Glycomics and Proteomics Simultaneously (GAPS): A rapid and novel sample preparation pipeline for complex glycoproteins
Inventors: Richard Lee, Hui Zhou
Invention Types: Diagnostic/Prognostic, Research Tool
Research Areas: Urology
Keywords: Assay, Proteomics, ReagentFor More Information Contact: Khunkhun, Rajinder
As one of the most abundant post-translational modification of proteins, glycosylation is involved in numerous biologic functions. Glycoproteins also comprise the vast majority of current clinical protein biomarkers. Additionally, therapeutic proteins, such as monoclonal antibodies (mAbs), are often glycosylated, and represent the most promising sector of new drug development with more than 50% of them being mAbs. In 2009, eight of the top ten best-selling biologics were glycoproteins. In addition, precise manipulation and optimization of the glycosylation of existing therapeutics will result in a flourish of biosimiliar and biobetter biologics in the near future.
As per current regulatory guidelines, both the protein and glycan structure of a biologic must be characterized in-depth because any subtle variation on either may induce significant and unexpected clinical outcomes. The inherent heterogeneity and complexity of glycans often create nearly insurmountable analytical challenges. Improved methods and strategies for interrogating the complex glycoproteins are solely needed.
Recently, Dr. Richard S. Lee and Dr. Hui Zhou in the Department of Urology devised an innovative sample preparation pipeline (GAPS) to capture both glycans and deglycosylated proteins, allowing the characterization of glycans, glycosylation sites, and proteins in a single workflow.||
GAPS is a novel sample preparation pipeline that is specifically designed for glycoproteins. It has the potential to become the standard procedure in the following areas:
• Quality control of therapeutic glycoproteins
• Research and development of biologics
• Biomarker discovery and validation
• Research in proteomics, glycomics, and glycoproteomics
The current methods to analyze glycoproteins have several limitations. They are 1) incapable of recovering both the protein and glycan fractions for analysis; 2) are laborious, inefficient, and time consuming; and 3) are not compatible with complex mixtures. In contrast, the GAPS pipeline provides the following performance and analytical advantages:
• Performed in a single device, minimizing sample transfer and sample loss.
• Increased capability for multiplexing and high-throughput.
• Rapid and efficient:
o 3-4 hours for purified glycoproteins
o 6-7 hours for complex samples (i.e. urine)
• Applicable to a wide range of samples including purified glycoprotein (mAbs), protein mixtures, and complex body fluids (i.e. urine, and plasma).
• Easily integrated into current shotgun-based proteomics pipelines, a promising feature for biomarker discovery and validation.
• Intact glycans are completely recovered without bias, irrespective of their size, structure and complexity
• Glycans are recovered purified, thus, without the need for extra purification, they can be readily analyzed directly by mass spectrometry, or can be further derivatized by per-methylation or reductive fluorophore labeling.
• Provides improved coverage of glycoproteins, offering:
o A significant analytical advantage for peptide mapping of therapeutics or other proteins of interest.
o Improved ability to accurately quantify glycoproteins in complex samples, (such as urine or plasma)
o Unambiguous identification of glycosylation sites, even in complex mixtures, without the need for sugar-enriching techniques.
o The only method that can identify partially glycosylated sites on a large scale.
Licensing and/or sponsored research/collaboration opportunity available
Key Publications: Not yet published
Related Publications: Zhou H, Briscoe AC, Froehlich JW, Lee RS, PNGase F catalyzes de-N-glycosylation in a domestic microwave. Anal. Biochem. 2012, 427(1), 33-35.
IPStatus: Pat. Pend.