Dataset Information

Characterization of Polyamidoamino (PAMAM) Dendrimers Using In-Line Reversed Phase LC Electrospray Ionization Mass Spectrometry.

ABSTRACT: Generation 3 (G3) PAMAM dendrimers are symmetrical, highly branched polymers widely reported in the scientific literature as therapeutic agents themselves or as carrier scaffolds for various therapeutic agents. A large number of analytical techniques have been applied to study PAMAM dendrimers, but one that has been missing is in-line reversed phase LC electrospray ionization mass spectrometry (RP/LC/ESI/MS). To translate PAMAM dendrimers into therapeutic agents, a better understanding of their purity, stability and structure is required, and in-line RP/LC/ESI/MS is widely applied to all three of these analytical questions. In this study, we developed a robust in-line RP/LC/ESI/MS method for assessing stability, purity and structure of the G3 PAMAM dendrimers, and we also examined the reasons why previous attempts at method development failed. Using the RP/LC/ESI/MS method we uncovered several unique aspects of the chemistry of G3 PAMAM dendrimers. They are interconverted between two isomeric forms by dialysis, and under higher concentration levels there is an inter-molecular displacement reaction resulting, which degrades PAMAM dendrimers. Purification of G3 dendrimers by RP/LC was also previously unreported; so we slightly modified the LC/MS method for isolating individual components from a complex dendrimer mixture. Thus, we have developed a robust, comprehensive method for characterizing PAMAM dendrimers and their degradation.

SUBMITTER: Lloyd JR

PROVIDER: S-EPMC4792036 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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Characterization of Polyamidoamino (PAMAM) Dendrimers Using In-Line Reversed Phase LC Electrospray Ionization Mass Spectrometry.

Lloyd John R JR Jayasekara P Suresh PS Jacobson Kenneth A KA

Analytical methods : advancing methods and applications 20151207 2

Generation 3 (G3) PAMAM dendrimers are symmetrical, highly branched polymers widely reported in the scientific literature as therapeutic agents themselves or as carrier scaffolds for various therapeutic agents. A large number of analytical techniques have been applied to study PAMAM dendrimers, but one that has been missing is in-line reversed phase LC electrospray ionization mass spectrometry (RP/LC/ESI/MS). To translate PAMAM dendrimers into therapeutic agents, a better understanding of their ...[more]

PMID: 26997980

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Project description:We present the first comprehensive capillary electrophoresis electrospray ionization mass spectrometry (CESI-MS) analysis of post-translational modifications derived from H1 and core histones. Using a capillary electrophoresis system equipped with a sheathless high-sensitivity porous sprayer and nano-liquid chromatography electrospray ionization mass spectrometry (nano-LC-ESI-MS) as two complementary techniques, we characterized H1 histones isolated from rat testis. Without any pre-separation of the perchloric acid extraction, a total of 70 different modified peptides, including 50 phosphopeptides, were identified in the rat linker histones H1.0, H1a-H1e, and H1t. Out of the 70 modified H1 histone peptides, 27 peptides could be identified with CESI-MS only, and 11 solely with LC-ESI-MS. Immobilized metal-affinity chromatography enrichment prior to MS analysis yielded a total of 55 phosphopeptides; 22 of these peptides could be identified only by CESI-MS, and 19 only by LC-ESI-MS, showing the complementarity of the two techniques. We mapped 42 H1 modification sites, including 31 phosphorylation sites, of which 8 were novel sites. For the analysis of core histones, we chose a different strategy. In a first step, the sulfuric-acid-extracted core histones were pre-separated using reverse-phase high-performance liquid chromatography. Individual rat testis core histone fractions obtained in this way were digested and analyzed via bottom-up CESI-MS. This approach yielded the identification of 42 different modification sites including acetylation (lysine and N(α)-terminal); mono-, di-, and trimethylation; and phosphorylation. When we applied CESI-MS for the analysis of intact core histone subtypes from butyrate-treated mouse tumor cells, we were able to rapidly detect their degree of modification, and we found this method very useful for the separation of isobaric trimethyl and acetyl modifications. Taken together, our results highlight the need for additional techniques for the comprehensive analysis of post-translational modifications. CESI-MS is a promising new proteomics tool as demonstrated by this, the first comprehensive analysis of histone modifications, using rat testis as an example.

Dataset Information

Characterization of Polyamidoamino (PAMAM) Dendrimers Using In-Line Reversed Phase LC Electrospray Ionization Mass Spectrometry.

Publications

Characterization of Polyamidoamino (PAMAM) Dendrimers Using In-Line Reversed Phase LC Electrospray Ionization Mass Spectrometry.

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