Enrichment Of Low-abundance Proteins Research Articles

Monolayer magnetic nanospheres have selective binding that allow the concentration of low-abundance proteins from blood serum

Here, the efficiency of magnetic nanosphere (Fe3O4) coated with L-histidine (L-his) was evaluated to enrich proteins of low-abundance in human serum. The chemometric analysis provided optimal conditions to concentrate low-abundance proteins, reducing the dynamic range of protein levels in the serum sample. Three independent factors (temperature, mass ratio and pH) were tested. The optimal condition achieved was using temperature (42 °C), ratio (3:1, nanomaterial/sample) and pH (5.5). The kinetic study revealed that the adsorption of proteins on the surface of the monolayer magnetic nanosphere follows a pseudo-second order model with R2 = 0.9153. Adsorption isotherm experiments revealed high capacity of protein adsorption on the surface of the nanosphere, indicating a maximum theoretical adsorption capacity (qmax) of 123.45 mg g−1. The efficiency of enrichment of low-abundance proteins in the serum sample was evaluated by gel electrophoresis (SDS-PAGE). Furthermore, when compared to the method using organic solvent (acetonitrile), Fe3O4@L-his showed adequate efficiency to simplify the serum sample. Using MALDI-TOF/TOF, approximately 50 proteins were identified using Fe3O4@L-his. As a proof of concept for this study, patients with bipolar disorder and healthy controls were discriminated using the magnetic nanosphere as an adsorbent in the sample preparation step. These results show that Fe3O4@L-his can be a fast, robust, and simple alternative to enrich low-abundance proteins in human serum, emerging as an important strategy in the search for biomarkers of human diseases.

Low-Abundance Protein Enrichment for Medical Applications: The Involvement of Combinatorial Peptide Library Technique.

The discovery of low- and very low-abundance proteins in medical applications is considered a key success factor in various important domains. To reach this category of proteins, it is essential to adopt procedures consisting of the selective enrichment of species that are present at extremely low concentrations. In the past few years pathways towards this objective have been proposed. In this review, a general landscape of the enrichment technology situation is made first with the presentation and the use of combinatorial peptide libraries. Then, a description of this peculiar technology for the identification of early-stage biomarkers for well-known pathologies with concrete examples is given. In another field of medical applications, the determination of host cell protein traces potentially present in recombinant therapeutic proteins, such as antibodies, is discussed along with their potentially deleterious effects on the health of patients on the one hand, and on the stability of these biodrugs on the other hand. Various additional applications of medical interest are disclosed for biological fluids investigations where the target proteins are present at very low concentrations (e.g., protein allergens).

An experimental workflow for enrichment of low abundant proteins from human serum for the discovery of serum biomarkers.

Serum contains proteins that possess important information about diseases and their progression. Unfortunately, these proteins, which carry the information in the serum are in low abundance and are masked by other serum proteins that are in high abundance. Such masking prevents their identification and quantification. Therefore, removal of high abundance proteins is required to enrich, identify, and quantify the low abundance proteins. Immunodepletion methods are often used for this purpose, but there are limitations in their use because of off-target effects and high costs. Here we presented a robust, reproducible and cost-effective experimental workflow to remove immunoglobulins and albumin from serum with high efficiency. The workflow did not suffer from such limitations and enabled identification of 681 low abundance proteins that were otherwise undetectable in the serum. The identified low abundance proteins belonged to 21 different protein classes, namely the immunity-related proteins, modulators of protein-binding activity, and protein-modifying enzymes. They also played roles in various metabolic events, such as integrin signalling, inflammation-mediated signalling, and cadherin signalling. The presented workflow can be adapted to remove abundant proteins from other types of biological material and to provide considerable enrichment for low-abundance proteins.

