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Download or read book Capillary Isoelectric Focusing-based Multidimensional Peptide/protein Separations for Proteomics Analysis written by and published by . This book was released on 2005 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:
Author : Jinzhi Chen
Publisher :
ISBN 13 :
Total Pages : 214 pages
Book Rating : 4.:/5 (547 download)
Download or read book Capillary Isoelectric Focusing-based Multidimensional Concentration/separation Platform for Ultrasensitive Proteome Analysis written by Jinzhi Chen and published by . This book was released on 2003 with total page 214 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Author : David Garfin
Publisher : Elsevier
ISBN 13 : 0080533620
Total Pages : 358 pages
Book Rating : 4.0/5 (85 download)
Download or read book Handbook of Isoelectric Focusing and Proteomics written by David Garfin and published by Elsevier. This book was released on 2005-09-29 with total page 358 pages. Available in PDF, EPUB and Kindle. Book excerpt: Isoelectric focusing (IEF) is a high-resolution, stand-alone technique that can be used as an analytical method or tool for protein purification. The only current book on the market, the Handbook of Isoelectric Focusing and Proteomics is the ideal 'one-stop' source for germane information in this discipline. This highly practical book also contains chapters on alternative methods that may pave the way in the search for efficient techniques for fractionating and purifying proteins. Complete with the history of IEF focusing to authors' insights and practical tips, this book is a must for anyone working in proteomics. * Is the only current book available on the subject * Includes author insights and practical tips* Is an ideal single source for students and researchers working in proteomics
Author : Mark A. Strege
Publisher : Springer Science & Business Media
ISBN 13 : 159259798X
Total Pages : 340 pages
Book Rating : 4.5/5 (925 download)
Download or read book Capillary Electrophoresis of Proteins and Peptides written by Mark A. Strege and published by Springer Science & Business Media. This book was released on 2008-02-04 with total page 340 pages. Available in PDF, EPUB and Kindle. Book excerpt: Throughout the more than 20 years that have followed the beginnings of capillary electrophoresis (CE), its application to the analysis of proteins and peptides has continued to be reliable, versatile, and productive. Over time, CE has matured to become a superb complement to HLPC, and in many cases has also evolved as an automated and quantitative replacement for conventional slab gel electrophoresis methods such as SDS-PAGE and isoelectric focusing. Within Capillary Electrophoresis of Proteins and Peptides, we have assembled contributions from researchers who are applying state-of-the-art CE for protein and peptide analysis, including topics that we believe are of great potential both in the present and for the future. In comparison to traditional separation methods, CE represents a miniaturized analysis technique (especially in its microchip-based format) that is highly dependent upon the basic fundamentals of effective sample recovery and high sensitivity detection. With these issues in mind, Chapters 1–4 describe recently developed approaches for both capillary coatings and analyte detection via laser-induced fluorescence. Since the discipline of biotechnology has established itself as a primary platform for the application of CE to the analysis of proteins and peptides, Chapters 5–7 demonstrate a variety of examples of the specific techniques that have been applied for the development of biopharmaceuticals and their commercialization. The methods covered here include also the analysis of oligosaccharides from glycoproteins.
Author : Feng Zhou
Publisher : ProQuest
ISBN 13 : 9780549388937
Total Pages : pages
Book Rating : 4.3/5 (889 download)
Download or read book Protein Characterization by Capillary Isoelectric Focusing Electrophoresis, Reversed Phase Liquid Chromatography and Mass Spectrometry written by Feng Zhou and published by ProQuest. This book was released on 2008 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: With the development of genomics, separation technology and mass spectrometry, proteomics has become an important tool in biological research. Characterization of posttranslational modification (PTM) by mass spectrometry is attracting an increasing attention because of its importance in signal transduction and the difficulty in obtaining this information from genomic methods. Bottom-up methods, which analyze the peptides from enzymatic digestion of proteins, are widely used for proteomic research. However, bottom-up methods may lose some important information when characterizing PTM since not all peptides are detected. In contrast, top-down methods directly characterize intact proteins, which makes it possible to retain PTM information. This dissertation focuses on the characterization of proteins by coupling high throughput methods, capillary isoelectric focusing electrophoresis (CIEF) and electrospray ionization (ESI) mass spectrometry (MS). In this work, a CIEF-RPLC-ESIMS system is designed and constructed for 2-D profiling of protein mixture. The microdialysis membrane based cathodic cell used in this system not only serves as the cathodic cell in CIEF separation, but also transfers the focused proteins separated by CIEF to RPLC. After eliminating the interference of ampholyte, a few fmol of protein can be detected with this system. The detection limit of this system is one order of magnitude lower than 2-D PAGE. By taking the advantage of high resolving power with mass spectrometry and multidimensional separation, the theoretical peak capacity of this system can reach 1x10 6, which is two orders of magnitude higher than theoretical peak capacity of 2-D PAGE. This system is firstly tested with a standard protein mixture. After slight modification, this system is used to profile the intact protein from a yeast enzyme concentrate and a lysate of the green sulfur bacterium Chlorobium tepidum. The pI vs. MW profile obtained from CIEF-RPLC-MS compares favorably with theoretical data derived from C. tepidum genome and experimental data obtained from 2-D-PAGE.
