Depletion Of High-abundance Proteins Research Articles

Online monitoring of immunoaffinity-based depletion of high-abundance blood proteins by UV spectrophotometry using enhanced green fluorescence protein and FITC-labeled human serum albumin

BackgroundThe removal of high-abundance proteins from plasma is an efficient approach to investigating flow-through proteins for biomarker discovery studies. Most depletion methods are based on multiple immunoaffinity methods available commercially including LC columns and spin columns. Despite its usefulness, high-abundance depletion has an intrinsic problem, the sponge effect, which should be assessed during depletion experiments. Concurrently, the yield of depletion of high-abundance proteins must be monitored during the use of the depletion column. To date, there is no reasonable technique for measuring the recovery of flow-through proteins after depletion and assessing the capacity for capture of high-abundance proteins.ResultsIn this study, we developed a method of measuring recovery yields of a multiple affinity removal system column easily and rapidly using enhanced green fluorescence protein as an indicator of flow-through proteins. Also, we monitored the capture efficiency through depletion of a high-abundance protein, albumin labeled with fluorescein isothiocyanate.ConclusionThis simple method can be applied easily to common high-abundance protein depletion methods, effectively reducing experimental variations in biomarker discovery studies.

High‐abundance protein depletion: Comparison of methods for human plasma biomarker discovery

Affinity depletion of abundant proteins from human plasma has become a routine sample preparation strategy in proteomics used prior to protein identification and quantitation. To date, there have been limited published studies comparing the performance of commercially available depletion products. We conducted a thorough evaluation of six depletion columns using 2-DE combined with sophisticated image analysis software, examining the following criteria: (i) efficiency of high-abundance protein depletion, (ii) non-specific removal of other than the targeted proteins and (iii) total number of protein spots detected on the gels following depletion. From all the products investigated, the Seppro IgY system provided the best results. It displayed the greatest number of protein spots on the depleted plasma gels, minimal non-specific binding and high efficiency of abundant protein removal. Nevertheless, the increase in the number of detected spots compared with the second best performing and cheaper multiple affinity removal column (MARC) was not shown to be statistically significant. The ProteoPrep spin column, considered to be the "deepest" depletion technique available at the time of conducting the study, surprisingly displayed significantly fewer spots on the flow-through fraction gels compared with both the Seppro and the MARC. The spin column format and low plasma capacity were also found to be impractical for 2-DE. To conclude, we succeeded in providing an overview of the depletion columns performances with regard to the three examined areas. Our study will serve as a reference to other scientists when deciding on the optimal product for their particular projects.

Poly(glycidyl methacrylate) beads embedded cryogels for pseudo-specific affinity depletion of albumin and immunoglobulin G

Depletion of high abundant proteins like albumin and immunoglobulin G (IgG) can be beneficial in the analysis of serum proteins. For this purpose, Cibacron Blue F3GA and iminodiacetic acid (IDA)-Cu2+ containing poly(glycidyl methacrylate) (PGMA) beads (1.6µm in diameter) were embedded into the poly(hydroxyethyl methacrylate) (PHEMA) cryogel. The PGMA beads were prepared by dispersion polymerization. The PGMA beads were modified with Cibacron Blue F3GA and iminodiacetic acid (IDA)-Cu2+ for simultaneous albumin and IgG depletion, respectively. The PHEMA cryogel was synthesized by free radical polymerization in the presence of the modified PGMA beads. The PHEMA and PHEMA/PGMA composite cryogels were characterized by swelling measurements and scanning electron microscopy (SEM). Protein depletion studies were carried out in a continuous experimental set-up in a stacked column. Albumin adsorption capacity of the PGMA-Cibacron Blue F3GA beads embedded PHEMA cryogel (PHEMA/PGMA-Cibacron Blue F3GA) was 342mg/g and IgG adsorption capacity of the PGMA-IDA-Cu2+ beads embedded PHEMA cryogel (PHEMA/PGMA-IDA-Cu2+) was 257mg/g. The composite cryogels depleted albumin and IgG from human serum with 89.4% and 93.6% efficiency, respectively. High desorption values (over 90% for both modified cryogels) were achieved with 0.05M phosphate buffer containing1.0M NaCl.

