Serum exosomes
Chettimada, Sukrutha, et al. "Exosome markers associated with immune activation and oxidative stress in HIV patients on antiretroviral therapy." Scientific Reports 8.1 (2018): 7227.
Biotech Support Group reports on a recent journal article using AlbuSorb™ to improve serum exosome purification for subsequent proteomic analysis. The authors investigated the role that exosomes play in HIV pathogenesis, as little is known about exosome cargo in relation to immune responses and oxidative stress. For this, plasma exosomes in HIV patients and their relationship to immunological and oxidative stress markers were characterized by electron microscopy, nanoparticle tracking, immunoblotting, and LC-MS/MS proteomics. For LC-MS/MS proteomics, the article states “To obtain purified exosome fractions for proteomic analysis,… albumin was depleted with 2 rounds of albumin depletion using AlbuSorb™ - Albumin Depletion Kit (Biotech Support Group)”. Using untargeted proteomic analysis, Exosomal Notch4 was increased in HIV-positive subjects versus controls and correlated with immune activation markers. The results from this study suggest that exosomes in antiretroviral therapy-treated HIV patients carry proteins related to immune activation and oxidative stress, have immunomodulatory effects on myeloid cells, and may have pro-inflammatory and redox effects during pathogenesis. “This reference shows that while exosomes are large hydrophobic vesicles, separating them from surrounding protein content, can be difficult. Nevertheless, AlbuSorb™ is demonstrated here to be useful in depleting albumin and enriching exosomes. Thus LC-MS/MS proteomic analysis of the exosomes is not compromised by interference from serum albumin.” states Swapan Roy, Ph.D., President and Founder of Biotech Support Group.
Urine
Zubiri, Irene, et al. "Diabetic nephropathy induces changes in the proteome of human urinary exosomes as revealed by label-free comparative analysis." Journal of proteomics 96 (2014): 92-102.
Urine exosome isolation via ultracentrifugation followed by albumin depletion provides researchers with knowledge of renal regulation and could lead to the identification of biomarkers for diabetic nephropathy and diabetic mellitus. By removing albumin using AlbuSorb™ authors identified more urinary proteins such as flotillin-2, lamp-1, PODXL, tsg-101 from exosome fractions of urine samples. Upon depletion of high abundance proteins such as albumin, urine exosomes from diabetic and health controls were analyzed by LC-MS/MS and selected reaction monitoring (SRM). The research cites AMBP, MLL3, VDAC1 as proteins in urinary exosomes of diabetic nephropathy patients.
Cerebrospinal Fluid
Gwenael Pottiez, Pawel Ciborowski. Proteomic Profiling of Cerebrospinal Fluid Expression Profiling In Neuroscience. Neuromethods.2012;64:245-270
Authors Pottiez et al published a chapter in the book Expression Profiling in Neuroscience, Neuromethods titled, Proteomic Profiling of Cerebrospinal Fluid, on proteomic profiling platforms which analyze cerebrospinal fluid (CSF) for protein biomarkers and developing protein profiles of CSF for early identification of neurological diseases. Authors provide examples of affinity-based systems for removing most abundant proteins and cite Albusorb™ albumin depletion kit from Biotech Support Group. Moreover variations of protein concentration yielded by immunodepletion of CSF samples from nondemented (ND) patients and patients with HIV-associated dementia (HAD) are recorded.
Synovial fluid
Happonen, K. E., Fürst, C. M., Saxne, T., Heinegård, D., & Blom, A. M. (2012). PRELP protein inhibits the formation of the complement membrane attack complex. Journal of Biological Chemistry, 287(11), 8092-8100.