Author : Mahmoud H. Hamdan
Publisher : John Wiley & Sons
ISBN 13 : 0471709107
Total Pages : 448 pages
Book Rating : 4.4/5 (717 download)
Download or read book Proteomics Today written by Mahmoud H. Hamdan and published by John Wiley & Sons. This book was released on 2005-05-13 with total page 448 pages. Available in PDF, EPUB and Kindle. Book excerpt: The last few years have seen an unprecedented drive toward theapplication of proteomics to resolving challenging biomedical andbiochemical tasks. Separation techniques combined with modern massspectrometry are playing a central role in this drive. This bookdiscusses the increasingly important role of mass spectrometry inproteomic research, and emphasizes recent advances in the existingtechnology and describes the advantages and pitfalls as well. * Provides a scientifically valid method for analyzing theapproximatey 500,000 proteins that are encoded in the humangenome * Explains the hows and whys of using mass spectrometry inproteomic analysis * Brings together the latest approaches combining separationtechniques and mass spectrometry and their application in proteomeanalysis * Comments on future challenges and how they may be addressed * Includes sections on troubleshooting
Author : Pawel Ciborowski
Publisher : Elsevier
ISBN 13 : 0444636900
Total Pages : 300 pages
Book Rating : 4.4/5 (446 download)
Download or read book Proteomic Profiling and Analytical Chemistry written by Pawel Ciborowski and published by Elsevier. This book was released on 2016-03-02 with total page 300 pages. Available in PDF, EPUB and Kindle. Book excerpt: Proteomic Profiling and Analytical Chemistry: The Crossroads, Second Edition helps scientists without a strong background in analytical chemistry to understand principles of the multistep proteomic experiment necessary for its successful completion. It also helps researchers who do have an analytical chemistry background to break into the proteomics field. Highlighting points of junction between proteomics and analytical chemistry, this resource links experimental design with analytical measurements, data analysis, and quality control. This targeted point of view will help both biologists and chemists to better understand all components of a complex proteomic study. The book provides detailed coverage of experimental aspects such as sample preparation, protein extraction and precipitation, gel electrophoresis, microarrays, dynamics of fluorescent dyes, and more. The key feature of this book is a direct link between multistep proteomic strategy and quality control routinely applied in analytical chemistry. This second edition features a new chapter on SWATH-MS, substantial updates to all chapters, including proteomic database search and analytical quantification, expanded discussion of post-hoc statistical tests, and additional content on validation in proteomics. Covers the analytical consequences of protein and peptide modifications that may have a profound effect on how and what researchers actually measure Includes practical examples illustrating the importance of problems in quantitation and validation of biomarkers Helps in designing and executing proteomic experiments with sound analytics
Author : Yanwei Zhan
Publisher :
ISBN 13 :
Total Pages : 74 pages
Book Rating : 4.:/5 (613 download)
Download or read book Development of Isoelectric Focusing Techniques for Protein Analyses written by Yanwei Zhan and published by . This book was released on 2008 with total page 74 pages. Available in PDF, EPUB and Kindle. Book excerpt: Isoelectric focusing (IEF) is a powerful approach in separations of zwitterionic substances such as proteins, peptides and amino acids. It is important in proteomic research. Generally, in IEF, carrier ampholytes (CAs) are necessary to establish a stable pH gradient. However, CAs also bring attendant problems such as a decrease in detection sensitivity and suppression of ionization of analytes in mass spectrometry (MS) detection. It is desirable to build a pH gradient without using CAs. A simple slab based design was developed to establish a pH gradient using the electrolysis of water and the strength of free flow electrophoresis (FFE). The simple and robust CA free FFE-IEF design was applied in protein fractionation. In capillary format, capillary isoelectric focusing (CIEF), coupled to MS is a promising hyphenated technique for biomolecular analysis based on the combination of the high separation power of CE and the high specificity of MS. Coupling of the instruments is usually achieved with a coaxial sheath liquid interface, which decreases the detection sensitivity because of the dilution of sample by the sheath liquid. In this project, nano-electrospray, a sheathless interface, was used for coupling. Additionally, another major challenge is the presence of CAs which suppresses the ionization of analytes and contaminates