Date of Award

1988

Embargo Period

8-1-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College

College of Graduate Studies

First Advisor

Daniel R. Knapp

Second Advisor

Harry S. Margolius

Third Advisor

Kwong-Yok Tsang

Fourth Advisor

Thomas Walle

Fifth Advisor

Mark G. Currie

Sixth Advisor

Lee Chao

Abstract

Dimethylpyrimidylornithyl-N-trifluoroethyl-O-TBDMS (DMPO-N-TFE-O-TBDMS) polyaminoalcohol derivatives of arginyl peptides were evaluated for their utility in peptide analysis by capillary gas chromatography-mass spectrometry (GC-MS). These derivatives were hydrolytically stable and sufficiently volatile for capillary GC-MS analysis of low nanomole amounts of peptides as large as RPPGF. Complete amino acid sequence information was generated under electron impact ionization conditions, including several ions diagnostic for arginine residues, with molecular weight information imparted by the [M-15]+ and [M-57]+ pair of ions characteristic of TBDMS derivatives. Analysis of the sequential intermediate derivatives by FAB-MS facilitated optimization of problematic derivatization steps. As little as 20 picomoles of peptide could be detected by capillary GC-MS-selected ion monitoring, but problems with the reproducibility of the diborane reduction reaction limited its utility for quantitative applications. DMPO-N,O-di-TBDMS polyaminoalcohol derivatives were found to provide similar structural information, with similar reproducibility problems at low levels. A procedure for selectively extracting arginyl peptides using immobilized phenylboronic acid columns exhibited limited binding with numerous co-extracted contaminants, and was judged to be impractical for biological samples. A reverse phase ion pairing liquid chromatographic procedure employing alternating ion pairing reagents successfully isolated 25-35 nanomole amounts of RPP, RPPG and RPPGF from spiked human urine. None of these peptides were detected at this level in unspiked urine. Further studies focused on developing sensitive and reliable means of analyzing RPPGF as the DMPO-N-trifluoroacetyl-O-methyl ester derivative by direct insertion probe MS, thus avoiding the problematic diborane reduction reaction. Under electron impact ionization conditions, this derivative generated an unusually intense molecular ion suitable for low and high resolution selected ion monitoring. Molecular specificity was obtained by employing collisionally activated dissociation linked field selected reaction monitoring mass spectrometry, monitoring the formation of a specific fragment ion from the molecular ion in the first field free region of a double focusing mass spectrometer. With low level samples, the products at each stage of the derivatization were purified by reverse or normal phase HPLC. A 12 nanomole sample of RPPGF isolated from spiked human urine was successfully analyzed and definitively identified by these methods.

Rights

All rights reserved. Copyright is held by the author.

Download

Share

COinS