Abstract

Next-Generation protein sequencing (NGPS) creates newfound ways of fully identifying every protein species in a single biological organism. It is an effort to use technology to determine proteomic data. The purpose of this research project is to use the current technology to sequence proteins and potentially find treatments for some diseases that are common today. Through NGPS, scientists can identify low abundant proteins including those that go through post-translational modifications (PTM) [1]. NGPS will allow us to fully determine protein sequences from protein samples using mass spectrometry with the ultimate goal of being able to determine the primary sequence of the protein in the given sample [1]. Antibodies are a specific class of proteins that aid our bodies in the immune response. Due to their variability in the complementary-determining region (CDR), NGPS will be used to determine the heavy and light chain sequences [2]. The goal of this technology is to fully determine the primary sequence of a protein in a given sample. The randomness of an antibody’s variable (V), diversity (D), and joining (J) genes (VDJ recombination) makes each protein unique. VDJ recombination refers to the process of T cells and B cells randomly assembling different gene segments. This process allows the antibody to make specific receptors that can recognize different molecules presented on the surface of antigens. Proteases are enzymes that break down proteins and peptides. By using different proteases with varying cutting rules, we can digest the antibody and run it through high mass accuracy determining instrument [1]. NGPS allows us to utilize mass spectrometry technology to measure proteins or polypeptides. Because of these measurements, post-translation modifications, including glycosylation, can be detected, unlike in DNA sequencing technology. Protein sequencing has the opportunity to play a major role in the fight against the COVID-19 outbreak and serve as curative measures for the treatment and Type 2 Diabetes [3]. Proteomics can serve as the basis of vaccine development as well as monitoring treatment. Utilizing techniques such as mass spectrometry could reveal the structure of the virus and ultimately allow for engineered tissues to produce the protein in large amounts in a lab setting. Currently, many companies are utilizing highly sensitized technology to carry out the goals of NGPS. The Oxford Nanopore is a company that uses technology to develop and explore more ways to undergo protein analysis. The methods used by this company involve using protein nanopores to mutate residues in pores to determine the overall sequence. The company utilizes modified aptamers that are drawn to the nanopore current. These aptamers can bind with some, but not all pores, allowing for the differentiation between target and non-target proteins. Nicoya Life Sciences is another company that uses Open Surface Plasmon Resonance (SPR) to detect molecular interactions. SPR uses an analyte (a mobile molecule) to bind to a ligand and observe changes in the refractive index. SPR allows researchers the ability to characterize the binding kinetics and affinities of monoclonal antibodies. SPR is an extremely promising technique to sequence proteins due to its flexibility in being able to work with a variety of molecules including lipids, nucleic acids, cells, viruses, nanoparticles, proteins, antibodies, carbohydrates, and more. The original goal behind NGPS was to establish a method to sequence proteins to aid in the early detection of common diseases such as Type 2 Diabetes. After significant research, it is now known that NGPS can be done in a variety of ways to accomplish a common goal—sequencing proteins and understanding how amino acids affect the human body. In the case of diseased states, NGPS can help researchers find ways to diagnose, treat, and cure diseases early on. Focusing on antibodies allows us to manipulate the body’s immune response to rid the host of pathogens. NGPS, however, is advancing at a much slower rate than anticipated by companies due to its many limitations including not being able to sequence large peptides, difficulties in material and composition of the sample, and needing to label small peptides to begin degradation. Ideally, finding a way to combine the high accuracy and specificity of certain techniques, the ability to detect low abundant proteins in others, and the flexibility of Open SPR would allow researchers and companies to create the standard for NGPS. Creating effective antibodies is precisely why NGPS has such great potential today. Ultimately, I found that as a standalone, Open SPR is the most effective method. However, as the research shows, there are limitations with each method, including Open SPR. The conclusion shows that it is necessary to find a combination of these techniques and create an accurate method, potentially using different technologies, to establish the most effective way to sequence proteins.

Thesis Completion

2021

Semester

Summer

Thesis Chair/Advisor

Schroeder, Kersten

Degree

Bachelor of Science (B.S.)

College

College of Medicine

Department

Burnett School of Biomedical Sciences

Degree Program

Biomedical Sciences

Language

English

Access Status

Open Access

Length of Campus-only Access

1 year

Release Date

2-1-2023

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