Extraction and Solubilization of Proteins for Proteomic Studies Richard M. Leimgruber Preparation of Bacterial Samples for 2-D PAGE Brian Berg Vandahl, Gunna Christiansen, and Svend Birkelund Preparation of Yeast Samples for 2-D PAGE Joakim Norbeck Preparation of Mammalian Tissue Samples for Two-Dimensional Electrophoresis Frank A. Witzmann Differential Detergent Fractionation of Eukaryotic Cells Melinda L. Ramsby and Gregory S. Makowski Serum or Plasma Sample Preparation for Two-Dimensional Gel Electrophoresis Anthony G. Sullivan, Stephen Russell, Henry Brzeski, Richard I. Somiari, and Craig D. Shriver Preparation of Plant Protein Samples for 2-D PAGE David W. M. Leung Laser-Assisted Microdissection in Proteomic Analyses Darrell L. Ellsworth, Stephen Russell, Brenda Deyarmin, Anthony G. Sullivan, Henry Brzeski, Richard I. Somiari, and Craig D. Shriver Purification of Cellular and Organelle Populations by Fluorescence-Activated Cell Sorting for Proteome Analysis William L. Godfrey, Colette J. Rudd, Sujata Iyer, and Diether Recktenwald Purification of Nucleoli From Lymphoma Cells and Solubilization of Nucleolar Proteins for 2-DE Separation Regis Dieckmann, Yohann Coute, Denis Hochstrasser, Jean-Jacques Diaz, and Jean-Charles Sanchez Prefractionation of Complex Protein Mixture for 2-D PAGE Using Reversed-Phase Liquid Chromatography Volker Badock and Albrecht Otto Fractionation of Complex Proteomes by Microscale Solution Isoelectrofocusing Using ZOOM(TM) IEF Fractionators to Improve Protein Profiling Xun Zuo, Ki-Boom Lee, and David W. Speicher Large-Format 2-D Polyacrylamide Gel Electrophoresis Henry Brzeski, Stephen Russell, Anthony G. Sullivan, Richard I. Somiari, and Craig D. Shiver Analysis of Membrane Proteins by Two-Dimensional Gels MichaelFountoulakis 2-D PAGE of High-Molecular-Mass Proteins Masamichi Oh-Ishi and Tadakazu Maeda Using Ultra-Zoom Gels for High-Resolution Two-Dimensional Polyacrylamide Gel Electrophoresis Sjouke Hoving, Hans Voshol, and Jan van Oostrum NEpHGE and pI Strip Proteomic 2-D Gel Electrophoretic Mapping of Lipid-Rich Membranes Steven E. Pfeiffer, Yoshihide Yamaguchi, Cecilia B. Marta, Rashmi Bansal, and Christopher M. Taylor Silver Staining of 2-D Gels Julia Poland, Thierry Rabilloud, and Pranav Sinha Zn2+ Reverse Staining Technique Carlos Fernandez-Patron Multiplexed Proteomics Technology for the Fluorescence Detection of Glycoprotein Levels and Protein Expression Levels Using Pro-Q(R) Emerald and SYPRO(R) Ruby Dyes Birte Schulenberg and Wayne F. Patton Multiplexed Proteomics Technology for the Fluorescence Detection of Phosphorylation and Protein Expression Levels Using Pro-Q(R) Diamond and SYPRO(R) Ruby Dyes Birte Schulenberg, Terrie Goodman, Thomas H. Steinberg, and Wayne F. Patton Sensitive Quantitative Fluorescence Detection of Proteins in Gels Using SYPRO(R) Ruby Protein Gel Stain Birte Schulenberg, Nancy Ahnert, and Wayne F. Patton Rapid, Sensitive Detection of Proteins in Minigels With Fluorescent Dyes: Coomassie Fluor Orange, SYPRO(R) Orange, SYPRO Red, and SYPRO Tangerine Protein Gel Stains Thomas H. Steinberg, Courtenay R. Hart, and Wayne F. Patton Differential In-Gel Electrophoresis in a High-Throughput Environment Richard I. Somiari, Stephen Russell, Stella B. Somiari, Anthony G. Sullivan, Darrell L. Ellsworth, Henry Brzeski, and Craig D. Shriver Statistical Analysis of 2-D Gel Patterns Francoise Seillier-Moiseiwitsch 2-DE Databases on the World Wide Web Christine Hoogland, Khaled Mostaguir, and Ron D. Appel Computer Analysis of 2-D Images Patricia M. Palagi,

The proteomics protocols handbook

The focus of this article is to review the recent advances in proteome analysis of human body fluids, including plasma/serum, urine, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, synovial fluid, nipple aspirate fluid, tear fluid, and amniotic fluid, as well as its applications to human disease biomarker discovery. We aim to summarize the proteomics technologies currently used for global identification and quantification of body fluid proteins, and elaborate the putative biomarkers discovered for a variety of human diseases through human body fluid proteome (HBFP) analysis. Some critical concerns and perspectives in this emerging field are also discussed. With the advances made in proteomics technologies, the impact of HBFP analysis in the search for clinically relevant disease biomarkers would be realized in the future.

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Human body fluid proteome analysis

Biomarkers are measurable indicators of a biological state. As our understanding of diseases meliorates, it is generally accepted that early diagnosis renders the best chance to cure a disease. In the context of proteomics, the discovery phase of identifying bonafide biomarkers and the ensuing validation phase involving large cohort of patient samples are impeded by the complexity of bodily fluid samples. High abundant proteins found in blood plasma make it difficult for the detection of low abundant proteins that may be potential biomarkers. Extracellular vesicles (EVs) have reignited interest in the field of biomarker discovery. EVs contain a tissue-type signature wherein a rich cargo of proteins and RNA are selectively packaged. In addition, as EVs are membranous structures, the luminal contents are protected from degradation by extracellular proteases and are highly stable in storage conditions. Interestingly, an appealing feature of EV-based biomarker analysis is the significant reduction in the sample complexity compared to whole bodily fluids. With these prescribed attributes, which are the rate-limiting factors of traditional biomarker analysis, there is immense potential for the use of EVs for biomarker detection in clinical settings. This review will discuss the current issues with biomarker analysis and the potential use of EVs as reservoirs of disease biomarkers.

