Modern Mass Spectrometry Combined with Principal Component Analysis for Rapid Characterization of Bacterial Species
| Autor: | Shiang-jiun Lin, 林香君 |
|---|---|
| Rok vydání: | 2016 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 104 Recently, mass spectrometric profiling has been applied to distinguish different bacterial species through fingerprinting. The most successful example is using matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF/MS) to detect lipid/protein components released from bacteria for classification. Ambient mass spectrometry has dramatically reduced analysis time for organic and biological compound analysis, as the method requires minimum or no sample pretreatment. Its most notable advantages are the abilities to manipulate samples under atmospheric conditions for high-throughput analysis, and fast sample replacement. Herein, a new strategy that combines thermal desorption electrospray ionization mass spectrometry (TD-ESI/MS), an ambient mass spectrometric technique, with principal component analysis (PCA) was developed to classify various bacterial species based on the lipid profiles obtained by TD-ESI/MS. The biological compounds in the cultures of twelve species of bacteria (six gram positive and six gram negative) were collected and analyzed using TD-ESI/MS and MALDI-TOF, respectively. The TD-ESI source comprised of a metal sampling probe, thermal desorption unit, and electrospray ionization interface was coupled to a triple quadrupole mass spectrometer for MS analyses. The lipid ions detected in each bacterial species by TD-ESI/MS were analyzed by PCA. In addition, the bacterial cells were extracted with organic solvents to obtain peptide/protein profiling with MALDI-TOF/MS. The lipid and protein profiles were further processed by principal component analysis (PCA).To directly collect single colonies on the petri dish, a stainless steel inoculating loop and an acupuncture needle were tested to generate the optimal mass spectrum by TD-ESI/MS. As a result, memory effects were mostly avoided with an acupuncture needle acting as a very small sampling probe to collect the colonies. An acupuncture needle was also used to determine the reproducibility of the tests, with the relative standard deviation (RSD) resulting in values below 7.11 % (n=10). A number of lipid ion peaks distributed between m/z 400-1000 were detected from all of the single colonies. Lipopolysacchrides, characteristic biomarkers for gram negative bacteria, were detected at m/z 550-800. Other lipids include diacylglycerols, triacylglycerols, phosphocholines, and sphingomyelin were also detected. The representative TD-ESI mass spectra were processed by PCA to successfully classify 12 bacterial species. In addition, analyses of homogeneous bacteria were performed by MALDI-TOF/MS at the range of m/z 2000-20000 (peptide/protein profiling) and m/z 400-1500 (lipid profiling). The representative mass spectra were analyzed by PCA to assist the classification of bacterial species. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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