the MS. In order to complete this project, a microcross union was chosen to couple CIEF with MS. A makeup solution was introduced to dilute the concentration of CAs after IEF to assist the ionization for MS detection. The makeup solution could replace the sheath liquid and could be maintained at a low flow rate so that nanoelectrospray could be performed. Monoliths can be described as integrated continuous porous separation media for micro scale separation columns. CAs were immobilized at different positions in the column according to their pIs, generating a monolithic immobilized pH gradient (M-IPG). In this project, carrier ampholytes was immobilized in poly (GMA-co-EDMA) based monolithic capillary and poly (GMA-co-acrylamide) based monolithic capillary to form a pH gradient. Two proteins were separated by IEF, which was implemented in poly (GMA-co-acrylamide) based monolithic capillary without CAs. The interface to MS was performed following the use of a microcross union as described previously. No typical noise of CAs was observed in the MS spectrum.
Author : Tian Xu
Publisher :
ISBN 13 :
Total Pages : 0 pages
Book Rating : 4.3/5 (776 download)
Download or read book Advancing Capillary Electrophoresis-mass Spectrometry for Top-down Proteomics written by Tian Xu and published by . This book was released on 2023 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Top-down proteomics (TDP) enables the proteome profiling of biological subjects at the proteoform level and understanding of differential functions associated with proteoform heterogeneity, such as sequence variation, post-translational modifications (PTMs), etc. Drastic advances on TDP technologies (e.g. sample preparation, separation/fractionation, fragmentation, bioinformatics, etc.) have been achieved in the past decades. Further improvements in separation remain desired for better analysis throughput and deeper proteome coverage. Capillary electrophoresis (CE), including capillary zone electrophoresis (CZE) and capillary isoelectric focusing (cIEF), provide superior separation performance for proteoforms. This dissertation focuses on the advancement of CE-MS-based tools on throughput, separation resolution, and capacity for TDP and utility of these tools for biological applications.In Chapter 2, we developed high-throughput and high-capacity cIEF-MS/MS platforms. The high-throughput platform enables efficient identification and quantification of proteoforms (less than one hour per run), whereas the high-capacity cIEF-MS/MS provides large number of proteoform identifications (IDs, more than 700 proteoforms in a single shot analysis) which is valuable for deep TDP. In Chapter 3, we further improved the stability and robustness of cIEF-MS platform using optimized linear polyacrylamide (LPA) capillary coating and catholyte with lower pH (pH~10). The work achieved high-resolution characterization and accurate isoelectric point (pI) determination of charge variants (~0.1 pI difference) of monoclonal antibodies (mAbs). In Chapter 4, we developed a nondenaturing cIEF-MS platform for ultrahigh resolution characterization of microheterogeneity of a variety of protein complexes. Typically, pI determinations of variants in protein complexes allow us to decipher how sequence or PTM variations modulate the pIs of the protein complexes. In Chapter 5, while CZE-MS/MS is a well-developed approach, for the first time, we coupled FAIMS to CZE-MS/MS to facilitate online gas-phase fractionation of proteoforms. The FAIMS greatly enhanced the sensitivity of the system and expanded the number of proteoform IDs, especially large proteoform IDs. The work renders CZE-FAIMS-MS/MS as a new powerful multidimensional platform for deep TDP.In Chapters 6 and 7, we applied cIEF-MS/MS and CZE-MS/MS for studying the sexual dimorphism of zebrafish brains and proteoform-level differences between metastatic and nonmetastatic colorectal cancer (CRC) cells, respectively. In Chapter 6, quantitative TDP of thousands of proteoforms from male and female zebrafish brains by cIEF-MS/MS based approach discovered various overexpressed proteoforms in male or female brains that are closely associated with hormone activity. In Chapter 7, We performed deep TDP study of non-metastatic and metastatic CRC cells (SW480 and SW620) using CZE-MS/MS based multidimensional platform and identified more than 20,000 proteoforms of over 2,000 proteins from the two cell lines, which presents around 5-folds higher number of proteoform IDs in comparison with previous TDP studies of human cancer cells. The work revealed significant discrepancies between the two isogenic cell lines regarding proteoform and single amino acid variant (SAAV) profiles. Quantitative data disclosed differentially expressed proteoforms between the two cell lines and their corresponding genes were connected to cancer pathways and networks.
