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.
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.
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: m/z: 188/114) chromatograms from N8 -acetylspermindine and x is the concentration of N8 - acetylspermindine. Acetylation of spermidine (SPD) and N8-acetylspermidine (N8AcSPD) with d6-acetic anhydride produces the d9 labeled triacetylated derivative of SPD and d6 labled triacetylated spermidine derivative of N8AcSPD. These triacetylated forms are retained on a C18 column. MS/MS gives characteristic m/z 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 d6- and dg-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.
","abstract_html":"<p>This thesis describes the development of a way to study the N<sup>8</sup> - 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 N<sup>8</sup> - acetylspermidine deacetylase enzyme mixture. By measuring the decrease of N<sup>8</sup> - acetylspermidine and the increase of spetmidine, N<sup>8</sup> -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 N<sup>8</sup> -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 N<sup>8</sup> -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 N<sup>8</sup> -acetylspermindine and x is the concentration of N<sup>8</sup> - acetylspermindine. Acetylation of spermidine (SPD) and N<sup>8</sup>-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<sub>6-</sub> and d<sub>g</sub>-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 N<sup>8</sup> - acetylspermidine deacetylase enzyme activity.</p>","abstract_has_math":false,"creators":["Tang, Jennifer Huiqin"],"institution":null,"degree_name":"Master of Science (M.S.)","degree_level":"Thesis - Pacific Access Restricted","degree_discipline":"Pharmaceutical and Chemical Sciences","degree_department":null,"school":null,"contributors":["Patrick R. Jones"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2005,"date_issued":"2005-01-01T08:00:00Z","date_published":"2005-01-01T08:00:00Z","updated_at":"2026-07-24T05:36:35Z","subjects":["Enzyme-linked immunosorbent assay","Enzymatic analysis","Medicinal and Pharmaceutical Chemistry","Medicine and Health Sciences","Pharmacy and Pharmaceutical Sciences"],"languages":[],"rights":[],"rights_urls":["http://rightsstatements.org/vocab/InC/1.0/"],"identifier_entries":[]},"links":{"outbound_url":"https://scholarlycommons.pacific.edu/uop_etds/607","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Patrick R. Jones"]},{"key":"dc:creator","label":"Author","values":["Tang, Jennifer Huiqin"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.available","label":"Dc Date Available","values":["2018-06-29T08:49:46Z"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Pharmaceutical and Chemical Sciences"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Thesis - Pacific Access Restricted"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Master of Science (M.S.)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Enzyme-linked immunosorbent assay","Enzymatic analysis","Medicinal and Pharmaceutical Chemistry","Medicine and Health Sciences","Pharmacy and Pharmaceutical Sciences"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:rights","label":"Dc Rights","values":["http://rightsstatements.org/vocab/InC/1.0/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://scholarlycommons.pacific.edu/uop_etds/607"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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.
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.
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: m/z: 188/114) chromatograms from N8 -acetylspermindine and x is the concentration of N8 - acetylspermindine. Acetylation of spermidine (SPD) and N8-acetylspermidine (N8AcSPD) with d6-acetic anhydride produces the d9 labeled triacetylated derivative of SPD and d6 labled triacetylated spermidine derivative of N8AcSPD. These triacetylated forms are retained on a C18 column. MS/MS gives characteristic m/z 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 d6- and dg-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.
"]},{"key":"dc:source","label":"Dc Source","values":["91"]},{"key":"dc:title","label":"Title","values":["DEVELOPMENT OF A LC/MS/MS ENZYME METHOD FOR N8 - ACETYLSPERMIDINE MEASUREMENTS IN ENZYME ASSAYS"]}]}],"canonical_facts":{"dc:contributor":["Patrick R. Jones"],"dc:creator":["Tang, Jennifer Huiqin"],"dc:date.available":["2018-06-29T08:49:46Z"],"dc:description.abstract":["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.
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.
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: m/z: 188/114) chromatograms from N8 -acetylspermindine and x is the concentration of N8 - acetylspermindine. Acetylation of spermidine (SPD) and N8-acetylspermidine (N8AcSPD) with d6-acetic anhydride produces the d9 labeled triacetylated derivative of SPD and d6 labled triacetylated spermidine derivative of N8AcSPD. These triacetylated forms are retained on a C18 column. MS/MS gives characteristic m/z 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 d6- and dg-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.
"],"dc:identifier":["https://scholarlycommons.pacific.edu/uop_etds/607"],"dc:rights":["http://rightsstatements.org/vocab/InC/1.0/"],"dc:source":["91"],"dc:subject":["Enzyme-linked immunosorbent assay","Enzymatic analysis","Medicinal and Pharmaceutical Chemistry","Medicine and Health Sciences","Pharmacy and Pharmaceutical Sciences"],"dc:title":["DEVELOPMENT OF A LC/MS/MS ENZYME METHOD FOR N8 - ACETYLSPERMIDINE MEASUREMENTS IN ENZYME ASSAYS"],"thesis:degree_discipline":["Pharmaceutical and Chemical Sciences"],"thesis:degree_level":["Thesis - Pacific Access Restricted"],"thesis:degree_name":["Master of Science (M.S.)"]},"updated_at":"2026-07-24T05:36:35Z"}