New methods for drugs analysis, disease diagnosis, and protein analysis by using modern mass spectrometry are developed in this thesis. In drugs analysis, we develop a rapid assessment of molecular similarity between an extremely complex innovator product and a candidate biosimilar by mass spectrometry. Protease digestion combined with Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry were successfully used to peptides mapping and de novo peptides sequencing. Overall ion signals obtained by MALDI-TOF/MS were processed by two multivariate statistics including principle component analysis (PCA) and hierarchical clustering analysis. Based on the variances of the peptide profile, innovator product and normally synthesized biosimilar were grouped on the PCA score plot, while impure biosimilar or abnormally synthesized biosimilar were distinctly separated. The results of hierarchical cluster analysis also revealed high conformity between innovator product and normally synthesized biosimilar. Abnormally synthesized products, as a set of quality controller, could be discriminated from innovator product favorably. Another quality control strategy developed in this study is building classification model, batches of product is successfully evaluated. Furthermore, the similarity of molecular weight distribution between these complex drugs is determined.
Another target of drug analysis is to develop a vital analytical method to directly detect the peptides synthesized on the resin. Except an organic solvent is transformed to disperse the resin-peptides samples, no other sample pretreatments are required before the MS detection. When using conventional destructive analytical methods to characterize masses of compounds on the solid supports, acid hydrolysis or acid cleavage of the peptide molecules depart from insoluble resin is required. In consequence, side-reactions such as de-blocked or de-protected cause additional fragments in the system, and determination of the intermediates or products are confusing and difficult. Unlike these acid release methods, the molecular weight information of the intact peptide molecules can be obtained in our direct analyses system, and sample consumption is also great reduced. Moreover, our strategy performed the analysis in the ambient environment is more straightforward for real-time monitor reaction and quality control than those techniques in high vacuum system. This direct non-destructive on-line monitoring method would allow following step by step peptide solid-phase synthesis be well quality controlled.
In the disease diagnose part, MALDI-TOF mass spectrometry combined with statistics were used to perform semi-quantitative albuminuria diagnoses. Based on the fact that the contributions of singly, doubly, triply, and quadruply charged albumin ions from the samples were inflected according to the concentration change. The severity of albuminuria of patients can be estimated by 2D peaks distribution (peaks ranking by p-value) or supervised principal component analysis (PCA).
In protein analysis study, we use liquid electrospray laser desorption ionization mass spectrometry (liquid-ELDI/MS) to directly characterized the proteins stored in different solutions containing acids, bases, buffers, organic solvents, or detergents without extra sample pretreatment. A drop of the sample solution was applied on a stainless steel plate., and then the surface of the sample drop was irradiated with a pulsed laser. The laser energy absorbed by the metal plate and surrounding solvent led to the desorption of protein molecules or the formation of fine droplets containing protein molecules. The desorbed protein species were then post-ionized within an electrospray plume to generate the ESI-like protein ions. Since no pH or composition adjustment of the sample solution is needed, this technique is useful for rapid and high throughput screening of the proteins in a solution to check their integrality after storage or prior to further biochemical treatment. In addition,, native and denatured conformation of the proteins can be distinctly examined by using acid-free and organic solvent-reduced ESI solutions in liquid-ELDI.
| Identifer | oai:union.ndltd.org:NSYSU/oai:NSYSU:etd-0725111-224828 |
| Date | 25 July 2011 |
| Creators | Cho, Yi-tzu |
| Contributors | Po-Chiao Lin, Shiuh-Jen Jiang, Jentaie Shiea, Yeung-hau Ho, Wei-Lung Tseng |
| Publisher | NSYSU |
| Source Sets | NSYSU Electronic Thesis and Dissertation Archive |
| Language | Cholon |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lib.nsysu.edu.tw/ETD-db/ETD-search/view_etd?URN=etd-0725111-224828 |
| Rights | not_available, Copyright information available at source archive |
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