Proteomic-Based Approach for Biomarker Discovery to Predict Silent Cerebral Infarct in Patients with Sickle Cell Disease.

Abstract 2579Poster Board II-556Silent cerebral infarction (SCI) is part of a spectrum of cerebrovascular disease, occurs in approximately 22% of children with sickle cell disease (SCD) and is associated with decreased cognitive function. While several plasma biomarkers have been shown to be elevated in acute stroke, to our knowledge none have been evaluated in SCD or SCI. The aim of this study was to develop a reliable pipeline to identify low abundance plasma proteins that correlate with SCI in patients with SCD. We used a proteomic discovery approach involving three sequential separation steps in order to compare the plasma proteomes in a discovery set of 15 children with SCD (7 children with SCI and 8 children without SCI). Baseline steady state plasma samples obtained from the Silent Infarct Transfusion (SIT) Trial Biologic Repository were matched for age and WBC. In the first dimension, hemoglobin was depleted with nickel-nitrilotriacetic acid (Ni-NTA). Subsequently, second dimension separation and enrichment was achieved by immunoaffinity depletion of the 12 most abundant proteins (ProteomeLab IgY-12 LC10) followed by third dimension separation by reverse phase liquid chromatography fractionation (RPLC) using a C18 column and a linear acetonitrile gradient. Collected fractions were subjected to tryptic digestion and analyzed using label-free quantification on a LTQ-Orbitrap (Thermo) mass spectrometer. The MS/MS data were analyzed using the Proteomics Analyzer Software System (PASS) version 4.0.10 (Integrated Analysis Inc, Bethesda, MD) with X! Tandem searches (www.thegpm.org; version 2008.12.01) using the human IPI database. Identified proteins were compared to databases of brain specific and brain enriched proteins to identify candidate biomarker proteins for SCI. After hemoglobin depletion, 71% of total protein was removed. On average, protein recovery after the LC-12 column was 4% of the total Ni-NTA depleted protein. Of the 9800 proteins that were identified in the plasma proteome of children with SCD, 23 were brain specific proteins. Evaluation of the relative abundance by spectral counts (SC) revealed 3 brain-related proteins that were over-represented in patients with SCI: microtubule-associated protein tau (a neurofibrillary tangle protein implicated in Alzheimer disease, frontotemporal dementia and parkinsonism), neuroligin-3 (a neuronal cell surface protein proposed to be involved in cell-cell-interactions via binding to beta-neurexins and implicated in autism and Asperger syndrome), and nucleosome-remodeling factor subunit BPTF (a histone-binding component of nucleosome-remodeling factor that is abundantly expressed in the fetal brain and re-expressed in neurodegenerative diseases). Reticulon-4, a potent neurite growth inhibitor involved in the restriction of axonal regeneration after injury, was under-represented in patients with SCI. After depletion of hemoglobin and other high abundant proteins, we were able to develop a database of plasma proteins in children with SCD and to identify brain specific proteins as potential surrogate markers of brain injury. These markers may be implicated in the pathophysiology of SCI. Although validation studies are necessary to determine the relevance of these candidate biomarkers in SCI and SCD, our methodology appears to be a practical approach to proteomic discovery in patients with hemolytic anemia.ProteinTotal samples (SCI+)Total samples (SCI−)Avg SC per protein (SCI+)Avg SC per protein (SCI−)SC ratio (SCI+/ (SCI−)Microtubule-associated protein tau432.51.31.9Neuroligin-3536.63.71.8Nucleosome-remodeling factor subunit BPTF422.51.51.7Reticulon-4641.33.00.4Total samples indicates number of samples in which protein was found; AvgSC, average spectral counts; SCI+, patients with silent cerebral infarct; SCI-, patients without silent cerebral infarct Disclosures:Casella:Boehringer Ingelheim: Honoraria, Research Funding, Travel funding.

LC-MS Based Detection of Differential Protein Expression.

