Serum
MONMOUTH JUNCTION, NJ -- Biotech Support Group reports on a new application on AlbuVoid™, an albumin depletion reagent that selectively voids albumin and enriches the low abundance proteome. The application report obtains unique protein identification’s from human and rat sera upon depleting albumin using AlbuVoid™ in addition to providing a serum proteomic workflow. Subsequent to using AlbuVoid™ isobaric labeling, glycopeptide enrichment with Concanavalin-A (ConA) 4B, PNGase treatment to remove glycosylation, liquid chromatography mass spectrometry (LC-MS/MS) and computational peptide or protein annotation is possible. The AlbuVoid™ proteomics workflow on albumin depletion has compatibility with quantitative label (i.e., isobaric tags for relative and absolute quantitation (iTRAQ)) and label-free LC-MS methods. The application report provides data on immobilized ConA to enrich glycopeptides and enzymatic cleaving of glyco-bond producing glycoprotein fraction specific peptides. On bead digestions minimize proteolytic hydrolysis inconsistencies and more glycoproteins in total protein sample are observed. In-gel digests, solution digests and C18 desalting are not required and minimum mis-cleavages occur.

Download the application report and obtain the serum proteomic workflow using AlbuVoid™ on Biotech Support Group website.

Kuruc Matt "Application Report -
AlbuVoid™ & On-Bead Digestion: Tackling the challenges of serum proteomics."

Discovery of Functional Serum Biomarkers Using AlbuVoid™ Enrichment and the ArrayBridge PEP Profiling Platform.

We report that by using AlbuVoid™ as an upfront serum enrichment product, the features displayed from the PEP technology is substantially improved compared to the virtually featureless landscape observed without any such enrichment. As an example using Hexokinase profiling, the final result is that with AlbuVoid™ enrichment, feature differences can be established between normal and cancer serum samples; such as would not be the case without AlbuVoid™ enrichment. Personal communication, Xing Wang, Ph.D., ArrayBridge (St. Louis, MO), manuscripts in process.

Knowledge surrounding the functional annotation of the proteome is vitally important as the landscape of protein conformations is highly variable, each conformation may contribute to a unique functional activity. Sequence annotation alone cannot capture this vital information, so new strategies are necessary. The challenge has been to overcome the analytical bias towards the most abundant proteins, and the complexities of mining the data to a manageable number of biomarker proteins that can be analyzed in more depth. So reconciling protein identifications to actual enzyme activities or functions has been subject to limitations in proteome separation and assay technologies. To overcome these inefficiencies in functional annotation, a top-down approach, starting with function, and ending with sequence and structural annotation is now available. The PEP technology, developed by ArrayBridge, uses a modified Two-dimensional Gel Electrophoresis to separate the proteome, without substantially compromising function. The isolated proteins are then electro-eluted from the PEP plate, and enzyme activities are measured systematically. This method thus provides a new functional dimension to explore the human serum proteome.

Genetic Engineering News Omics Tutorial Describes Biotech Support Group and ArrayBridge Joint Functional Proteomic Data
A Genetic Engineering News article describes a joint collaboration with ArrayBridge (St. Louis, MO). The article describes the combination of first low abundance protein enrichment/high abundance protein depletion with the Biotech Support Group product – AlbuVoid™, followed by a modified 2-dimensional electrophoretic separation, and transfer via the ArrayBridge PEP plate into microplates. From there, the resolved functionally active proteins were measured and characterized.

The citation is:
Xing Wang, Ph.D., Zhenyu Sun, M.D., Xiaofeng Chen, M.D., Xiong Su, Ph.D., Gan Wang, Ph.D., Matthew Kuruc. Genetic Engineering News Jan 1, 2015 (Vol. 35, No. 1) OMICS Tutorial Discovery of Functional Serum Biomarkers, Exploring Cancer's Signature in the Sensitive Functional Domain of the Human Proteome
http://www.genengnews.com/gen-articles/discovery-of-functional-serum-bio...