Global industrial enzymes market to surpass $10.5 bn by 2028

The salt-soluble globulins, glycinins (11S globulin), and β-conglycinins (7S globulin), are the most abundant seed proteins of soybean seeds. Together, these two groups of proteins account for 60–70% of total soybean seed proteins. Proteomic assessment of the less abundant soybean seed proteins using general isolation protocols is challenging due to the overwhelming abundance of storage proteins. Development of a simple, fast, and inexpensive method to remove most storage proteins from a seed extract will significantly enhance the study of the nonabundant proteins within seeds. We have developed two simple methods for the depletion of abundant seed proteins resulting in the enrichment of low abundance proteins from soybean seeds. Here, we provide a detailed procedure for the isolation, separation, identification, and quantification of low abundance seed proteins of soybean.

Elastomers production increases liquid sodium silicate demand

The salt-soluble globulins, glycinins (11S globulin), and β-conglycinins (7S globulin), are the most abundant seed proteins of soybean seeds. Together, these two groups of proteins account for 60–70% of total soybean seed proteins. Proteomic assessment of the less abundant soybean seed proteins using general isolation protocols is challenging due to the overwhelming abundance of storage proteins. Development of a simple, fast, and inexpensive method to remove most storage proteins from a seed extract will significantly enhance the study of the nonabundant proteins within seeds. We have developed two simple methods for the depletion of abundant seed proteins resulting in the enrichment of low abundance proteins from soybean seeds. Here, we provide a detailed procedure for the isolation, separation, identification, and quantification of low abundance seed proteins of soybean.

Soybean seed proteomics: Methods for the isolation, detection, and identification of low abundance proteins.

The salt-soluble globulins, glycinins (11S globulin), and β-conglycinins (7S globulin), are the most abundant seed proteins of soybean seeds. Together, these two groups of proteins account for 60-70% of total soybean seed proteins. Proteomic assessment of the less abundant soybean seed proteins using general isolation protocols is challenging due to the overwhelming abundance of storage proteins. Development of a simple, fast, and inexpensive method to remove most storage proteins from a seed extract will significantly enhance the study of the nonabundant proteins within seeds. We have developed two simple methods for the depletion of abundant seed proteins resulting in the enrichment of low abundance proteins from soybean seeds. Here, we provide a detailed procedure for the isolation, separation, identification, and quantification of low abundance seed proteins of soybean.

Systemic Effects of Hemorrhagic Snake Venom Metalloproteinases: Untargeted Peptidomics to Explore the Pathodegradome of Plasma Proteins.

Hemorrhage induced by snake venom metalloproteinases (SVMPs) is a complex phenomenon that involves capillary disruption and blood extravasation. HF3 (hemorrhagic factor 3) is an extremely hemorrhagic SVMP of Bothrops jararaca venom. Studies using proteomic approaches revealed targets of HF3 among intracellular and extracellular proteins. However, the role of the cleavage of plasma proteins in the context of the hemorrhage remains not fully understood. The main goal of this study was to analyze the degradome of HF3 in human plasma. For this purpose, approaches for the depletion of the most abundant proteins, and for the enrichment of low abundant proteins of human plasma, were used to minimize the dynamic range of protein concentration, in order to assess the proteolytic activity of HF3 on a wide spectrum of proteins, and to detect the degradation products using mass spectrometry-based untargeted peptidomics. The results revealed the hydrolysis products generated by HF3 and allowed the identification of cleavage sites. A total of 61 plasma proteins were identified as cleaved by HF3. Some of these proteins corroborate previous studies, and others are new HF3 targets, including proteins of the coagulation cascade, of the complement system, proteins acting on the modulation of inflammation, and plasma proteinase inhibitors. Overall, the data indicate that HF3 escapes inhibition and sculpts the plasma proteome by degrading key proteins and generating peptides that may act synergistically in the hemorrhagic process.

Improved Protein Coverage in Bottom-Up Proteomes Analysis Using Fluoroalcohol-Mediated Supramolecular Biphasic Systems With Mixed Amphiphiles for Sample Extraction, Fractionation, and Enrichment.