While several techniques are available in proteomics, LC-MS based analysis of complex protein/peptide mixtures has turned out to be a mainstream analytical technique for quantitative proteomics. Significant technical advances at both sample preparation/separation and mass spectrometry levels have revolutionized comprehensive proteome analysis. Moreover, automation and robotics for sample handling process permit multiple sampling with high throughput.For LC-MS based quantitative proteomics, sample preparation turns out to be critical step, as it can significantly influence sensitivity of downstream analysis. Several sample preparation strategies exist, including depletion of high abundant proteins or enrichment steps that facilitate protein quantification but with a compromise of focusing on a smaller subset of a proteome. While several experimental strategies have emerged, certain limitations such as physiochemical properties of a peptide/protein, protein turnover in a sample, analytical platform used for sample analysis and data processing, still imply challenges to quantitative proteomics. Other aspects that make analysis of a proteome a challenging task include dynamic nature of a proteome, need for efficient and fast analysis of protein due to its constant modifications inside a cell, concentration range of proteins that exceed dynamic range of a single analytical method, and absence of appropriate bioinformatics tools for analysis of large volume and high dimensional data.This paper gives an overview of various LC-MS methods currently used in quantitative proteomics and their potential for detecting differential protein expression. Fundamental steps such as sample preparation, LC separation, mass spectrometry, quantitative assessment and protein identification are discussed.For quantitative assessment of protein expression, both label and label free approaches are evaluated for their set of merits and demerits. While most of these methods edge on providing "relative abundance" information, absolute quantification is achieved with limitation as it caters to fewer proteins. Isotope labeling is extensively used for quantifying differentially expressed proteins, but is severely limited by successful incorporation of its heavy label. Lengthy labeling protocols restrict the number of samples that can be labeled and processed. Alternatively, label free approach appears promising as it can process many samples with any number of comparisons possible but entails reproducible experimental data for its application.

Multicolumn Separation Platform for Simultaneous Depletion and Prefractionation Prior to 2-DE for Facilitating In-Depth Serum Proteomics Profiling

In this report, we describe an integrated fluidic platform composed of tandem affinity columns for the depletion of high-abundance proteins from human serum and on-line fractionation/concentration of medium- and low-abundance proteins by tandem immobilized metal-ion affinity chromatography (IMAC) columns and reversed phase (RP) column for in-depth proteomics analysis. The depletion columns were based on monolithic polymethacrylate with surface immobilized protein A, protein G', and antibodies for depleting the top 8 high-abundance proteins. The IMAC fractionation/concentration columns consisted of monolithic stationary phases with surface bound iminodiacetic acid (IDA) chelated with Zn2+, Ni2+ and Cu2+, while the RP column was packed with nonpolar polymer beads. The integrated multicolumn fluidic platform was very effective in reducing simultaneously both the dynamic range differences among the protein constituents of serum and the complexity of the proteomics samples, thus, facilitating the in-depth proteomics analysis by 2-DE followed by MALDI-TOF and LC-MS/MS. In fact, the number of detected spots was approximately 1450 using SYPRO fluorescent stain from which 384 spots were subsequently detected by Coomassie Blue. Since the investigation was simply a proof of concept, 295 proteins were readily identified in some selected spots by MALDI-TOF and LC-MS/MS.

Selective separation and enrichment of proteins in aqueous two-phase extraction system

A simple aqueous two-phase extraction system (ATPS) of PEG/phosphate was proposed for selective separation and enrichment of proteins. The combination of ATPE with HPLC was applied to identify the partition of proteins in two phases. Five proteins (bovine serum albumin, Cytochrome C, lysozyme, myoglobin, and trypsin) were used as model proteins to study the effect of phosphate concentration and pH on proteins partition. The PEG/phosphate system was firstly applied to real human saliva and plasma samples, some proteins showed obviously different partition in two phases. The primary results manifest the selective separation and enrichment of proteins in ATPS provided the potential for high abundance proteins depletion in proteomics.

