The authors thank Dr. Kent Seeley, Director of The Center for Drug Discovery and Innovation (CDDI) Proteomics Core Facility at University of South Florida for the use of this facility. This work was supported by NIH/NIDCD grant R01 DC004295 to B.H.A.S.

Ethics Statement
Experiments using mice tissue were approved by the University of South Florida Institutional Animal Care and Use Committee (Protocols 3931R, 3482R) as set forth under the guidelines of the National Institutes of Health.
1. Protein Extraction
<ol>
	<li>Isolate cochlear sensory epithelium from 16 30-day-old (P30) CBA/J mice and store at -80 &#176;C.</li>
	<li>On the day of the experiment, wash tissue with 500 &#181;l of 1x phosphate buffered saline (PB...

<ol>
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The authors declare no competing interests.

The key steps to maximizing protein identification from the cochlear sensory epithelium are: 1) use of multiple endoproteinases for digestion, 2) use of multiple separation techniques, and 3) utilization of a high-resolution mass spectrometer. The application of multiple enzymes increases the number of peptides and improves protein sequence coverage, hence improving the number of identified proteins from the cochlear tissue. Trypsin, the most commonly used protease provides efficient and specific cleavage of proteins, ge...

Proteomics is the study of complex biological systems by analyzing protein expression, function, modifications, and interactions1. Several methods have been utilized for proteome analysis of the inner ear, including antibody microarray2, two-dimensional gel electrophoresis3-5, and DIGE6. However, only a limited number of proteins have been identified and characterized2,7-10, compared to the over 10,000 genes and expressed sequence tags (ESTs) identified in the inner ear11,12, MS is the most commonly used and comprehensive technique in proteomics for protein characterization. Analysis of complex proteomic samples, such as the cochlea, can be challenging. However, the combination of multiple separation techniques with MS enables the identification of a greater number of peptides and proteins, due to an increased dynamic concentration range and peak capacity13. Multidimensional chromatography reduces highly complex protein mixtures by allowing the use of different adsorption mechanisms. There are two commonly used MS proteome analysis approaches, shotgun and bottom-up proteomics. In shotgun proteomics, a mixture of intact proteins is enzymatically digested and separated using multidimensional chromatography with strong cation-exchange chromatography (SCX) followed by reversed-phase liquid chromatography (RPLC)14,15. The separated peptides are subjected to tandem MS and database searching15. A major advantage of this technique is that thousands of proteins can be identified in a single analysis and the technique is better suited to membrane proteins.
In the bottom-up approach, the protein mixture is separated, usually by one- or two-dimensional electrophoresis, and the individual protein bands or spots cut out and digested with an enzyme such as trypsin, usually resulting in multiple peptides. However, another more recently developed electrophoretic approach, used in bottom-up proteomics, is GELFrEE. This technique fractionates protein samples in liquid-phase and makes them less complex prior to analysis. This technique is reproducible, offers high protein recovery, and reduces the distribution of high abundant proteins in complex protein samples16. Peptides, resulting from separated proteins, are analyzed by MS, by using peptide mass fingerprinting or tandem MS (MS/MS), to create sequence tags for database searching17-19. Some of the major advantages of using the bottom-up approach are the ability to obtain high-resolution separations and comprehensive protein coverage. Bottom-up proteomics is the most widely used technique in proteomics20, hence, several bioinformatics tools are available. In addition, proteins can be separated in a complex mixture before digestion, so there is a greater chance of identification.
One of the major challenges in using the inner ear for proteomic analysis is its small size, restricted accessibility, and cell type diversity21. In addition, key proteins that distinguish its functionality, such as ion channels, transporters and receptors, are membrane proteins, which can be difficult to isolate22. Thus, filter-aided sample preparation (FASP) is advantageous for proteomic analyses of tissues that are limited for protein extraction and that require detergents to solubilize membranes23. This filtering allows for the MS analysis of membrane and soluble proteins and for the ability to isolate peptides from low molecular weight contaminants23,24.
The present protocol describes commonly used proteomic approaches that are combined and modified to analyze both soluble and membrane proteins and to maximize the number of protein IDs from the cochlear sensory epithelium. We will describe using shotgun proteomics with FASP multi-digestion, ion exchange chromatography, high resolution MS, and data analysis. In addition, we will describe bottom-up proteomics with GELFrEE, FASP multi-digestion, high resolution MS, and data analysis.