Author :
Publisher :
ISBN 13 :
Total Pages : 0 pages
Book Rating : 4.:/5 (86 download)
Download or read book Novel Micro-separation Techniques and Their Couplings to Mass Spectrometry for the Analysis of Complex Peptides and Proteins written by and published by . This book was released on 2013 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Mass spectrometry (MS)-based technology has evolved as a powerful tool for the analysis of a wide array of biomolecules. However, its application to complex peptidomics and proteomics samples, for example neuropeptides, has proven to be challenging due to their extreme chemical complexities and low abundances in biological tissue samples. This PhD dissertation is focused on addressing some of these challenges by developing novel micro-separation tools and coupling them with MS to achieve enhanced MS sensitivity and increased peptide and protein coverage with lower sample consumption and reduced experimental time. Both chromatography-based and electrophoresis-based separations as well as their couplings to MS have been explored in this work, including monolithic material-based chromatography, improved capillary electrophoresis (CE) separation and novel coupling of separations to mass spectrometric imaging. N-acryloxysuccinimide-based monolithic column has been fabricated with specific antibodies immobilized onto the column to enable the extraction of targeted neuropeptide family from biological matrices. Alternatively, enzymes were immobilized onto the column for highly efficient on-column enzymatic digestion for proteomics studies. A methacrylate-based monolithic column with C12 functional groups was developed and coupled to both electrospray ionization (ESI) MS and matrix-assisted laser desorption/ionization (MALDI) MS for reversed-phase liquid chromatography (RPLC). For capillary electrophoresis, two improved capillary isoelectric focusing (CIEF) systems have been developed, including membrane-assisted CIEF and immobilized pH-gradient CIEF to obtain reduced sample loss and enhanced MS signal for neuropeptide analysis. An improved sheathless pressure-assisted CE (PACE) system was also developed for coupling to MALDI MS. For novel separation-MS imaging coupling, three generations of interfaces have been designed to couple CE, LC and multi-dimensional separations to MS imaging (MSI) for quantitative analysis of complex peptides. Collectively, this work results in development of the new generation of micro-separation and sample preparation tools and their coupling to various mass spectrometric ionization and detection modes for sensitive and rapid analysis of complex peptides and proteins. The technological innovations have significantly expanded the toolbox for peptidomics/proteomics analysis and will enable more in-depth investigations on peptide function and regulation.
Author : John M. Walker
Publisher : Springer Science & Business Media
ISBN 13 : 1603273751
Total Pages : 1105 pages
Book Rating : 4.6/5 (32 download)
Download or read book Molecular Biomethods Handbook written by John M. Walker and published by Springer Science & Business Media. This book was released on 2008-11-04 with total page 1105 pages. Available in PDF, EPUB and Kindle. Book excerpt: Recent advances in the biosciences have led to a range of powerful new technologies, particularly nucleic acid, protein and cell-based methodologies. The most recent insights have come to affect how scientists investigate and define cellular processes at the molecular level. This book expands upon the techniques included in the first edition, providing theory, outlines of practical procedures, and applications for a range of techniques. Written by a well-established panel of research scientists, the book provides an up-to-date collection of methods used regularly in the authors’ own research programs.
