of cellular heterogeneity with high throughput, increased multiplexity, and improved sensitivity. In this review, the principles, developments, advantages, and limitations of these advanced technologies in analysis of single‐cell proteins, along with their biological applications to study cellular heterogeneity, are described. At last, the remaining challenges, possible strategies, and future opportunities that will facilitate the improvement and broad applications of single‐cell proteomic technologies in cell biology and medical research are discussed.     

Interactive map projection algorithm for illustrating protein surfaces

An interactive map projection algorithm and cluster analysis program are described which can be used in the display and analysis of protein surfaces. The application of the techniques to the analysis of protein charge distributions is described, and a brief discussion presented on various other applications.     

DARPins and other repeat protein scaffolds: advances in engineering and applications

Antibodies have long been regarded as the only class of binding proteins. With the emergence of protein engineering techniques, new binding proteins based on alternative scaffolds have been designed. Additionally, modern technologies for selection and evolution from libraries are independent of the antibody scaffold and could thus be readily used for obtaining specific binding proteins. One important group of alternative scaffolds is based on repeat proteins. Nature is widely using these proteins to modulate protein-protein interactions, and even in the adaptive immune system of jawless vertebrates; the step to their application as an alternative to antibodies seems therefore logical. In this review, progress on DARPins and other repeat protein scaffolds will be discussed. Advances in their design as well as novel applications will be highlighted.     

Co-expression for intracellular processing in microbial protein production

The biological activity of a recombinant protein is highly dependent on its biophysical properties including post-translational modifications, solubility, and stability. Production of active recombinant proteins requires careful design of the expression strategy and purification schemes. This is often achieved by proper modification of the target protein during and/or after protein synthesis in the host cells. Such co-translational or post-translational processing of recombinant proteins is typically enabled by co-expressing the required enzymes, folding chaperones, co-factors and/or processing enzymes in the host. Various applications of the co-expression technology in protein production are discussed in this review with representative examples described.     

Characterization of loaded liposomes by size exclusion chromatography

This review focuses on the use of conventional (SEC) and high performance (HPSEC) size exclusion chromatography for the analysis of liposomes. The suitability of both techniques is examined regarding the field of liposome applications. The potentiality of conventional SEC is strongly improved by using a HPLC system associated to gel columns with a size selectivity range allowing liposome characterization in addition to particle fractionation. Practical aspects of size exclusion chromatography are described and a methodology based on HPSEC coupled to multidetection modes for on-line analysis of liposomes via label or substance encapsulation is presented. Examples of conventional SEC and HPSEC applications are described which concern polydispersity, size and encapsulation stability, bilayer permeabilization, liposome formation and reconstitution, incorporation of amphiphilic molecules. Size exclusion chromatography is a simple and powerful technique for investigation of encapsulation, insertion/interaction of substances from small solutes (ions, surfactants, drugs, etc.) up to large molecules (proteins, peptides and nucleic acids) in liposomes.     

邻位连接技术：一种新的高灵敏度蛋白质检测技术

蛋白质组学(proteomics)诞生以来,高效准确的蛋白质检测技术受到越来越多的关注.最近 ,一种高灵敏度的蛋白质检测技术,邻位连接技术(proximity ligation assay, PLA)被建 立.该技术采用核酸适体(aptamer)或单/多克隆抗体 核酸复合物作为邻位连接探针(proximity probes).当一对邻位探针同时识别同一个目标蛋白分子时,它们将在空间位置上相互临近,通过连接反应形成一段可扩增的DNA标签序列,该标签序列能够反映待测蛋白的种类及浓度.该技术将对蛋白质的检测转变为对DNA核酸序列的检测,实现了特殊蛋白质的检测,定量及定位.文章从该方法的产生背景,发展过程,原理以及探针制备等方面对该方法进行了系统的介绍,列举了该方法的几种重要应用,并对该方法在蛋白质组学研究领域的应用前景进行了展望.