मानव स्वदेशी लार लॉलीपॉप की तरह एक ultrafiltration जांच का प्रयोग Peptidome नमूनाकरण: सरल और क्लीनिकल मास स्पेक्ट्रोमेट्री के लिए पेप्टाइड जांच बढ़ाएँ
Summary
भविष्य नैदानिक आवेदन के लिए लार के नमूने को ध्यान में रखते हुए, लॉलीपॉप की तरह एक ultrafiltration जांच (LLUF) के मानव मौखिक गुहा में फिट था गढ़े. NanoLC-LTQ मास स्पेक्ट्रोमेट्री से पचाया नहीं लार की प्रत्यक्ष विश्लेषण LLUF जांच की क्षमता के लिए बड़ी प्रोटीन और उच्च बहुतायत प्रोटीन हटाने के लिए, और कम प्रचुर पेप्टाइड्स अधिक से detectable का प्रदर्शन किया.
Abstract
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.
Protocol
Discussion
हमने पाया है कि कई पेप्टाइड टुकड़े पचाया नहीं मानव लार में मौजूद हैं. इन पेप्टाइड टुकड़ों प्रोलाइन युक्त प्रोटीन, actin, अल्फा amylase, एक ग्लोबिन अल्फा, बीटा ग्लोबिन, 1 histain, केरातिन 1, 7 mucin, polymeric इम्मुनोग्लोबुलिन रिस…
Disclosures
The authors have nothing to disclose.
Acknowledgements
यह काम राष्ट्रीय संस्थानों स्वास्थ्य अनुदान (R01-01-AI067395, R21-R022754-01, और R21 I58002-01) द्वारा समर्थित किया गया. हम सी. Niemeyer पांडुलिपि के महत्वपूर्ण पढ़ने के लिए धन्यवाद.
Materials
| Name of the reagent | Company | Catalog number | Comments |
| Polyethersulfone membranes | Pall Corporation | 30 kDa MWCO | |
| Teflon fluorinated ethylene propylene tube | Upchurch Scientific | ||
| Blue dextran | Sigma | ||
| Nano LC system | Eksigent | ||
| C18 trap column | Agilent | 5065-9913 | |
| LTQ linear ion-trap mass spectrometer | Thermo Fisher | ||
| Sorcerer 2 | Sage-N Research | ||
| Acetonitrile-0.1% formic acid | J.T. Baker | 9832-03 | LC/MS grade |
| Water-0.1% formic acid | J.T. Baker | 9834-03 | LC/MS grade |
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