Proteome analysis of malignant pleural effusion

e22194 Background: Because pleural effusion contains proteins of potential diagnostic value, a comprehensive proteomic analysis of the pleural effusion is worthy to discover a new biomarker. Malignant pleural effusion and tuberculous pleural effusion are sometimes diagnositc challenge due to their similarity like lymphocyte-dominant effusion. Herein, we tried to identify differentially expressed proteins in both effusion using proteomic anlaysis. Methods: Twenty microliters of pleural effusions(PEs) from 3 patients with non-small cell lung cancer(NSCLC) and 3 patients with tuberculous pleurisy(TBC) were used for proteome analysis. After depletion of high abundant proteins including albumin, IgG with MARS Hu-6(Agilent), proteins were separated by SDS-PAGE and subject to in-gel tryptic digestion. The resulting peptides were analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS). The MS/MS spectra were analyzed by Spectrum Mill against normal and reversed human protein databases. Results: In the total of 6 samples, 90 proteins were identified with more than 2 peptides and less than 1% of false positive rate. Among the identified pleural proteins, 57 proteins were detected both in PEs of NSCLS and TBC, 19 and 14 proteins were identified only in the PE of NSCLC and TBC, respectively. We analysed molecular functions, molecular composition and molecular processes of identified proteins with FindGo software. Among the identified proteins, we found the biomarker candidates that significantly have different expression levels in malignant effusion; apolipoprotein B precursor, vitronectin, complement factor B, histidine-rich glycoprotein precursor, coagulation factor II precursor variant. Conclusions: We found that several pleural effusion proteins may serve as potential biomarker candidates for differential diagnosis between maligant and tuberculous pleural effusion. We'll confirm these proteins through the proteomic method(MRM) and immunological method(Western bolt). No significant financial relationships to disclose.

Liquid chromatography–mass spectrometry: a tool for proteome analysis and biomarker discovery and validation

Mass spectrometry (MS) coupled to liquid chromatography (LC) represents a widely used analytical tool for studying proteins, and discovering various diseases biomarkers. To enable interested readers to grasp fundamental and experimental concepts of liquid chromatography separation for protein analytes and their accurate detection when interfaced with the powerful technique of mass spectrometry. Methods for enhancing LC separation, the utility of an atmospheric pressure soft ionization method, particularly electrospray ionization, as well as the interphase of improved chromatographic columns with the various modes of mass spectrometry, and the characteristics of the various mass analyzers, will be elucidated in order to achieve sensitive and specific detection of protein markers. With a coherent biomarker pipeline, the successful throughput from a discovery phase to a robust validation method has nevertheless been disappointing. The development of faster, cost-effective and sensitive multidimensional MS methods, improvements in sample preparation and processing procedures such as high abundance protein depletion and faster chromatographic columns and modes, in addition to optimization of MS-based detection methods, are expected to improve further the analytical performance of proteomic techniques for biomarker discovery in complicated, but convenient, non- or minimally invasive clinical samples such as blood and urine, and ultimately to improve the biomarker validation process.

Proteomics-based identification of α1-antitrypsin and haptoglobin precursors as novel serum markers in infiltrating ductal breast carcinomas

Background The identification of pathological markers of breast cancer for either diagnosis, treatment response or for survival is of critical importance. Methods Serum protein profiling using 2-DE separations coupled to matrix-assisted laser desorption ionization mass spectrometry has been used to explore protein alterations in patients with infiltrating ductal breast carcinomas (IDCA). Sera from 39 breast cancer patients and 40 healthy controls were selected for screening study using 2-DE combined with MS. The protein expression patterns obtained after the depletion of high abundance proteins was determined by coomassie blue G-250 stain after 2-DE electrophoresis. Results Six proteins that expressed differentially in the IDCA group were found. The expression levels of four isoforms corresponding to haptoglobin precursor and two isoforms of α1-antitrypsin precursor (α1-AT) were upregulated in sera from breast cancer patients. There was an increased expression of both proteins in the sera of patients with various tumor stages (I, II, III) in comparison to healthy women. Applying immunohistochemistry, we further validated α1-AT immunoreactivity in 51 formalin-fixed paraffin-embedded sections of breast tumors. Enhanced expression of α1-AT like activity has been found in IDCA breast tumors, as well as, in different histological types of breast cancer. No significant association has been found with lymph node occurrence, while in high tumor categories a tendency to an increased expression of α1-AT has been found, thereby suggesting a possible role of this protein in tumor growth. Conclusions These proteins may constitute new and useful markers of breast cancer that offer a clue to a better understanding of inflammatory pathways and carcinogenesis events linked to breast cancer progression.