Author : Xiaojing Shen
Publisher :
ISBN 13 :
Total Pages : 194 pages
Book Rating : 4.6/5 (985 download)
Download or read book Leveraging Capillary Zone Electrophoresis-mass Spectrometry for Multi-level Proteomics written by Xiaojing Shen and published by . This book was released on 2020 with total page 194 pages. Available in PDF, EPUB and Kindle. Book excerpt: Mass spectrometry (MS) coupled with online liquid-phase separation is the major tool for large-scale bottom-up proteomics (peptide-centric), top-down proteomics (proteoform-centric), and native proteomics (protein complex-centric). While liquid chromatography (LC)-MS is the dominant method for proteomics at different levels, capillary zone electrophoresis (CZE)-MS has emerged as a valuable and complementary technique, which provides high-capacity separation and highly sensitive detection of peptides, proteoforms and even protein complexes under native conditions. This work focuses on developing novel CZE-MS/MS methods for multi-level proteomics (bottom-up, top-down, and native).In Chapter 2, a high-throughput bottom-up proteomics workflow was developed by coupling immobilized trypsin-based speedy protein digestion with fast CZE-MS/MS. Immobilized trypsin produced almost the same digestion performance as free trypsin for complex proteomes with about 50-times higher speed (15 min vs. 12 h). Integration of immobilized trypsin (IM)-based rapid protein cleavage and fast CZE-MS/MS enables the identification of thousands of proteins from the mouse brain proteome in only 3 h, which is significantly faster than the typical LC-MS-based bottom-up proteomics workflow (3 h vs. >12 h). The high-throughput workflow was expected to be useful for bottom-up proteomics of human clinical samples (e.g., serum and urine).Chapter 3 presents the first example of CZE-MS/MS with activated ion-electron capture dissociation (AI-ECD) on a high-end quadrupole-time-of-flight (Q-TOF) mass spectrometer for top-down proteomics, enabling high-resolution separation, highly sensitive detection, and extensive gas-phase backbone cleavages of proteoforms. The CZE-AI-ECD method will be useful to the top-down proteomics community for the comprehensive characterization of proteoforms in complex proteomes. Chapter 4 and 5 focus on the development of novel CZE-MS methods for native proteomics, delineating proteins and protein complexes under native conditions. In Chapter 4, a native CZE-MS/MS platform with an Orbitrap mass spectrometer was established for native proteomics of a complex proteome (E. coli), leading to the identification of 23 protein complexes in discovery mode. The work represents the first example of native proteomics via coupling online liquid-phase separation to native MS and MS/MS. The characterization of large protein complexes (up to 200 kDa) was also achieved with a new CZE-MS system on a high-end Q-TOF mass spectrometer.In Chapter 5, a novel native capillary isoelectric focusing (cIEF)-assisted CZE-MS method is presented for the characterization of monoclonal antibodies (mAbs) with large sample loading capacity and high separation resolution. Using the method, the potential separations of different conformations of the SigmaMAb and the detection of its various glyco-proteoforms and homodimer were documented. The method separated the NISTmAb into three peaks with a microliter sample loading volume, corresponding to its different proteoforms. In addition, eight glyco-proteoforms of the NISTmAb and its homodimer were detected. The results demonstrate the potential of the native cIEF-assisted CZE-MS method for advancing the characterization of large proteins (i.e., mAbs) and protein complexes under native conditions.
Author : P. G. Righetti
Publisher : Elsevier
ISBN 13 : 0444813012
Total Pages : 415 pages
Book Rating : 4.4/5 (448 download)
Download or read book Immobilized PH Gradients: Theory and Methodology written by P. G. Righetti and published by Elsevier. This book was released on 1990 with total page 415 pages. Available in PDF, EPUB and Kindle. Book excerpt: Immobilized pH gradients (IPG) represent the most advanced development of isoelectric focusing (IEF). Originally developed to overcome all the problems of IEF in soluble amphoteric buffers (CA) (such as pH gradient instability, complexation with CA chemicals, unreproducibility of pH gradients, protein precipitation at the pI), it turned out to be an entirely new technique, quite different in principle and operation from conventional IEF. The book is thus meant to bring the reader up to date with this fast developing field. The book is divided into six chapters containing information on: detailed treatment of all the chemistry of the Immobiline chemicals; theory of pH gradient generation (computer simulations, tables with all the possible pH recipes); all analytical aspects of IPGs, including staining techniques, blotting etc.; two-dimensional maps, with a detailed treatise of advantages and limitations; preparative aspects of IPGs, including comparison with other preparative electrophoretic techniques; some examples of applications, including genetic and forensic analysis, blood polymorphism etc.The book is extensive and up-to-date, while also extensively covering the theory. Clearly written, with easily accessible information, the volume is a worthy asset to the Lab. Techniques series.
