DEVELOPMENT OF A LC/MS/MS ENZYME METHOD FOR N8 - ACETYLSPERMIDINE MEASUREMENTS IN ENZYME ASSAYS

<p>This thesis describes the development of a way to study the N⁸ - acetylspermidine deacetylase enzyme activity. The method created in this thesis emphasizes sensitivity, accuracy and safety.</p> <p>In this study, HeLa cells were cultured and extracted to yield a crude N⁸ - acetylspermidine deacetylase enzyme mixture. By measuring the decrease of N⁸ - acetylspermidine and the increase of spetmidine, N⁸ -acetylspermidine deacetylase enzyme activity can be determined using either a Varian 1200L LC/MS/MS or an API 3000 LC-ES (+)/MS/MS. An acetylation-derivatization method was developed because N⁸ -acetylspermidine and spermidine are hard to purify from a biological sample since they are not retained on a CIS solid phase extraction column or on a RP HPLC (high performance liquid chromatography) reverse phase column due to their small molecular weight and high polarity.</p> <p>The quantitation of N⁸ -acetylspem1indine over the range 2ng/ul to 5pg/ul was fit by linear regression as y = 1.064x + 0.218 with an R-squared value of 0.9996, where y is the peak area of the fragment-ion SRM (selected reaction monitoring: <em>m/z</em>: 188/114) chromatograms from N⁸ -acetylspermindine and x is the concentration of N⁸ - acetylspermindine. Acetylation of spermidine (SPD) and N⁸-acetylspermidine (N8AcSPD) with <em>d6</em>-acetic anhydride produces the <em>d9</em> labeled triacetylated derivative of SPD and <em>d6 </em>labled triacetylated spermidine derivative of N8AcSPD. These triacetylated forms are retained on a C18 column. MS/MS gives characteristic <em>m/z</em> fragment ions for the derivatized species: N8AcSPD (278 to 215), NlAcSPD (278 to 218) and SPD (281 to 218). The fragment-ion SRM (selected reaction monitoring) chromatograms are used for the quantitation. A plot of peak area ratios for known mixtures of N8AcSPD and total SPD versus the molar ratios of N8AcSPD and total SPD was found to fit a linear regression line withy= 0.705x + 0.035 with an R-squared value of 0.9919. Quantitation of d_6- and d_g-tri-acetylspermidine by LC/MS/MS is possible at the low levels of materials found in cell extracts since the separation method results in a lower limit of quantitation. This approach enables the study of N⁸ - acetylspermidine deacetylase enzyme activity.</p>

Name thesis:degree_name

Master of Science (M.S.)

Level thesis:degree_level

Thesis - Pacific Access Restricted

Discipline thesis:degree_discipline

Pharmaceutical and Chemical Sciences

Year dc:date.available

2005

Author dc:creator

• Tang, Jennifer Huiqin

Contributors dc:contributor

• Patrick R. Jones

• Enzyme-linked immunosorbent assay
• Enzymatic analysis
• Medicinal and Pharmaceutical Chemistry
• Medicine and Health Sciences
• Pharmacy and Pharmaceutical Sciences

Licence

• http://rightsstatements.org/vocab/InC/1.0/

Repository record dc:identifier

https://scholarlycommons.pacific.edu/uop_etds/607

OAI identifier oai:identifier

oai:scholarlycommons.pacific.edu:uop_etds-1606