The art of observing rare protein species in proteomes with peptide ligand libraries

The present review deals with the use of combinatorial ligand libraries, composed by hexapeptides, in the capture and concentration of the low-abundance proteome. This method, first reported in 2005, is compared with other current methodologies aimed at exploring the "deep proteome", such as: depletion of high-abundance proteins (especially in sera and cerebrospinal fluid) by individual or combined antibodies (up to 20 against the most abundant species); narrow (1-pH-unit) IPGs (zoom gels) and prefractionation with multicompartment electrolyzers or with Off-Gel electrophoresis. The physico-chemical properties of the hexapeptide library are also explored, namely in assessing the proper length of the baits and the behavior of shorter oligopeptides, down to capture elicited by single amino acids. A number of examples on the use of this library is given, such as the analysis of biological fluids (human sera, urine, bile, cerebrospinal fluid) and of cell lysates (platelets, red blood cells). In all cases, it was possible to detected from three to five times as many proteins as compared to control, untreated samples. Perhaps the most spectacular results were obtained with the erythrocyte proteome, where 1570 proteins could be identified in the "minority" proteome, representing only 2% of the total cell lysate. Another interesting area of application regards the concentration and detection of trace impurities contaminating r-DNA proteins meant for human consumption: several host proteins, never reported before, could be revealed for the first time. Other nonhuman samples are currently under investigation, such as egg-white (where no less than 148 unique gene products could be identified), egg yolk (with 255 unique species) and latex from Hevea brasiliensis. It is anticipated that the ligand library could be a most useful tool for detecting biomarkers for different pathologies and for drug treatment. As a future outlook, one could envision the synthesis of specialized libraries able to capture given classes of proteins (e.g., phospho- and glyco-proteins). Additionally, the library could be used in association with other techniques currently in vogue, such as zoom gels, Off-Gels, and the like.

Affinity prefractionation for MS‐based plasma proteomics

The plasma proteome has proven to be one of the most challenging proteomes to profile using currently available proteomics technologies. A plethora of methodologies have been used to profile human plasma in order to discover potential biomarkers for disease and for therapy optimization. Affinity-based prefractionation coupled to MS has been shown to be one of the most successful ways to dig deeper into the plasma proteome. Depletion of high abundant plasma proteins is becoming an initial method of choice in any plasma profiling project. However, several other affinity-based enrichment methods have been published in recent years. Here we review both protein and peptide affinity prefractionation methods coupled with MS-based proteomics. Analysis of the proportion of cellular and extracellular annotated proteins of publicly available MS plasma proteomics data is performed to estimate the analytical depth of various prefractionation methods.

Generation of an affinity matrix useful in the purification of natural inhibitors of plasmepsin II, an antimalarial‐drug target

Various approaches for removal of high-abundance components in body fluids are currently available. While most methods are constructed for plasma depletion, there is a need for body-fluid-specific strategies. The aim of the present study was to design an affinity matrix suitable for the depletion of high-abundance proteins in CSF (cerebrospinal fluid). Hence, molecules with specific affinity towards proteins present at high concentration in CSF were desired. Affibody molecules are specific binders of small size that have shown high stability under various conditions and are therefore good candidates for such a matrix. The protein composition in CSF resembles that in plasma. However, 20% of the proteins are brain-derived and are therefore present in higher proportions in CSF than in plasma, whereas larger plasma-derived proteins are less abundant in CSF. Therefore five high-abundance CSF proteins were chosen for the design of a CSF-specific depletion setup. Affibody molecules with specificity towards HSA (human serum albumin), IgG, transferrin and transthyretin were combined in an affinity column. In addition, polyclonal antibodies against cystatin C were coupled to chromatographic beads and packed in a separate column. Highly reproducible and efficient removal of the five target proteins was observed. The proportion of depleted proteins were estimated to be 99, 95, 74, 92 and 83% for HSA, IgG, transferrin, transthyretin and cystatin C respectively. SDS/PAGE analysis was used for monitoring and identifying proteins in native CSF, depleted CSF samples and the captured fractions. Moreover, shotgun proteomics was used for protein identification in native as well as depleted CSF and the achieved data were compared. Enhanced identification of lower-abundance components was observed in the depleted fraction, in terms of more detected peptides per protein.