<table border="0" cellpadding="0" cellspacing="0" width="749">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:264px;">8% Tris-acetate cartridge&#160;</td>
			<td style="width:159px;">Protein Discovery</td>
			<td style="width:133px;">42103</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetone</td>
			<td>Sigma-Aldrich</td>
			<td>179124</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetonitrile</td>
			<td>Honeywell</td>
			<td>015-1L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AEBSF</td>
			<td>Calbiochem</td>
			<td>101500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium formate</td>
			<td>Fisher Scientific</td>
			<td>AC16861</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Aprotinin</td>
			<td>Calbiochem</td>
			<td>616370</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ASB-14&#160;</td>
			<td>Calbiochem</td>
			<td>182750-5GM</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine serum albumin</td>
			<td>BioRad</td>
			<td>500-0112</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">C18 column&#160;</td>
			<td>New Objective</td>
			<td>A25112</td>
			<td>75 &#956;m &#215; 10 cm&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DC Protein Assay</td>
			<td>BioRad</td>
			<td>500-0116</td>
			<td>Microplate Assay Protocol</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E9884</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Endoproteinase Lys-C</td>
			<td>Sigma-Aldrich</td>
			<td>P3428</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FASP Protein Digestion Kit</td>
			<td>Protein Discovery</td>
			<td>44250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Formic acid&#160;</td>
			<td>Fluka</td>
			<td>94318</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GELFrEE Fractionation System</td>
			<td>Protein Discovery</td>
			<td>42001</td>
			<td>GELFrEE 8100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Leupeptin</td>
			<td>Calbiochem</td>
			<td>108975</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MacroSpin Column</td>
			<td>The Nest Group</td>
			<td>SMM SS18V</td>
			<td>Silica C18</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microcystin</td>
			<td>Calbiochem</td>
			<td>475815</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pepstatin</td>
			<td>Sigma-Aldrich</td>
			<td>P5318</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polysulfoethyl A Column</td>
			<td>The Nest Group</td>
			<td>202SE0503</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium dodecyl sulfate (SDS)</td>
			<td>Sigma-Aldrich</td>
			<td>L3771</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sonic Dismembrator&#160;</td>
			<td>Thermo Fisher</td>
			<td>15-338-53</td>
			<td>Model 100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin</td>
			<td>Sigma-Aldrich</td>
			<td>T6567</td>
			<td>Proteomics Grade</td>
		</tr>
	</tbody>
</table>

bottom-up and shotgun proteomics to identify a comprehensive cochlear proteome

Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that transduce the mechanical energy of sound into an electro-chemical energy processed by the peripheral and central nervous systems. Several proteomic techniques have been developed to study the cochlear inner ear, such as two-dimensional difference gel electrophoresis (2D-DIGE), antibody microarray, and mass spectrometry (MS). MS is the most comprehensive and versatile tool in proteomics and in conjunction with separation methods can provide an in-depth proteome of biological samples. Separation methods combined with MS has the ability to enrich protein samples, detect low molecular weight and hydrophobic proteins, and identify low abundant proteins by reducing the proteome dynamic range. Different digestion strategies can be applied to whole lysate or to fractionated protein lysate to enhance peptide and protein sequence coverage. Utilization of different separation techniques, including strong cation exchange (SCX), reversed-phase (RP), and gel-eluted liquid fraction entrapment electrophoresis (GELFrEE) can be applied to reduce sample complexity prior to MS analysis for protein identification.

To obtain the most comprehensive proteome of the cochlear sensory epithelium, quick tissue dissection is required prior to protein extraction and sample preparation. Two proteomic techniques can be used, shotgun and bottom-up proteomics. To prepare samples for shotgun proteomics, FASP digestion procedure was used as illustrated in Figure 1. The FASP method allows for concentration of proteins, removal of detergents, and digestion of proteins using multiple enzymes. There were two double digestion procedu...

Watch this Scientific Journal Video about Bottom-up and Shotgun Proteomics to Identify a Comprehensive Cochlear Proteome at JoVE.com

Bottom-up and Shotgun Proteomics to Identify a Comprehensive Cochlear Proteome

Proteome analysis of the cochlear sensory epithelium can be challenging due to its small size and because membrane proteins are difficult to isolate and identify. Both membrane and soluble proteins can be identified by combining multiple preparative methods and separation techniques along with high-resolution mass spectrometry.

Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that ...

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18.6K ビュー.  University of South Florida. 人工内耳感覚上皮のプロテオーム解析は、その小さなサイズのために、膜タンパク質が単離および同定することが困難であるため、挑戦的であることができる。膜および可溶性タンパク質の両方は、高分解能質量分析と一緒に複数の調製方法及び分離技術を組み合わせることによって同定することができる。 