A new class of supramolecular biphasic systems containing fluoroalcohol-induced coacervates (FAiC) provides concomitant fractionation of complex protein mixtures, high solubilizing power for extraction of various types of proteins, especially those with high hydrophobicity (such as membrane proteins), and enrichment of low-abundance proteins. Subsequently, the use of FAiC biphasic systems (BPS) in the bottom-up proteomics workflow resulted in significantly higher coverage for the whole proteome, various subproteomes, especially those embedded or associated with membranes, post-translationally modified proteins, and low-abundance proteins (LAPs) as compared to the conventional methodologies. In this work, we used a new type of FAiC-BPS composed of mixed amphiphiles, a zwitterionic surfactant 3-(N,N-dimethylmyristyl ammonia) propane sulfonate (DMMAPS), a quaternary ammonium salt (QUATS), and hexafluoroisopropanol (HFIP) as the coacervator for extraction, fractionation, and enrichment of yeast proteome in bottom-up proteomics. The coverage of the lower-abundance proteins (abundance below 2000 molecules/cell) improved by more than 100% using DMMAPS and DMMAPS + QUATS systems as compared to the conventional methods using urea or detergent solutions for protein solubilization. Additionally, these coacervate systems show increased coverage of integral membrane proteins and proteins with α-helices by up to 24 and 555%, respectively.

Extending the Depth of Human Plasma Proteome Coverage Using Simple Fractionation Techniques.

Human plasma is one of the most widely used tissues in clinical analysis, and plasma-based biomarkers are used for monitoring patient health status and/or response to medical treatment to avoid unnecessary invasive biopsy. Data-driven plasma proteomics has suffered from a lack of throughput and detection sensitivity, largely due to the complexity of the plasma proteome and in particular the enormous quantitative dynamic range, estimated to be between 9 and 13 orders of magnitude between the lowest and the highest abundance protein. A major challenge is to identify workflows that can achieve depth of plasma proteome coverage while minimizing the complexity of the sample workup and maximizing the sample throughput. In this study, we have performed intensive depletion of high-abundant plasma proteins or enrichment of low-abundant proteins using the Agilent multiple affinity removal liquid chromatography (LC) column-Human 6 (Hu6), the Agilent multiple affinity removal LC column-Human 14 (Hu14), and ProteoMiner followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) and C18 prefractionation techniques. We compared the performance of each of these fractionation approaches to identify the method that satisfies requirements for analysis of clinical samples and to include good plasma proteome coverage in combination with reasonable sample output. In this study, we report that one-dimensional (1D) gel-based prefractionation allows parallel sample processing and no loss of proteome coverage, compared with serial chromatographic separation, and significantly accelerates analysis time, particularly important for large clinical projects. Furthermore, we show that a variety of methodologies can achieve similarly high plasma proteome coverage, allowing flexibility in method selection based on project-specific needs. These considerations are important in the effort to accelerate plasma proteomics research so as to provide efficient, reliable, and accurate diagnoses, population-based health screening, clinical research studies, and other clinical work.

Experimental conditions influence the formation and composition of the corona around gold nanoparticles

BackgroundOvarian cancer is one of the deadliest gynecological malignancies. While the overall survival of ovarian cancer patients has slightly improved in recent years in the developed world, it remains clinically challenging due to its frequent late diagnosis and the lack of reliable diagnostic and/or prognostic markers. The aim of this study was to identify potential new molecular target proteins (NMTPs) responsible for the poor outcomes. When nanoparticles (NP) are exposed to biological fluids, a protein coat, termed the protein corona (PC), forms around the NP, and the PC represents a tool to identify NMTPs. This study investigates the influence of pre-processing conditions, such as lysis conditions and serum/plasma treatment, on the PC composition and the resulting identification of NMTPs.ResultsUsing gel electrophoresis, pre-processing conditions, including cell-lysis techniques and enrichment of low-abundance proteins (LAPs) by immunocentrifugation of serum/plasma, were shown to alter the relative amounts and compositions of proteins. PCs formed when 20 nm gold-NPs (GNPs) were incubated with lysate proteins from either RIPA- or urea lysis. Proteomic analysis of these PCs showed 2–22-fold enrichment of NMTPs in PCs from urea lysates as compared to RIPA lysates. Enriched NMTPs were then classified as cellular components, biological and molecular functions-associated proteins. The impact of enriched LAPs (eLAPs) on both PC composition and NMTP identification was shown by comparative proteomic analysis of original plasma, eLAPs, and PCs derived from eLAPs; eLAPs-PCs enhanced the abundance of NMTPs approximately 13%. Several NMTPs, including gasdermin-B, dermcidin, and kallistatin, were identified by this method demonstrating the potential use of this PC approach for molecular target discovery.ConclusionThe current study showed that the pre-processing conditions modulate PC composition and can be used to enhance identification of NMTPs.