Serum Profiling Making Mark On Predictive Medicine
Vicki Glaser.Genetic Engineering & Biotechnology News. 2011;31(7):1-55.
AlbuVoid™'s application for albumin depletion is cited for inhibiting albumin binding and collecting the fraction containing albumin in Genetic Engineering & Biotechnology News.

Plasma
Espes, Daniel, Joey Lau, and Per-Ola Carlsson. "Increased circulating levels of betatrophin in individuals with long-standing type 1 diabetes." Diabetologia(2013): 1-4.
Authors Espes et al published an article in the journal Diabetologia which discovered an increase in hormone betatrophin in type 1 diabetics as compared to health individuals. Betatrophin causes an increase of pancreatic β cell replication and regulates glucose levels. Found in liver and adipose tissue, the hormone also increases β cell mass expansion. Analysis of betatrophin by mass spectrometry (MS) or gel electrophoresis could require enriching the protein mixture and depleting high abundance proteins to display less abundant proteins. The article titled, “Increased circulating levels of betatrophin in individuals with long-standing type 1 diabetes” utilized AlbuVoid™’s albumin depletion protocol for removing albumin from plasma samples. The article quotes AlbuVoid™ from Biotech Support Group, “Plasma samples were depleted of albumin using AlbuVoid Albumin Depletion Kit (Biotech Support Group, Monmouth Junction, NJ, USA). Data were normalized for total protein content.” Betatrophin increases proliferation of beta cell and this study identified double the concentration of betatrophin as measured by western immunoblot using a betatrophin primary antibody in type 1 diabetics. Moreover factors such as age in healthy controls displayed a direct relationship with increase in betatrophin whereas triacylglycerol, LDL-cholesterol, HDL-cholesterol levels and insulin were not affected. Increasing concentrations of betatrophin did not prevent against the loss of C-peptide suggesting type 1 diabetes on betatropin treatment would benefit from combination treatment. The authors concluded, “An intervention in patients with type 1 diabetes with betatrophin treatment might require supraphysiological dosing as well as combination with immune regulatory treatment.”

