In The February Issue:
- New Survey: Sample Preparation for Mass Spectrometry
- Two New Scientists Join Base Pair
- Aptamers for Affinity Purification
New Survey: Sample Prep for Mass Spec
Base Pair is currently developing aptamer-based pull-down assays for purification or depletion of specific analytes.
We’d like to learn more about current challenges in sample preparation for mass spectrometry. Please take this brief, 10-question survey and tell us about your experience with mass spectrometry. Each respondent will be entered to win a $25 Starbucks gift card.
Base Pair Welcomes Pamela Lockyer and Alfred Daramola
Base Pair is excited to welcome Pamela Lockyer in our newly-created Senior Operations Associate position. After receiving her Bachelor of Science at the University of Central Arkansas, Pamela studied osteoporosis and metabolic bone diseases at the University of Arkansas for Medical Sciences (UAMS) As a graduate student at the University of North Carolina, Pamela worked on several projects related to the role of signaling pathways during cardiovascular inflammation and regulation of cardiomyocyte response by the ubiquitin-proteasome system (UPS) following ischemia/reperfusion injury and heart failure.
“I’m very excited about joining Base Pair Biotechnologies and look forward to assisting customers doing exciting work with aptamers.” –Pamela Lockyer
Base Pair is also pleased to welcome Alfred Daramola as an intern Associate Scientist. Alfred is a fall 2017 graduate of biotechnology from the University of Houston-Clear Lake (UHCL), where he was a teaching assistant in molecular genetics and studied functional enzymes from the toluene catabolism pathway in Pseudomonas Putida. Alfred is from Lagos, Nigeria where he received his BS in biochemistry studying the effects of herbal remedies used in treating skin rash, a side effect of the HIV drug Nevirapine.
Aptamers for Affinity Purification
Affinity purification involves the use of selective binding to remove targets of interest or contaminants from a complex sample. It is widely used to prepare reagents for use in research, diagnostics, and pharmaceuticals. In many cases affinity chromatography is performed, with the selective binding partner immobilized on a chromatographic support. While a wide range of affinity ligands, including antibodies and recombinant proteins, are commonly used, there are a growing number of studies employing aptamers. Affinity products are designed to maximize specificity, binding capacity, and yield. At the same time, they must be resistant to contamination and harsh sterilization procedures.
Aptamer Advantages in Affinity Purification
Aptamers offer several key advantages in affinity purification. Due to their small size (about 1/10th the size of an antibody), aptamers can be more densely bound to a support, increasing binding capacity. While antibodies and recombinant proteins are often produced via cell culture, aptamers are chemically synthesized, eliminating potential viral, bacterial, or endotoxin contamination. Chemical synthesis eliminates the lot-to-lot variability associated with biologicals and reduces the cost for large-scale production. Nucleic acid aptamers are not susceptible to proteases and can be easily bound to a wide range of materials without affecting target selectivity. Aptamers can be selected to differentiate between highly similar molecules or to bind proteins with specific post-translational modifications. They can also be selected for binding and dissociation in desired sample and elution conditions (2,4).
Isolation/Purification of Target from Complex Samples
Isolating a single target from a complex sample can be challenging and is often a multi-step process with increasing specificity for the desired target. Preliminary studies have demonstrated the potential utility of aptamers for single-step purification from complex samples. Researchers in Germany purified Vascular Endothelial Growth Factor (VEGF121) using aptamers covalently immobilized on magnetic beads. The aptamer was selective for the receptor-binding domain of VEGF121, enabling recognition of all isoforms of the protein. More than 75% of all bound VEGF was eluted with a NaCl salt solution. (4) Researchers in France utilized DNA aptamers in affinity chromatography to independently isolate three different serum proteins; Factor VII, Factor H, and Factor IX. The specificity, binding capacity, and purity achieved matched or exceeded the performance of current methods. The aptamer-based columns also maintained performance following repeated treatment with 1M sodium hydroxide (2). Researchers in Japan purified IgG from human serum using an Fc aptamer resin. Both capacity and purity were comparable to Protein A. The Fc aptamer was also successful in the purification of several therapeutic antibodies from CHO culture supernatant. Elution was performed with neutral buffers containing 10mM EDTA or 0.2M KCl. The aptamer resin was regenerated using 6M urea or heating at 85°C for 5 minutes in pure water (5).
Removal of Contaminants
Just as aptamers can be used to purify a single analyte of interest from a complex sample, they can also be used to remove contaminants for purification of a solution. Aptamers have been used to remove arsenite and arsenate from groundwater and to remove uranium from radioactive waste water. They have also been used successfully to remove specific bacteria, including e. coli, and bacterial toxins from soil, water, and food. Aptamers can also be used to remove specific high-abundance proteins or contaminants, such as viruses, from blood samples (1).
Purification for Mass Spectrometry in Biomarker Discovery
Whole-cell SELEX, or the selection of aptamers against a specific cell type, is increasingly being used to discover new cell-surface biomarkers. Once an aptamer against a specific cell type is selected, it is then used as an affinity ligand to capture the cell surface target from a cell lysate. Following isolation and digestion, mass spectrometry can be utilized to identify the unknown target. Researchers at the University of Florida utilized a T-cell acute lymphoblastic leukemia cell line for whole cell SELEX. One of the selected aptamers showed strong selectivity for leukemia cells. Biotinylated aptamer and magnetic beads were used to capture the aptamer-target complex from a leukemia cell lysate. Proteins were eluted and analyzed by SDS-PAGE. Aptamer-purified protein bands were digested and analyzed by LC-MS/MS to identify the leukemia cell target as protein tyrosine kinase-7, or PTK7 (E). Researchers in China recently used a similar procedure involving 58f cell lysates and aptamer-bead complexes to isolate an unknown target protein for an aptamer to nasopharyngeal carcinoma cells. Following gel and mass spectrometry analysis they identified CD109 as a potential biomarker for nasopharyngeal carcinoma (3).
Custom Aptamer Selection for Purification & Pull-Down
Base Pair has developed aptamers for a wide range of targets including small molecule drugs, antibodies, proteins, peptides, and many other types of targets. Selection strategies can be designed to capture target proteins in complex sample matrices for purification or depletion.
Contact Base Pair today for more information on selection of aptamers for applications involving pull-down of proteins, antibodies, small molecules, and other targets.
References:
1. Bilibana, M.P., et al. Aptamers as the agent in decontamination assays (Apta-Decontamination Assays): from the environment to the potential application in vivo. Journal of Nucleic Acids. 2017. Article ID 3712070
2. Forier, C., et al. DNA aptamer affinity ligands for highly selective purification of plasma-related proteins from multiple sources. Journal of Chromatography A. 2017. 1489:39-50.
3. Jia, W., et al. CD109 is identified as a potential nasopharyngeal carcinoma biomarker using aptamer selected by cell-SELEX. 2016. 7(34).
4. Lonne, M., et al. Development of an aptamer-based affinity purification method for vascular endothelial growth factor. Biotechnology Reports 2015. 8:16-23.
5. Miyakawa, S., et al. Structural and molecular basis for hyperspecificity of RNA aptamers to human immunoglobulin G. 2008. RNA. 14(6):1154-1163.
6. Shangguan, D., et al. Cell-specific aptamer probes for membrane protein elucidation in cancer cells. Journal of Proteome Research. 2008. 7(5):2133-2139.