Reducing agent-mediated precipitation of high-abundance plasma proteins

Depletion of high-abundance proteins is regarded as a critical sample preparation step for most plasma proteomic analyses and profiling strategies. This report describes a process that rapidly and reproducibly precipitates high-abundance disulfide-rich proteins, including albumin and transferrin, from serum and plasma. A low volume of concentrated reducing agent, viz. dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP), was added directly to plasma followed by a brief incubation at ambient temperature. Removal of the precipitate via centrifugation and identification of the protein content revealed an albumin-enriched pellet. Direct analysis of the supernatant by MALDI-TOF-MS afforded peptidome and small protein profiles with enhanced features and minimal ionization of full-length albumin. The reproducible and quantitative nature of the method has been demonstrated by monitoring the plasma levels of an antiangiogenic protein biologic, rKringle5 (rK5). The 10.5-kDa analyte was only reliably detected in plasma after treatment with reducing agent, ionizing linearly from 150 to 1200 fmol (on-target) with a mean CV of 7%. This method distinguishes itself from immunoaffinity resin-based approaches since it can be scaled to large milliliter quantities and it is compatible with plasma from all species tested.

Liquid‐phase‐based separation systems for depletion, prefractionation and enrichment of proteins in biological fluids for in‐depth proteomics analysis

This review article is concerned with (i) proteomic sample preparation including among other things the depletion of high-abundance proteins and the concentration of low-abundance proteins and (ii) their subsequent prefractionation for further analysis. Subjects (i) and (ii) are essentials for in-depth proteomic analysis by multidimensional liquid-phase separation techniques (e.g. multidimensional electrophoresis and LC in various formats) followed by mass spectrometric identification and bioinformatic. More than 90 papers published in the period covering early 2000 to the present have been reviewed. It should be noted that this review article is by no means exhaustive, and the aim was to rather provide a concise description of the latest developments in the field.

Approaching clinical proteomics: current state and future fields of application in fluid proteomics

The field of clinical proteomics offers opportunities to identify new disease biomarkers in body fluids, cells and tissues. These biomarkers can be used in clinical applications for diagnosis, stratification of patients for specific treatment, or therapy monitoring. New protein array formats and improved spectrometry technologies have brought these analyses to a level with potential for use in clinical diagnostics. The nature of the human body fluid proteome with its large dynamic range of protein concentrations presents problems with quantitation. The extreme complexity of the proteome in body fluids presents enormous challenges and requires the establishment of standard operating procedures for handling of specimens, increasing sensitivity for detection and bioinformatical tools for distribution of proteomic data into the public domain. From studies of in vitro diagnostics, especially in clinical chemistry, it is evident that most errors occur in the preanalytical phase and during implementation of the diagnostic strategy. This is also true for clinical proteomics, and especially for fluid proteomics because of the multiple pretreatment processes. These processes include depletion of high-abundance proteins from plasma or enrichment processes for urine where biological variation or differences in proteolytic activities in the sample along with preanalytical variables such as inter- and intra-assay variability will likely influence the results of proteomics studies. However, before proteomic analysis can be introduced at a broader level into the clinical setting, standardization of the preanalytical phase including patient preparation, sample collection, sample preparation, sample storage, measurement and data analysis needs to be improved. In this review, we discuss the recent technological advances and applications that fulfil the criteria for clinical proteomics, with the focus on fluid proteomics. These advances relate to preanalytical factors, analytical standardization and quality-control measures required for effective implementation into routine laboratory testing in order to generate clinically useful information. With new disease biomarker candidates, it will be crucial to design and perform clinical studies that can identify novel diagnostic strategies based on these techniques, and to validate their impact on clinical decision-making.

Depletion of High-Abundance Serum Proteins from Human Uterine Lavages Enhances Detection of Lower-Abundance Proteins

Two-dimentional electrophoresis, 2-DE, of human uterine lavages demonstrated the abundance of serum proteins. Two methods of serum protein removal were assessed. Multiple affinity removal system was most effective in depleting abundant serum proteins. 2D-DiGE comparing depleted versus nondepleted lavages resulted in increased protein detection following depletion, further enhanced by assessing the protein profile over a narrower pI 4-7 range. Demonstrating depletion of abundant serum proteins is important for sensitive proteomic analysis.