Cell Culture
Narain, Niven Rajin, Rangaprasad Sarangarajan, Vivek K. Vishnudas, and Michael Andrew Kiebish. "USE OF MARKERS IN THE IDENTIFICATION OF CARDIOTOXIC AGENTS AND IN THE DIAGNOSIS AND MONITORING OF CARDIOMYOPATHY AND CARDIOVASCULAR DISEASE." U.S. Patent 20,140,100,128, issued April 10, 2014.
Albumin depletion using AlbuVoid™ column for protocols of cardiovascular disease biomarker sample preparation. Narain, Niven Rajin et al authored a patent on methods for detecting biomarkers of cardiovascular diseases, monitoring disease progression and treatment of cardiovascular disease. Cells are treated with a test agent and modulation of the agent is correlated to level of cardiovascular disease biomarker in cells. The cardiovascular biomarker in certain situations is related to diseases such as cardiomyopathy, arrhythmias, QT duration, hypertension, thromboembolic complications, atrial fibrillation, LV myocardial dysfunction, heart valve damage and heart failure. The patent also cites protein purification and isolation methods from cells or tissues. Contaminating proteins, cellular material and culture medium are separated from the protein or biologically active portions. Marker proteins and active portions of polypeptide fragments are used as immunogens against antibodies. The formation of protein or peptides using recombinant DNA techniques is also cited. Lipids, protein, nucleic acid in cell models are indicative of diseases and proper sample preparation allows for the analysis of biomarkers of arachidonic acid metabolites, plasmalogens, oxidized lipid metabolites, docosahexanoic acid. Biomarker detection involves quantitative polymerase chain reaction, qPCR, proteomic techniques, RNA isolation, cDNA synthesis and using qPCR arrays for disease profiling. For large-scale high-throughput quantitative proteomic analysis mass spectrometry and iTRAQ® analysis is used in conjunction with 2D-LC MALDI MS/MS for peptide quantification. Abundance ratios are compared to samples from multiple iTRAQ® experiments .Samples are fractionated by 2D-LC, strong cation exchange (SCX) followed by reverse-phase HPLC and LC-MS analysis. Lipids are detected using methods such as LC-MS, NMR, quad mass spec, solid-phase extraction (SPE) chromatography, electrospray ionization (ESI) and MALDI mass spectrometry. Cell lysis using a buffer and protease inhibitor, centrifugation, protein concentration measurement using Bradford assay, protein reduction, alkylation and digestion with trypsin is described for protein extraction. Secretome sample preparation involves preparing a medium of cells cultured in media not containing serum which is concentrated, reduced, alkylated, desalted and digested. Another medium of cells is cultured in serum containing medium, reduced using columns and reconstituted using PBS. Serum albumin is depleted from these samples using Biotech Support Group's AlbuVoid™ column.
The patent quotes "In one embodiment, the cells can be cultured in serum containing medium: The volume of the medium can be reduced using 3k MWCO Vivaspin columns (GE Healthcare Life Sciences), then can be reconstituted with 1×PBS (Invitrogen®). Serum albumin can be depleted from all samples using AlbuVoid column (Biotech Support Group, LLC) following the manufacturer's instructions with the modifications of buffer-exchange to optimize for condition medium application." Samples are labeled, vacuumed and analyzed by LC-MS/MS. The labeled peptide mixtures are separated by 2D-nanoLC and electrospray tandem mass spectrometry is used for analysis. Peptide mixtures are separated using a polysulfoethyl aspartamide column and eluted in a C18 trap column. Proteins and peptides are queried using bioinformatics software.
The invention also cites the opportunity to develop kits which detect antibody & oligonucleotides, bind to antibody-protein complexes and anneal complimentary nucleic acids from biological samples. In addition to the discovery of lipid and kinase marker proteins the kits can be used for identifying control proteins such as albumin in blood samples. The reagent kits are also used for the development of a panel of reagents for detecting markers of cardiovascular diseases.

Patents
Patent Application Cites AlbuVoid™ For Secretome Sample Preparation & Neuroscience Research
Narain, Niven Rajin, and Paula Patricia Narain. COMPOSITIONS AND METHODS FOR DIAGNOSIS AND TREATMENT OF PERVASIVE DEVELOPMENTAL DISORDER United States Patent Application 20150023949, Pub. Date 01/22/2015.
The patent provides albumin sample preparation research application to track modulators of disease processes and experiment data on metabolomics, transcriptomics, single nucleotide polymorphisms using sample preparation and an artificial intelligence based informatics platform. Serum containing media are reduced using columns, reconstituted with buffers and serum albumin is depleted using Biotech Support Group's AlbuVoid™. Culture medium optimization and subsequent modifications to buffer exchange is performed. Protein extraction requires a lysis buffer with protease inhibitors, vortexing, ultrasonication, centrifugation and clean up of debris. Secretome sample preparation of cultures and conditioned media requires reduction using dithiothreitol (DTT), alkylation using iodoacetamide and desalting using acetone precipitation. Quantitation of proteins using Bradford assay is performed and reduction, alkylation, digestion using trypsin is performed. Pooled aliquots of tryptic digests are labeled using iTRAQ, and analyzed using liquid chromatography mass spectrometry.
Narain, Niven Rajin, Rangaprasad Sarangarajan, and Vivek K. Vishnudas. "INTERROGATORY CELL-BASED ASSAYS AND USES THEREOF." U.S. Patent No. 20,120,258,874. 11 Oct. 2012.
Serum albumin depletion for secretome sample preparation using AlbuVoid™ while developing a cellular modeling system.
Inventors Narain et al published United States Patent 20120258874 titled, “INTERROGATORY CELL-BASED ASSAYS AND USES THEREOF” describing the invention of a cellular modeling system which develops molecular signatures allowing scientists to gain insight into the mechanisms of disease by providing information on tissue microenvironment. The patent emphasizes a systems biology approach in which diseased cells are subjected to environmental stimuli, high throughput biological readouts and bioinformatic analysis. The invention created “hubs” which are drug discovery candidates obtained from a combination of “network biology, genomic, proteomic, metabolomic, transcriptomic, and bioinformatics tools and methodologies”. Thus information on disease diagnosis or intervention and insight into mechanisms of drug toxicity is obtained. Researchers used AlbuVoid™ for serum albumin depletion for secretome sample preparation. Albumin was depleted from cell culture in serum containing medium using AlbuVoid™ to isolate and identify low abundance proteins.
The patient cites AlbuVoid™ from Biotech Support Group
“In one embodiment, the cells can be cultured in serum containing medium: The volume of the medium can be reduced using 3k MWCO Vivaspin columns (GE Healthcare Life Sciences), then can be reconstituted withlxPBS (Invitrogen). Serum albumin can be depleted from all samples using AlbuVoid column (Biotech Support Group, LLC) following the manufacturer's instructions with the modifications of buffer-exchange to optimize for condition medium application.”

