Sampling human indigenous saliva peptidome using a lollipop-like ultrafiltration probe: Simplify and enhance peptide detection for clinical mass spectrometry

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題名:	Sampling human indigenous saliva peptidome using a lollipop-like ultrafiltration probe: Simplify and enhance peptide detection for clinical mass spectrometry
作者:	黃俊銘;Zhu, Wenhong;Gallo, Richard L.;Huang, Chun-Ming
貢獻者:	生醫理工學院生醫科學與工程學系
關鍵詞:	Adult;centrifugation;Chromatography, Liquid - methods;digestion;disease course;Female;gels;Humans;JoVE Medicine;liquid chromatography;Male;mass spectrometry;Medicine;Membranes, Artificial;monitoring;mouth;pathogens;peptides;Polymers;prediction;proteins;proteome;proteomics;saliva;Saliva - chemistry;Salivary Proteins and Peptides - analysis;Salivary Proteins and Peptides - chemistry;sampling;Sulfones;Tandem Mass Spectrometry - methods;therapeutics;trypsin;two-dimensional gel electrophoresis;ultrafiltration;Ultrafiltration - instrumentation;Ultrafiltration - methods;Young Adult
日期:	2012-08-07
上傳時間:	2026-04-23 11:20:33 (UTC+8)
出版者:	MYJoVE Corporation;United States: MyJove Corporation
摘要:	摘要： Although human saliva proteome and peptidome have been revealed 1-2 they were majorly identified from tryptic digests of saliva proteins. Identification of indigenous peptidome of human saliva without prior digestion with exogenous enzymes becomes imperative, since native peptides in human saliva provide potential values for diagnosing disease, predicting disease progression, and monitoring therapeutic efficacy. Appropriate sampling is a critical step for enhancement of identification of human indigenous saliva peptidome. Traditional methods of sampling human saliva involving centrifugation to remove debris 3-4 may be too time-consuming to be applicable for clinical use. Furthermore, debris removal by centrifugation may be unable to clean most of the infected pathogens and remove the high abundance proteins that often hinder the identification of low abundance peptidome.Conventional proteomic approaches that primarily utilize two-dimensional gel electrophoresis (2-DE) gels in conjugation with in-gel digestion are capable of identifying many saliva proteins 5-6. However, this approach is generally not sufficiently sensitive to detect low abundance peptides/proteins. Liquid chromatography-Mass spectrometry (LC-MS) based proteomics is an alternative that can identify proteins without prior 2-DE separation. Although this approach provides higher sensitivity, it generally needs prior sample pre-fractionation 7 and pre-digestion with trypsin, which makes it difficult for clinical use. To circumvent the hindrance in mass spectrometry due to sample preparation, we have developed a technique called capillary ultrafiltration (CUF) probes 8-11. Data from our laboratory demonstrated that the CUF probes are capable of capturing proteins in vivo from various microenvironments in animals in a dynamic and minimally invasive manner 8-11. No centrifugation is needed since a negative pressure is created by simply syringe withdrawing during sample collection. The CUF probes combined with LC-MS have successfully identified tryptic-digested proteins 8-11. In this study, we upgraded the ultrafiltration sampling technique by creating a lollipop-like ultrafiltration (LLUF) probe that can easily fit in the human oral cavity. The direct analysis by LC-MS without trypsin digestion showed that human saliva indigenously contains many peptide fragments derived from various proteins. Sampling saliva with LLUF probes avoided centrifugation but effectively removed many larger and high abundance proteins. Our mass spectrometric results illustrated that many low abundance peptides became detectable after filtering out larger proteins with LLUF probes. Detection of low abundance saliva peptides was independent of multiple-step sample separation with chromatography. For clinical application, the LLUF probes incorporated with LC-MS could potentially be used in the future to monitor disease progression from saliva. 其他題名： J Vis Exp 出版者： United States: MyJove Corporation 出版日期： 2012-08-07 出處： Journal of Visualized Experiments, 2012-08 (66), p.e4108 版權： Copyright © 2012, Journal of Visualized Experiments 版權： Copyright © 2012, Journal of Visualized Experiments 2012 識別號： ISSN: 1940-087X 識別號： EISSN: 1940-087X 識別號： DOI: 10.3791/4108 識別號： PMID: 22895356
顯示於類別:	[生醫科學與工程學系] 期刊論文

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