Approximately 65% of the total protein in normal synovial fluid consists of human serum albumin. Authors cite AlbuSorb™ from Biotech Support Group for albumin depletion from synovial fluid for detecting and measuring Proline arginine rich end leucine-rich repeat protein (PRELP). Scientists demonstrated that PRELP inhibits MAC by decreasing C9 polymerization, thereby preventing limiting complement attack on basement membranes. Proper identification of low-abundance proteins in synovial fluid that may prevent disease biomarkers discovery is enhanced by albumin depletion. After using Albusorb™, synovial fluid proteins on two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is studied. The ultimate goal of such application would be to comparatively find and analyze novel biomarkers of rheumatoid arthritis
Serum or plasma
Åvall, Karin. "In vivo and in vitro studies of apolipoprotein CIII in diabetes." (2017).
apoCIII can be influenced by insulin, PPAR activation, FXR activation, hyperglycemia, free fatty acids, NF-KB activation pathways.Sample preparation methods to treat insulin resistance, diabetes and obesity progress research on the discovery of diabetogenic factors. Insulin deficiency and insulin resistance can alter apoCIII gene expression, a marker linked to apoptosis and inflammation. High apoCIII in type 2 diabetes and islet function deterioration can be research areas linked to dyslipidemia. To progress diabetes research, albumin was depleted from serum or plasma to progress protein concentration research. ApoCIII, produced in the pancreatic islet, might be elevated in type 2 diabetes and can promote hypertriglyceridemia.
Serum
Roy, Swapan, and Matthew Kuruc. "Methods to Monitor the Functional Subproteomes of SERPIN Protease Inhibitors." Functional Proteomics. Humana Press, New York, NY, 2019. 41-54
Biotech Support Group contributed the chapter, “Methods to Monitor the Functional Subproteomes of SERPIN Protease Inhibitors,” Although SERPINs are found in a variety of functional on/off sub-forms, customarily they are observed and reported in aggregate. As a result, their influence on disease is missed. This chapter describes Albumin depletion products AlbuVoid™ & AlbuSorb™ and and demonstrates their utility to satisfy investigations of serum SERPINs at a functional level.
Berggren, Per-olof, and Lisa Juntti-berggren. "Methods for treating and/or limiting development of diabetes." U.S. Patent No. 20,170,037,118. 9 Feb. 2017.
In brief, the inventors disclose methods and compositions for limiting development of and/or treating diabetes, involving compounds of formula A-B, wherein A is a pancreatic β cell targeting moiety, and B is an inhibitor of expression and/or activity of Apolipoprotein CIII (apoCIII), protein kinase A (PKA), Src kinase, and/or β1 integrin. The patent states “…to evaluate the levels of apoCIII in sera, albumin is removed from serum samples using standard techniques, such as … AlbuSorb™ (Biotech Support Group)”. The inventors describe that ApoCIII can be identified using any suitable technique, including but not limited to MALDI mass spectrometry.
Swapan Roy, Matthew Kuruc. The Functional Subproteomes of Serpin Protease Inhibitors are Now Open for LC-MS Biomarker Discovery. MOJ Proteomics Bioinform 2016, 3(6): 00106
The authors consider that the conformational variants of the unique family of protease inhibitors annotated as SERPINs, are most often underrepresented in proteomic analyses. This limits understanding the complex regulation that this family of proteins presents to the networks within the protease web of interactions. Using bead-based separation provided by the NuGel™ family of proteomic enrichment products - notably AlbuVoid™ & AlbuSorb™, the authors demonstrate their utility to satisfy investigations of serum SERPINs. The authors also suggest their use to develop functional profiles of the SERPIN Proteoform, and how those can establish relationships to disease phenotypes, gene mutations, and deregulated mechanisms.