Application of MALDI/SELDI Mass Spectrometry to Cancer Biomarker Discovery and Validation

This article provides a focused perspective on the application of MALDI/SELDI mass spectrometry (MS)- based technologies to biomarker discovery and validation in various biological materials, which might eventually serve as the basis for new clinical tests to improve diagnosis, guide molecularly-targeted therapy, and monitor the activity and therapeutic response to various cancers. However, the contribution of MALDI/SELDI MS to these goals has been scant due partially to current unavailability of robust analytical methods and instruments to overcome the finite complexity of the proteome and peptidome. Moreover, with a coherent pipeline, the successful throughput from marker discovery to robust validation methods has been dismal. While the development of a faster and more sensitive MS methods will have a major impact on the ability to identify thousands of proteins in body fluids, improvement in sample preparation methods such as high abundance protein depletion and chromatography, and better sample processing methods have also played key roles. Further optimization of MS-based methods is expected to further improve their analytical performance. Because of the guarded skepticism voiced by several investigators about the ultimate value of the entire MS-based proteomics approach for biomarker discovery in complicated samples such as blood, exhaustive experimental methods that use multiple reaction monitoring targeted proteomic approaches, which include immunoassays coupled with MS spectrometry techniques need to be developed and standardized, initially for less complex body fluids, tissues, or even cells or tissue explants in animal models and later on translated to blood, in order to avoid the pitfalls of bias and/or overfitting that plagued earlier studies, and it is still hoped that eventually proteomic profiling in complex biological samples will become a fulfilled promise rather than just an unrealized hope for the scientific and clinical community. Keywords: Bioinformatics, blood, CSF, diagnoses, plasma, statistics, urine

Depletion of high-abundance proteins from human plasma using a combination of an affinity and pseudo-affinity column

Human serum albumin (HSA) and immunoglobulin G (IgG) represent over 75% of all proteins present in human plasma. These high-abundance proteins prevent the detection of low-abundance proteins which are potential markers for various diseases. The depletion of HSA and IgG is therefore essential for further proteome analysis. In this paper we describe the optimization of conditions for selective depletion of HSA and IgG using affinity and pseudo-affinity chromatography. A BIA Separations CIM (convective interaction media) Protein G disk was applied for the removal of IgG and the Mimetic Blue SA A6XL stationary phase for the removal of HSA. The binding and the elution buffer for CIM Protein G disk were chosen on the basis of the peak shape. The dynamic binding capacity was determined. It was shown to be dependent on the buffer system used and independent of the flow rate and of the concentration of IgG. Beside the binding capacity for the IgG standard, the binding capacity was also determined for IgG in human plasma. The Mimetic Blue SA A6XL column was characterized using human plasma. The selectivity of the depletion was dependent on the amount of human plasma that was loaded on the column. After the conditions on both supports had been optimized, the Mimetic Blue SA A6XL stationary phase was combined with the CIM Protein G disk in order to simultaneously deplete samples of human plasma. A centrifuge spin column that enables the removal of IgG and HSA from 20 μL of human plasma was designed. The results of the depletion were examined using sodium dodecyl sulfate polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis.

Cover Picture: Electrophoresis 21'2008

AbstractThis issue has an emphasis on “Proteomics and Related Topics”. It comprises 11 research articles including the “Fast Track” article on the topic of proteomics, glycoproteomics, proteins and peptides. The “Fast Track” article describes a CE‐LIF detection‐based assay for the simultaneous measurements of the electrophoretic mobility, catalytic activity and the variation of activity over time of the individual enzymes molecules of Escherichia coli beta‐galactosidase. The remaining 10 research articles of the Emphasis deal with the development of sensitive fluorescent staining for proteomic analysis, depletion of high abundance proteins form human serum, lectin affinity chromatography in the identification of rat urinary glycoproteome, lab‐on‐chip screening strategy of mouse serum samples prior to proteomics analysis, identification of proteins from membrane preparations, capillary coating for CE of proteins, characterization of rabbit liver apothioneins by CE‐ESI‐MS, quantitative analysis of recombinant protein charge heterogeneity by imaging CIEF, dye staining and immunodetection of proteins on a PVDF membrane, and separation of multiphosphorylated peptide isomers by CZE.