Serum
Zheng et al.
AlbuVoid™ Coupled to On-Bead Digestion - Tackling the Challenges of Serum Proteomics. J Proteomics Bioinform 2015, 8:9
For cancer research, serum and plasma are especially attractive sample types as collection of blood is common, simple and only minimally invasive. Yet serum samples can offer unique challenges in LC-MS proteomic analyses. The two biggest challenges being: 1) the high abundance of Albumin accounting for about 50% of the total protein mass and, 2) proteolytic resistance, in large part due to substantial amount of glycoprotein, a modification that manifests proteolytic resistance. In this short report, we describe new methods using a surface/bead based product, AlbuVoid™, which depletes Albumin through a negative selection or voidance strategy, retaining the vast amount of the remaining serum proteome on the bead. We then combine this novel enrichment, with a direct and seamless integration with Trypsin digestion, a method conventionally referred to as on-bead digestion. We evaluated the digestion time as a parameter to identify whether different sub-populations of peptides and proteins can be observed by LC-MS analyses. Using 2 different allotted digestion times - 4 hours, and overnight, each with a singular 3 hour gradient LC-MS run, between 400-500 total proteins were observed for both human and rat sera, with overlapping and distinct sub-populations observable at each digest time. These results support that the described methods gain efficiencies over other high abundance depletion and in-solution digestion workflows. We solicit that such workflows will minimize many of the inconsistencies of proteolytic hydrolysis for both discovery and quantitative serum proteomic applications.
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.
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.
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.
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.
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