Application Report. AlbuSorb™ Product Extension combines Albumin and Immunoglobulin Depletion in a Consumable Format
From a foundational NuGel™ platform chemistry, a library of bead architectures has been created to support proteomic enrichment. These beads are general non-specific protein adsorbents, or stated another way - beads with weak affinity or imperfect fit interactions. Two of our products support Albumin Removal: AlbuSorb™ for selective binding of Albumin & AlbuVoid™ for negative selection or voidance of Albumin with consequent enrichment of the remaining serum proteome on the bead. We now report on adding Immunoglobulin depletion as an extension to AlbuSorb™. A LC-MS/MS analysis on human serum revealed between 500-600 total proteins, many of which are qualitatively and quantitatively biased to sub-proteomes either depleted of Immunoglobulins, or not depleted of Immunoglobulins. AlbuSorb™ PLUS designates and distinguishes AlbuSorb™ without immunoglobulin depletion, from AlbuSorb™ with Immunoglobulin depletion. Here we compare proteomic data derived from AlbuSorb™, AlbuSorb™ PLUS (which includes Immunoglobulin depletion), and AlbuVoid™.
Holmberg, Rebecka, Essam Refai, Anders Höög, Rosanne M. Crooke, Mark Graham, Gunilla Olivecrona, Per-Olof Berggren, and Lisa Juntti-Berggren. "Lowering apolipoprotein CIII delays onset of type 1 diabetes." Proceedings of the National Academy of Sciences 108, no. 26 (2011): 10685-10689
The glycoprotein Apolipoprotein C-III (apoCIII) inhibits lipolysis and its expression is documented to play a vital role in the development of hypertriglyceridemia when increased. The aim of this study was to determine apoCIII’s increased levels in serum in T1D patients and that it affects the function and survival of pancreatic β cells in vitro. Authors Holmberg et al cite Albusorb™ from Biotech Support Group to remove albumin from rat serum samples. Scientists discovered that treating prediabetic animals with an antisense against apo CIII prolonged diabetes onset.
Tang MX, Ogawa K, Asamoto M. Effects of Nobiletin on PhIP-Induced Prostate and Colon Carcinogenesis in F344 Rats Nutrition and Cancer.2011;63(2):227-33
Authors showed how 0.05% citrus flavonoid nobiletin inhibited PhIP-induced rat prostate and colon carcinogenesis. Albusorb™ was used for albumin depletion from serum samples to enable leptin expression. Following this, serum samples were diluted and denatured in the presence of sodium dodecyl sulfate and 2-mercaptoethanol by heating at 100◦C for 5 min. Then, proteins in each sample were electrophoretically. To prevent, nonspecific binding on the membranes 5% skim milk at room temperature for 1 h, followed by incubation with a polyclonal rabbit antileptin antibody allowed for leptin expression by western blot.
Holmberg, Rebecka Apolipoprotein CIII and Ljungan virus in diabetes 2010. Doctoral Thesis
Lowering the levels of apolipoprotein CIII is beneficial to prevent development of type 1 diabetes. Albusorb™ was used on isolated islet cells from sera in prediabetic rats undergoing antisense treatment. Albusorb™ removed >90% albumin from serum samples. The method involved adding serum to a binding buffer with Albusorb™ powder followed by mixing and centrifugation. The supernatant was collected, freeze dried in 100ml 0.1% TFA and run on ACE C18 column 20-60%. The apolipoprotein CIII elutes were analyzed with by area under the curve measurements. Analysis of apolipoprotein CIII was done using MALDI mass spec.
Lu Q, Zheng X, McIntosh T Development of different analysis platforms with LC-MS for pharmacokinetic studies of protein drugs. Analytical Chemistry.2009;81(21):8715-23
Using Albusorb™'s albumin depletion method first and then digest the depleted albumin solution (flow through fraction) for the subsequent LC-MS analysis of peptides, either 1-dimensional LC or 2-dimensional LC (ion exchange and reversed phase) with MS analysis. In this paper, authors use AlbuSorb™ from Biotech Support Group in a sample of serum (i.e., 30 μL) containing the protein drug along with a binding buffer provided (i.e., 250 μL) and then 40 mg of Albusorb™ powder is added in a spin-tube. At room temperature, the sample was mixed for 5-10 min on a rotating shaker, the spin-tube was centrifuged for 2 min, and the supernatant was collected for further analysis.