Chloroplast Isolation and Enrichment of Low-Abundance Proteins by Affinity Chromatography for Identification in Complex Proteomes.

Comprehensive knowledge of the proteome is a crucial prerequisite to understand dynamic changes in biological systems. Particularly low-abundance proteins are of high relevance in these processes as these are often proteins involved in signal transduction and acclimation responses. Although technological advances resulted in a tremendous increase in protein identification sensitivity by mass spectrometry (MS), the dynamic range in protein abundance is still the most limiting problem for the detection of low-abundance proteins in complex proteomes. These proteins will typically escape detection in shotgun MS experiments due to the presence of high-abundance proteins. Therefore, specific enrichment strategies are still required to overcome this technical limitation of MS-based protein discovery. We have searched for novel signal transduction proteins, more specifically kinases and calcium-binding proteins, and here we describe different approaches for enrichment of these low-abundance proteins from isolated chloroplasts from pea and Arabidopsis for subsequent proteomic analysis by MS. These approaches could be extended to include other signal transduction proteins and target different organelles.

Plasma/serum proteomics: depletion strategies for reducing high-abundance proteins for biomarker discovery.

Plasma and serum are widely used for proteomics-based biomarker discovery. However, analysis of these biofluids is highly challenging due to the complexity and wide dynamic range of their proteomes. Notably, highly abundant proteins tend to obscure the detection of potential biomarkers that are usually of lower concentrations. Among the strategies to resolve this problem are: depletion of high-abundance proteins, enrichment of low abundant proteins of interestand prefractionation. In this review, we focus on current and emerging depletion techniques used to enhance the detection and identification of the less abundant proteins in plasma and serum. We discuss the applications and contributions of these methods to proteomics analysis of plasma and serum alongside their limitations and future perspectives.

Reproducible Large-Scale Synthesis of Surface Silanized Nanoparticles as an Enabling Nanoproteomics Platform: Enrichment of the Human Heart Phosphoproteome.

A reproducible synthetic strategy was developed for facile large-scale (200 mg) synthesis of surface silanized magnetite (Fe3O4) nanoparticles (NPs) for biological applications. After further coupling a phosphate-specific affinity ligand, these functionalized magnetic NPs were used for the highly specific enrichment of phosphoproteins from a complex biological mixture. Moreover, correlating the surface silane density of the silanized magnetite NPs to their resultant enrichment performance established a simple and reliable quality assurance control to ensure reproducible synthesis of these NPs routinely in large scale and optimal phosphoprotein enrichment performance from batch-to-batch. Furthermore, by successful exploitation of a top-down phosphoproteomics strategy that integrates this high throughput nanoproteomics platform with online liquid chromatography (LC) and tandem mass spectrometry (MS/MS), we were able to specifically enrich, identify, and characterize endogenous phosphoproteins from highly complex human cardiac tissue homogenate. This nanoproteomics platform possesses a unique combination of scalability, specificity, reproducibility, and efficiency for the capture and enrichment of low abundance proteins in general, thereby enabling downstream proteomics applications.