Poster
Improved proteomic enrichment and workflow strategies
US HUPO 2014, Poster 089
Proteomic sample preparation technology for depleting hemoglobin, and enriching the low abundance proteome from human erythrocyte lysates and determination of the position and nature of human protein N termini in different tissues and disease states. Biotech Support Group has developed a series of enrichment reagents that offer common features and key advantages. After separations, the sub-proteomes retain their structural and functional integrity. All the products are consumable and economical; not being derived from biological sources. We report here applications for these products to specifically deplete albumin by a voidance strategy - called AlbuVoid™, which consequently enriches for the low abundance serum protein content. Such a strategy compares favorably to high abundance immuno-depletion (Agilent column) methods. Also, we continue to advance simplified workflows that utilize on-bead digestion for LC-MS. Together, these methods create efficiencies necessary to support high throughput investigations in all areas of discovery, targeted, functional and chemical proteomics.
New On-Bead Digestion Protocols Improve LC-MS Workflows Of Albumin Depleted Samples US HUPO
2013
Translational Proteomics

Biotech Support Group LLC, reports on a new technical poster which describes the use of on-bead digestion protocols as a suitable method to generate peptides for LC-MS/MS analysis, after albumin depletion on its AlbuVoid™ product. Entitled “New On-Bead Digestion Protocols Improve LC-MS Workflows Of Albumin Depleted Samples”, it was presented at the US HUPO Conference in Baltimore, MD, March 10-11, 2013. It describes by several performance metrics, that the ‘on-bead’ digestion protocol is comparable to the product’s standard elution protocol. On-bead protocols provide advantaged speed, simplicity and reduced potential for keratin contamination. The poster also reports on the efficiency of AlbuVoid™ as a simple consumable product for albumin depletion, with 20-30% more protein/peptide identifications achievable relative to untreated plasma, under identical operating and informatic parameters. “With greater interest in the proteomics community for better workflows and performance for LC-MS analyses, direct proteolytic digestion of the bound protein content - commonly called on-bead digestion, has many desirable workflow advantages. With this report on AlbuVoid™, we have now demonstrated that the surface chemistry platform from which it was derived, our novel passivated porous silica –NuGel™, offers the rigidity and low water content ideal for protein concentration and proteolytic digestion”, states Swapan Roy, Ph.D, President & Founder of Biotech Support Group. “We envision designing new strategies for high abundance depletion that can utilize on-bead digestion workflows for highly complex bead-bound proteomes along with many chemical proteomic applications.”

AlbuVoid™ for Studying the Albumin Fraction for Drug and Protein Interactions & Analyzing the Unbound Fraction for Albuminome Subproteome Biomarker Discovery from Biotech Support Group

6 Reasons To Choose AlbuVoid™ For Novel Serum Biomarkers In Umbilical Cord Serum Proteomics And Stem Cell Treatments from Biotech Support Group