Assessing the Effects of Diurnal Variation on the Composition of Human Parotid Saliva: Quantitative Analysis of Native Peptides Using iTRAQ Reagents
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- 作者：Markus Hardt ; H. Ewa Witkowska ; Sally Webb ; Lindsay R. Thomas ; Scott E. Dixon ; Steven C. Hall ; and Susan J. Fisher
- 刊名：Analytical Chemistry
- 出版年：2005
- 出版时间：August 1, 2005
- 年：2005
- 卷：77
- 期：15
- 页码：4947 - 4954
- 全文大小：679K
- 年卷期：v.77,no.15(August 1, 2005)
- ISSN：1520-6882
文摘
Changes in salivary composition correlate with diseasesusceptibility, disease state, or both. However, use ofsaliva for diagnostic purposes is complicated by the gland-specific effects of circadian rhythm or diurnal variation.We recently characterized a suite of peptides in the
10-kDa fraction of human parotid saliva that includedmany novel species. In this study, we used novel iTRAQlabeling chemistry to investigate possible diurnal effectson peptide generation. We collected samples producedby gustatory stimulation as the ductal secretions at fourtime points under conditions that minimized proteolysis,pooled them according to collection time, and isolated theLMW fractions. Samples collected at each collection timewere derivatized with a different isobaric iTRAQ reagent.The labeled samples were combined, separated by reversed-phase HPLC, co-spotted with matrix on MALDItargets, and analyzed by MALDI TOF/TOF mass spectrometry. With this approach, we achieved relative quantification of the parotid peptides at four time points. Inseveral cases, abundance during the day changed dramatically. iTRAQ tagging improved the efficiency ofMS/MS fragmentation, which in turn allowed the identification of several novel peptides. Our results demonstrated both the utility of this method and the importanceof diurnal effects on the composition of the human parotidsaliva peptidome.
10-kDa fraction of human parotid saliva that includedmany novel species. In this study, we used novel iTRAQlabeling chemistry to investigate possible diurnal effectson peptide generation. We collected samples producedby gustatory stimulation as the ductal secretions at fourtime points under conditions that minimized proteolysis,pooled them according to collection time, and isolated theLMW fractions. Samples collected at each collection timewere derivatized with a different isobaric iTRAQ reagent.The labeled samples were combined, separated by reversed-phase HPLC, co-spotted with matrix on MALDItargets, and analyzed by MALDI TOF/TOF mass spectrometry. With this approach, we achieved relative quantification of the parotid peptides at four time points. Inseveral cases, abundance during the day changed dramatically. iTRAQ tagging improved the efficiency ofMS/MS fragmentation, which in turn allowed the identification of several novel peptides. Our results demonstrated both the utility of this method and the importanceof diurnal effects on the composition of the human parotidsaliva peptidome.