Construction of discontinuous capillary isoelectric focusing system and its application in pre-fractionation of exosomal proteins

Exosomal proteins play an important role in screening disease biomarker and clinic diagnosis. However, low concentration, complex composition and difficulty in extraction hindered the study of their structure and properties. In this study, a new protein pre-fractionation system, discontinuous monolithic immobilized pH gradient capillary isoelectric focusing (DM-IPG-CIEF), was constructed by reassembling pieces of monolithic IPG-CIEF column to create small cavity with well-defined pH range. The monolithic IPG-CIEF column exhibited good linearity (R2 = 0.9997) and preparation and operation reproducibility. Then removal of high-abundant proteins and enrichment of low-abundant proteins were performed by the DM-IPG-CIEF system successfully. The system was applied to pre-fractionation and enrichment of exosomal proteins extracted from HeLa cells. The exosomal proteins with pI in the range of 6.0–9.0 were focused in enrichment cavity and collected. According to nano-HPLC-MS/MS analysis, 130 proteins in this range were identified, indicating great potential of the DM-IPG-CIEF system in pretreatment of exosomal proteins.

Use of DNA-Aptamers for Enrichment of Low Abundant Proteins in Cellular Extracts for Quantitative Detection by Selected Reaction Monitoring

Use of DNA-Aptamers for Enrichment of Low Abundant Proteins in Cellular Extracts for Quantitative Detection by Selected Reaction Monitoring

Effects of Alanyl-Glutamine Treatment on the Peritoneal Dialysis Effluent Proteome Reveal Pathomechanism-Associated Molecular Signatures

Peritoneal dialysis (PD) is a modality of renal replacement therapy in which the high volumes of available PD effluent (PDE) represents a rich source of biomarkers for monitoring disease and therapy. Although this information could help guide the management of PD patients, little is known about the potential of PDE to define pathomechanism-associated molecular signatures in PD.We therefore subjected PDE to a high-performance multiplex proteomic analysis after depletion of highly-abundant plasma proteins and enrichment of low-abundance proteins. A combination of label-free and isobaric labeling strategies was applied to PDE samples from PD patients (n = 20) treated in an open-label, randomized, two-period, cross-over clinical trial with standard PD fluid or with a novel PD fluid supplemented with alanyl-glutamine (AlaGln).With this workflow we identified 2506 unique proteins in the PDE proteome, greatly increasing coverage beyond the 171 previously-reported proteins. The proteins identified range from high abundance plasma proteins to low abundance cellular proteins, and are linked to larger numbers of biological processes and pathways, some of which are novel for PDE. Interestingly, proteins linked to membrane remodeling and fibrosis are overrepresented in PDE compared with plasma, whereas the proteins underrepresented in PDE suggest decreases in host defense, immune-competence and response to stress. Treatment with AlaGln-supplemented PD fluid is associated with reduced activity of membrane injury-associated mechanisms and with restoration of biological processes involved in stress responses and host defense.Our study represents the first application of the PDE proteome in a randomized controlled prospective clinical trial of PD. This novel proteomic workflow allowed detection of low abundance biomarkers to define pathomechanism-associated molecular signatures in PD and their alterations by a novel therapeutic intervention.

Use of DNA-aptamers for enrichment of low abundant proteins in cellular extracts for quntitative detection by selected reaction monitoring

The relationship between the amount of a target protein in a complex biological sample and its amount measured by selected reaction monitoring (SRM) mass spectrometry upon the affinity enrichment of target protein with aptamers immobilized on a solid phase was studied. Human thrombin added in known concentrations to cellular extracts derived from bacterial cells was used as model target protein. It has been demonstrated that the affinity enrichment of thrombin in cellular extracts by means of the thrombin-binding aptamer immobilized on the surface of magnetic microbeads results in an approximately 10-fold increase of the concentration of target protein and a 100-fold decrease of the low limit of a target protein concentration range where its quantitative detection by SRM is possible without an interference from other peptides present in a tryptic digest.

One-Step Facile Synthesis of Aptamer-Modified Graphene Oxide for Highly Specific Enrichment of Human A-Thrombin in Plasma.

The enrichment of low-abundance proteins in complex biological samples plays an important role in clinical diagnostics and biomedical research. This work reports a novel one-step method for the synthesis of aptamer-modified graphene oxide (GO/Apt) nanocomposites, without introducing the use of gold, for the rapid and specific separation and enrichment of human α-thrombin from buffer solutions with highly concentrated interferences. The obtained GO/Apt nanocomposites had remarkable aptamer immobilization, up to 44.8 nmol/mg. Furthermore, GO/Apt nanocomposites exhibited significant specific enrichment efficiency for human α-thrombin (>90%), even under the presence of 3000-fold interference proteins, which was better than the performance of other nanomaterials. Finally, the GO/Apt nanocomposites were applied in the specific capturing of human α-thrombin in highly concentrated human plasma solutions with negligible nonspecific binding of other proteins, which demonstrated their prospects in rare protein analysis and biosensing applications.

