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Applications of 21 Tesla FT-ICR Top-down Proteomics in Clinical Research and Diagnosis

Title: Applications of 21 Tesla FT-ICR Top-down Proteomics in Clinical Research and Diagnosis.
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Name(s): He, Lidong, author
Marshall, Alan G., 1944-, professor directing dissertation
Tang, Hengli, university representative
Dorsey, John G., committee member
Hu, Yan-yan, committee member
Florida State University, degree granting institution
College of Arts and Sciences, degree granting college
Department of Chemistry and Biochemistry, degree granting department
Type of Resource: text
Genre: Text
Doctoral Thesis
Issuance: monographic
Date Issued: 2018
Publisher: Florida State University
Place of Publication: Tallahassee, Florida
Physical Form: computer
online resource
Extent: 1 online resource (149 pages)
Language(s): English
Abstract/Description: With recent progress in clinical proteomics, mass spectrometry (MS)-based methods have been widely implemented in diagnosis of diseases which offers high specificity that conventional clinical tests lack. The recent development of high resolution high mass accuracy mass spectrometry leads to full characterization of intact proteins (e.g., therapeutic monoclonal antibodies and endogenous protein biomarkers) in a top-down MS/MS fashion. Top-down MS/MS offers “birds’ eye” view of the proteins and yields more confident protein sequence assignment and post-translational modifications localization. This dissertation describes the latest top-down applications in disease precision diagnosis that can potentially lead to future personalized treatment. Chapter 2 describes a pilot study for characterization of monoclonal antibodies by top-down and middle-down approaches with the advantages of fast sample preparation with minimal artifacts, ultrahigh mass accuracy, and extensive residue cleavages by use of 21 tesla FT-ICR MS/MS. The ultrahigh mass accuracy yields an rms error of 0.2–0.4 ppm for antibody light chain, heavy chain, heavy chain Fc/2, and Fd subunits. The corresponding sequence coverages are 81%, 38%, 72%, and 65% with MS/MS rms error ~4 ppm. Extension to a monoclonal antibody in human serum as a monoclonal gammopathy model yielded 53% sequence coverage from two nano-LC MS/MS runs. A blind analysis of five therapeutic monoclonal antibodies at clinically relevant concentrations in human serum resulted in correct identification of all five antibodies. Nano-LC 21 T FT-ICR MS/MS provides nonpareil mass resolution, mass accuracy, and sequence coverage for mAbs, and sets a benchmark for MS/MS analysis of multiple mAbs in serum. This is the first time that extensive cleavages for both variable and constant regions have been achieved for mAbs in a human serum background. Chapter 3 describes a novel protein de novo sequencing method given that top-down MS/MS complete sequence coverage is virtually impossible. To characterize the “AA sequence gap” between two adjacent fragments, the number of gap AA sequences with identical masses for di-, tri-, and tetra-AA gaps grows exponentially with increasing number of gap amino acids. If peptide fragment mass could be measured exactly (in practice, to 0.00001 Da), it would then be possible to define the overall atomic composition for the group of amino acids spanning a product ion gap 3-4 amino acids long. I show that de novo top-down/middle-down MS/MS can determine the germline sequence category for a given monoclonal antibody and further serve to identify its novel mutations. Chapter 4 applies my developed top-down protein de novo sequencing in characterization of serum monoclonal immunoglobulins from plasma cell disorders. The current five-year survival rate for systemic AL amyloidosis or multiple myeloma is below 50%, indicating the urgent need for better diagnosis methods and treatment plans. Unlike genomic testing, which requires bone marrow aspiration and may fail to identify all monoclonal immunoglobulins produced by the body, the present method requires only a blood draw. In addition, circulating monoclonal immunoglobulins spanning the entire population are analyzed and reflect the selection of germline sequence by B cells. The monoclonal immunoglobulin light chain FR2-CDR2-FR3 was sequenced by de novo MS/MS and 100% matched the gene sequencing result except for two amino acids with isomeric counterparts, enabling accurate germline sequence classification. This work represents the first application of top/middle-down MS/MS for de novo sequencing of endogenous monoclonal immunoglobulins with polyclonal immunoglobulins background. Chapter 5 is focused on top-down MS/MS diagnosis of hemoglobin disorders. Hemoglobinopathies and thalassemias are the most common genetically determined disorders. Current diagnosis methods include cation exchange high performance liquid chromatography and electrophoresis for screening whose results can be ambiguous because of limited resolving power, and expensive and laborious genetic testing is needed for confirmation. I developed a top-down MS/MS approach with the advantages of fast data acquisition (3 min), ultrahigh mass accuracy, and extensive residue cleavages by use of 21 tesla FT-ICR MS/MS for hemoglobin variant sequence de novo characterization and thalassemia diagnosis. With my developed generic approach for hemoglobin variant de novo sequencing, all eighteen hemoglobin variants were correctly identified in blind analysis which include the first characterization of homozygous hemoglobin Himeji variant. It is the first time that the abundance ratio between intact δ and β subunits (δ/β) is used for beta thalassemia (including beta thalassemia trait/major) screening. Therefore, 21 T FT-ICR MS sets the benchmark for top-down MS/MS analysis of hemoglobin variants and thalassemia.
Identifier: 2018_Su_He_fsu_0071E_14616 (IID)
Submitted Note: A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy.
Degree Awarded: Summer Semester 2018.
Date of Defense: May 25, 2018.
Keywords: antibody, hemoglobin, mass spectrometry, MS/MS, proteomics, top-down
Bibliography Note: Includes bibliographical references.
Advisory Committee: Alan G. Marshall, Professor Directing Dissertation; Hengli Tang, University Representative; John G. Dorsey, Committee Member; Yanyan Hu, Committee Member.
Subject(s): Chemistry
Persistent Link to This Record: http://purl.flvc.org/fsu/fd/2018_Su_He_fsu_0071E_14616
Host Institution: FSU

Choose the citation style.
He, L. (2018). Applications of 21 Tesla FT-ICR Top-down Proteomics in Clinical Research and Diagnosis. Retrieved from http://purl.flvc.org/fsu/fd/2018_Su_He_fsu_0071E_14616