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. Authors Zubiri et al published an article in the Journal of Proteomics which cites AlbuSorb™ for high abundance protein depletion. Titled, 'Diabetic nephropathy induces changes in the proteome of human urinary exosomes as revealed by label-free comparative analysis', the article cites protocols of proteomic workflows for biomarker discovery involving isolating exosomes from urine. Proteinuria in urine samples causes contamination of urine samples for proteomics research. Sample preparation protocols with depletion could complement precipitation techniques as less contamination is present in exosomal fractions upon depletion, enrichment, concentration and sample clarification. Urine exosome isolation via ultracentrifugation upon performing depletion provides researchers with knowledge of renal regulation and could lead to the identification of biomarkers for diabetic nephropathy and diabetic mellitus. 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. Detailed protocols are provided on analysis of:
Protein based profiling of intact proteins in CSF: Gel analysis by two-dimensional gel electrophoresis with DIGE technology and protein identification by tandem mass spectrometry of in-gel-digested protein spots. Next database searches, statistical analysis and validation follows.
Peptide based profiling of CSF:Tryptic digestion of samples followed by implementing isobaric tags for relative and absolute quantitation (iTRAQ) technology for tagging and labeling peptides and measuring changes of protein content according to ratios of peptides. Next database searches, statistical analysis and validation follows.
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 Kaisa E. Happonen et al published an article titled, "PRELP inhibits the formation of the complement membrane attack complex" in the Journal of Biological Chemistry which cites 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. Abundant proteins HSA and IgG are depleted using Albusorb™, followed by size-exclusion chromatography (SEC) of intact proteins prior to preparation of samples for analysis by LC/LC-MS/MS. Next tandem mass spectrometry (MS/MS), coupled with multidimensional liquid chromatography (LC) and database searching is effective technique for protein identification and characterization. Examples of elevated biomarker candidates which are elevated in erosive or nonerosive rheumatoid arthritis (RA) are G3PDH,Peptidylprolyl isomerase, Cystatin B, Phosphoglycerate mutase 1, α2-plasmin inhibitor, S100A8 (calgranulin A), IgG1H Nie, Thymosin-β4. The ultimate goal of such application would be to comparatively find and analyze novel biomarkers of rheumatoid arthritis and search for the same biomarkers in synovial fluid of disease and non disease patient samples by proteomic techniques.
Serum
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 published an article titled, “Lowering apolipoprotein CIII delays onset of type 1 diabetes” in the journal Proceedings of the National Academy of Sciences in which Albusorb™ from Biotech Support Group is used to remove albumin from rat serum samples. Scientists setup experiments implementing the animal model diabetes-prone BB rat (DPBB) to ascertain if apoCIII increases contributed to calcium increase and B-cell death in vivo. To evaluate the levels of apoCIII in pos, neg, and control sera, scientists used Albusorb™ (Biotech Support Group) to remove albumin from serum samples. Proteins from the freeze-dried collected samples were eluted and dissolved in 100 μL 0.1% TFA. Area under curve on an ACE C18 10- × 0.21-cm column 20–60% where apoCIII elutes evaluated and ApoCIII was identified by MALDI mass spectrometry. 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.