In-depth proteomic analysis of Glycine max seeds during controlled deterioration treatment reveals a shift in seed metabolism

Seed aging is one of the major events, affecting the overall quality of agricultural seeds. To analyze the effect of seed aging, soybean seeds were exposed to controlled deterioration treatment (CDT) for 3 and 7days, followed by their physiological, biochemical, and proteomic analyses. Seed proteins were subjected to protamine sulfate precipitation for the enrichment of low-abundance proteins and utilized for proteome analysis. A total of 14 differential proteins were identified on 2-DE, whereas label-free quantification resulted in the identification of 1626 non-redundant proteins. Of these identified proteins, 146 showed significant changes in protein abundance, where 5 and 141 had increased and decreased abundances, respectively while 352 proteins were completely degraded during CDT. Gene ontology and KEGG analyses suggested the association of differential proteins with primary metabolism, ROS detoxification, translation elongation and initiation, protein folding, and proteolysis, where most, if not all, had decreased abundance during CDT. Western blotting confirmed reduced level of antioxidant enzymes (DHAR, APx1, MDAR, and SOD) upon CDT. This in-depth integrated study reveals a major downshift in seed metabolism upon CDT. Reported data here serve as a resource for its exploitation to metabolic engineering of seeds for multiple purposes, including increased seed viability, vigor, and quality. Biological significanceControlled deterioration treatment (CDT) is one of the major events that negatively affects the quality and nutrient composition of agricultural seeds. However, the molecular mechanism of CDT is largely unknown. A combination of gel-based and gel-free proteomic approach was utilized to investigate the effects of CDT in soybean seeds. Moreover, we utilized protamine sulfate precipitation method for enrichment of low-abundance proteins, which are generally masked due to the presence of high-abundance seed storage proteins. Reported data here serve as resource for its exploitation to metabolic engineering of seeds for multiple purposes, including increased seed viability, vigor, and quality.

Identification of functional metabolic biomarkers from lung cancer patient serum using PEP technology.

BackgroundReprogrammed metabolism is a new hallmark of cancer. In many types of cancer, most of the genes in the glycolytic pathway are overexpressed, reflecting an essential shift of metabolism during cancer development. The reprogrammed metabolism contributes to cancer development in multiple ways, from supplying the elevated energy requirement to creating a microenvironment suitable for tumor growth and suppressing the human immune surveillance system.MethodIn this study, a functional proteomics top-down approach was used to systematically monitor metabolic enzyme activities in resolved serum proteins produced by a modified 2-D gel separation and subsequent Protein Elution Plate, a method collectively called PEP.ResultsWe found that the enrichment of low abundance proteins with a bead based product called AlbuVoid™, is important to increase the number of observable features and to increase the level of signal achievable from the assay used. From our methods, significant metabolic enzyme activities were detected in both normal and lung cancer patient sera in many fractions after the elution of the 2-D gel separated proteins to the Protein Elution Plate (PEP). Eighteen fractions with the most dramatic metabolic enzyme activity difference between the normal and lung cancer patient sera were submitted for mass spectrometry protein identification. Proteins from the glycolytic metabolic pathway, such as GAPDH along with other proteins not previously annotated to the glycolytic pathway were identified. Further verification with commercially purified GAPDH showed that the addition of purified GAPDH to the metabolic enzyme assay system employed enhanced the enzyme activity, demonstrating that proteins identified from the PEP technology and mass spectrometry could be further verified with biological assay.ConclusionThis study identified several potential functional enzyme biomarkers from lung cancer patient serum, it provides an alternative and complementary approach to sequence annotation for the discovery of biomarkers in human diseases.