Name: sampling human indigenous saliva peptidome using a lollipop-like ultrafiltration probe: simplify and enhance peptide detection for clinical mass spectrometry
Uploaded: 2012-08-07T13:00:00
Description: Watch this Scientific Journal Video about Sampling Human Indigenous Saliva Peptidome Using a Lollipop-Like Ultrafiltration Probe: Simplify and Enhance Peptide Detection for Clinical Mass Spectrometry

This work was supported by National Institutes of Health Grants (R01-AI067395-01, R21-R022754-01, and R21-I58002-01). We thank C. Niemeyer for critical reading of the manuscript.

1. Creation of LLUF Probes
<ol>
 <li>The polyethersulfone membranes (2 cm2) were sealed with triangle polypropylene paddles (University of California, San Diego) by gluing membranes with epoxy on the borders of paddles. A negatively charged polyethersulfone membrane with a molecular weight cut-off (MWCO) at 30 kDa was used.</li>
 <li>A teflon fluorinated ethylene propylene tube (inner diameter/outer diameter, 0.35/0.50 cm) was attached to a cylinder exit of a triangle polypropylene paddle so the LLUF probe c...

<ol>
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We have found that many peptide fragments exist in human undigested saliva. These peptide fragments are derivatives from various forms of proline-rich proteins, actin, alpha amylase, alpha 1 globin, beta globin, histain 1, keratin 1, mucin 7, polymeric immunoglobulin receptor, satherin, S100A9. There could be many factors contributing to the production of peptides with undetermined cleavage sites. For example, some peptide fragments may be naturally present in human whole saliva. Many peptides with -PQ C-termini were ide...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalog number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Polyethersulfone membranes </td>
 <td>Pall Corporation </td>
 <td>&nbsp;</td>
 <td>30 kDa MWCO </td>
 </tr>
 <tr>
 <td>Teflon fluorinated ethylene propylene tube </td>
 <td>Upchurch Scientific </td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Blue dextran </td>
 <td>Sigma </td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Nano LC system</td>
 <td>Eksigent</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>C18 trap column </td>
 <td>Agilent</td>
 <td>5065-9913</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>LTQ linear ion-trap mass spectrometer </td>
 <td>Thermo Fisher</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sorcerer 2</td>
 <td>Sage-N Research</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Acetonitrile-0.1% formic acid</td>
 <td>J.T. Baker</td>
 <td>9832-03</td>
 <td>LC/MS grade</td>
 </tr>
 <tr>
 <td>Water-0.1% formic acid</td>
 <td>J.T. Baker</td>
 <td>9834-03</td>
 <td>LC/MS grade</td>
 </tr>
 </tbody>
</table>

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

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.

Watch this Scientific Journal Video about Sampling Human Indigenous Saliva Peptidome Using a Lollipop-Like Ultrafiltration Probe: Simplify and Enhance Peptide Detection for Clinical Mass Spectrometry 

Sampling Human Indigenous Saliva Peptidome Using a Lollipop-Like Ultrafiltration Probe: Simplify and Enhance Peptide Detection for Clinical Mass Spectrometry

Considering saliva sampling for future clinical application, a lollipop-like ultrafiltration (LLUF) probe was fabricated to fit in the human oral cavity. Direct analysis of undigested saliva by NanoLC-LTQ mass spectrometry demonstrated the ability of LLUF probes to remove large proteins and high abundance proteins, and make low-abundant peptides more detectable.

Although human saliva proteome and peptidome have been revealed 1-2 they were majorly identified from tryptic digests of saliva proteins. Identification ...

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In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Surgical Training for the Implantation of Neocortical Microelectrode Arrays Using a Formaldehyde-fixed Human Cadaver Model

This protocol describes a procedure to assist surgeons in training for the implantation of microelectrode arrays into the neocortex of the human brain. ...