/pdf/exosomes-in-bodily-fluids-are-a-highly-stable-resource-of-hkisy67u6q.pdf

Exosomes in bodily fluids are a highly stable resource of disease biomarkers.

Serum proteins may often serve as indicators of disease and is a rich source for biomarker discovery. However, the large dynamic range of proteins in serum makes the analysis very challenging because high-abundant proteins tend to mask those of lower abundance. A prefractionation step, such as depletion of a few high-abundant proteins before protein profiling, can assist in the discovery and detection of less abundant proteins that may prove to be informative biomarkers. In the present study, five different depletion columns were investigated considering efficiency, specificity, and reproducibility. Our research included quantitative determination of total protein, albumin, and immunoglobulin G (IgG) concentrations, one- and two-dimensional gels and mass spectrometric analysis of the serum samples before and after the depletion step. Our results showed that all five depletion columns tested removed albumin and IgG with high efficiency. We found that based on reproducibility and binding specificity, the Multiple Affinity Removal Column that removed a total of six high-abundant proteins (albumin, IgG, antitrypsin, IgA, transferring, and haptoglobin) offered the most promising depletion approach. Among the disposable (single-use) products, the ProteoExtract Albumin/IgG Removal kit displayed the best results. Depleted serum from the Multiple Affinity Removal column was further evaluated by 2-D gel electrophoresis (2-DE) analysis, and the results indicated increased resolution and improved intensity of low-abundant proteins in a reproducible fashion. Our study provides a comprehensive investigation of commercially available depletion columns and will be of high importance for future proteomic studies on serum samples.

Comparison of different depletion strategies for improved resolution in proteomic analysis of human serum samples.

Systematic detection of low-abundance proteins in human blood that may be putative disease biomarkers is complicated by an extremely wide range of protein abundances. Hence, depletion of major proteins is one potential strategy for enhancing detection sensitivity in serum or plasma. This study compared a recently commercialized HPLC column containing antibodies to six of the most abundant blood proteins ("Top-6 depletion") with either older Cibacron blue/Protein A or G depletion methods or no depletion. In addition, a prototype spin column version of the HPLC column and an alternative prototype two antibody spin column were evaluated. The HPLC polyclonal antibody column and its spin column version are very promising methods for substantially simplifying human serum or plasma samples. These columns show the lowest nonspecific binding of the depletion methods tested. In contrast other affinity methods, particularly dye-based resins, yielded many proteins in the bound fractions in addition to the targeted proteins. Depletion of six abundant proteins removed about 85% of the total protein from human serum or plasma, and this enabled 10- to 20-fold higher amounts of depleted serum or plasma samples to be applied to 2-D gels or alternative protein profiling methods such as protein array pixelation. However, the number of new spots detected on 2-D gels was modest, and most newly visualized spots were minor forms of relatively abundant proteins. The inability to detect low-abundance proteins near expected 2-D staining limits was probably due to both the highly heterogeneous nature of most plasma or serum proteins and masking of many low-abundance proteins by the next series of most abundant proteins. Hence, non2-D methods such as protein array pixelation are more promising strategies for detecting lower abundance proteins after depleting the six abundant proteins.

Depletion of multiple high-abundance proteins improves protein profiling capacities of human serum and plasma.

Proteomic technologies are being used to discover and identify disease-associated biomarkers. The application of these technologies in the search for potential diagnostic/prognostic biomarkers in the serum of patients has been limited by the presence of highly abundant albumin and immunoglobulins that constitute approximately 60-97% of the total serum proteins. The purpose of the study was to evaluate whether treatment of human serum with Affi-Gel Blue alone or in combination with Protein A (Aurum serum protein mini kit, Bio-Rad) before two-dimensional gel electrophoresis (2-DE) analysis removed high abundance proteins to allow the visualization of low abundant proteins. Serum samples were treated with either Affi-Gel Blue or Aurum kit and then subjected to 2-DE using 11 cm, pH 4-7 isoelectric focussing strips for the first dimension and 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis for second dimension. Protein spots were visualized using a fluorescent protein dye (SYPRO Ruby, Bio-Rad). Comparison between treatment methods showed significant removal of albumin by both Affi-Gel Blue and Aurum kit and considerable differences in the protein profile of the gels after each treatment. Direct comparison between treatments revealed twenty-eight protein spots unique to Affi-Gel Blue while only two spots were unique after Aurum kit treatment. Unique spots in Affi-Gel Blue and Aurum kit treated serum were not visualized in untreated serum. Sixteen hours of Affi-Gel Blue treatment resulted in enhanced visualization of fifty-three protein spots by two-fold, thirty-one by five-fold, twelve by ten-fold and six by twenty-fold. In parallel after Aurum kit treatment two-, five-, ten- and twenty-fold enhancements of thirty, thirteen, eight and five protein spots, respectively, were observed. The pattern of increased visualization of protein spots with both treatment methods was similar. In conclusion, treatment of serum samples with Affi-Gel Blue or Aurum kit before 2-DE analysis can be used to remove high abundance proteins in order to increase the detection sensitivity of proteins present in low abundance.

Gillian Barker

Papers

An approach to remove albumin for the proteomic analysis of low abundance biomarkers in human serum