Author : Jennifer E. Van Eyk
Publisher : John Wiley & Sons
ISBN 13 : 3527622160
Total Pages : 694 pages
Book Rating : 4.5/5 (276 download)
Download or read book Clinical Proteomics written by Jennifer E. Van Eyk and published by John Wiley & Sons. This book was released on 2008-09-08 with total page 694 pages. Available in PDF, EPUB and Kindle. Book excerpt: Unparalleled in its scope and depth, this book brings together proteomic approaches in diagnosis and treatment from all clinical fields, including clinical toxicology. The result is a new discipline in molecular medicine that will revolutionize the treatment and prevention of cancer, stroke and other severe diseases. Following an overview of clinical proteomics, the authors look at the technologies available, before moving on to cancer, cardiopulmonary disease, diabetes and stroke. A whole section is devoted to toxicity and the work is rounded off with a discussion of the future of clinical proteomics.
Author : Steven A. Cohen
Publisher : John Wiley & Sons
ISBN 13 : 0470276258
Total Pages : 490 pages
Book Rating : 4.4/5 (72 download)
Download or read book Multidimensional Liquid Chromatography written by Steven A. Cohen and published by John Wiley & Sons. This book was released on 2008-05-09 with total page 490 pages. Available in PDF, EPUB and Kindle. Book excerpt: Multidimensional Liquid Chromatography (MDLC) is a very powerful separation technique for analyzing exceptionally complex samples in one step. This authoritative reference presents a number of recent contributions that help define the current art and science of MDLC. Topics covered include instrumentation, theory, methods development, and applications of MDLC in the life sciences and in industrial chemistry. With the information to help you perform very difficult separations of complex samples, this reference includes chapters contributed by leading experts or teams of experts.
Author : Jing Wei
Publisher :
ISBN 13 :
Total Pages : 228 pages
Book Rating : 4.:/5 (47 download)
Download or read book Combination of Capillary Isoelectric Focusing and Liquid Chromatography with Electrospray Ionization Mass Spectrometry for Protein Characterization written by Jing Wei and published by . This book was released on 1998 with total page 228 pages. Available in PDF, EPUB and Kindle. Book excerpt: Combination of capillary isoelectric focusing (CIEF) and liquid chromatography (LC) with electrospray ionization mass spectrometry (ESIMS) is utilized for protein characterization. Both CIEF-ESIMS and LC-MS are studied for phosphoprotein characterization. At the intactprotein level, on-line CIEF-ESIMS is applied for phosphorylation analysis to obtain the two dimensional information: isoelectric point and molecular mass. At the peptide level, on-line LC-ESI-MS and LC-ESI neutral loss MS/MS were highly specific for the determination of sites of phosphorylation and successfully applied on the tryptic digest of tau proteins phosphorylated by different kinases. On-line combination of CIEF with ESI-TOF MS is demonstrated for high resolution analysis of some model proteins and human hemoglobin variants. The successful coupling of these two techniques optimizes the efficiency of CIEF separation since TOF MS is able to produce a full spectrum in the shortest response time among all kinds of mass analyzers.
Author : Tim Wehr
Publisher : CRC Press
ISBN 13 : 1482289857
Total Pages : 301 pages
Book Rating : 4.4/5 (822 download)
Download or read book Capillary Electrophoresis of Proteins written by Tim Wehr and published by CRC Press. This book was released on 1998-10-20 with total page 301 pages. Available in PDF, EPUB and Kindle. Book excerpt: "Provides practical information on the application of capillary electrophoresis (CE) to protein analysis, with an emphasis on developing and optimizing CE techniques in the laboratory. Includes separation methods bases on mass, charge, isoelectric point, molecular sieving, and affinity interactions."
