Name: two-dimensional gel electrophoresis coupled with mass spectrometry methods for an analysis of human pituitary adenoma tissue proteome
Uploaded: 2018-04-02T13:00:00
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This work was supported by the National Natural Science Foundation of China (Grant No. 81572278 and 81272798 to XZ), the grants from China &#34;863&#34; Plan Project (Grant No. 2014AA020610-1 to XZ), the Xiangya Hospital Funds for Talent Introduction (to XZ), and the Hunan Provincial Natural Science Foundation of China (Grant No. 14JJ7008 to XZ). The authors also acknowledge the scientific contribution of Dr. Dominic M. Desiderio in the University of Tennessee Health Science Center. X.Z. continuously developed and used 2DE-MS methods to analyze pituitary adenoma proteome starting from 2001, conceived the concept for the present manuscript, wrote and revised the manuscript, coordinated the pertinent work, and was responsible for the financial support and corresponding work. Y.L. participated in revision of the manuscript. Y.H participated in collection of references, and revision of the manuscript. All authors approved the final manuscript.

The present protocol follows the guidelines of the Xiangya Hospital Medical Ethics Committee of Central South University, China. A head cap and gloves should be worn for the entire experimental procedure to avoid keratin contamination from skin and hair8.
1. Preparation of Samples
<ol>
	<li>Collect PA tissues (0.2 - 0.5 mg) from the neurosurgical department. Immediately freeze in liquid nitrogen, and then transfer to -80 &#176;C for storage.</li>
	<li>Add 2 mL of 0.9%...

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reference+map+of+a+pituitary+adenoma+proteome.">A reference map of a pituitary adenoma proteome.</a> Proteomics. 3 (5), 699-713 (2003).</li><li>Desiderio, D. M., Zhan, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+study+of+the+human+pituitary+proteome:+The+characterization+of+differentially+expressed+proteins+in+an+adenoma+compared+to+a+control.">A study of the human pituitary proteome: The characterization of differentially expressed proteins in an adenoma compared to a control.</a> Cell. Mol. Biol. 49 (5), 689-712 (2003).</li><li>Zhan, X., Desiderio, D. 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2DE, coupled with MS methods including 2DE-MS PMF and 2DE-MS/MS, has been successfully used in our long-term program - the use of proteomics to study human NFPA proteomic variations and molecular network variations for the elucidation of molecular mechanisms and discovery of effective biomarkers for NFPAs. 2DE-based comparative proteomics with good reproducibility plays an important role in the identification of NFPA proteomic variations9,10,<sup class...

PA is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, which play important roles in the human endocrine system. PA includes clinically functional and nonfunctional PAs (FPA and NFPA)1,2. NFPA is difficult in early stage diagnosis and therapy because of only slightly elevated hormone levels (e.g., LH, and FSH) in blood compared to FPA, which has significantly increased levels of corresponding hormones in blood3,4,5. The clarification of molecular mechanisms and discovery of effective biomarkers has important clinical significance in the diagnosis, therapy, and prognosis of NFPA. Our long-term goal is to develop and use proteomic methods to study NFPA for the discovery of reliable biomarkers to clarify its molecular mechanisms, and recognize effective therapeutic targets as well as diagnostic and prognostic markers. 2-DE coupled with MS methods have been extensively used in our long-term research program regarding human PA proteome1,2,6,7, including establishment of proteome reference maps3,8, analysis of differentially expressed protein profiles9,10,11,12,13, hormone variants14,15, post-translational modifications such as phosphorylation14 and tyrosine nitration16,17,18, the proteomic variation of invasive relative to noninvasive NFPAs19, and the proteomic heterogeneity of NFPA subtypes13, which led to the discovery of multiple important pathway networks (mitochondrial dysfunction, cell cycle dysregulation, oxidative stress, and MAPK signaling system abnormality) that are altered in NFPA13,19,20.
2DE separates proteins according to their isoelectric point (pI) (isoelectric focusing, IEF) and molecular weight (via sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE)1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23. This is a common and classical separation technique in the field of proteomics, since the introduction of the concepts of the proteome and proteomics in 199524. MS is the crucial technique for finding out the identity of 2DE-separated proteins, including PMF and MS/MS strategies. The very rapid development of MS instruments, especially in the aspects of detection sensitivity and resolution, in combination with the improvement of LC system, greatly improves the identity of low or extremely low abundance proteins in a proteome to maximize the coverage of a proteome. It also challenges our traditional concepts that only one or two proteins are present in a 2D gel spot in an analysis of complex human tissue proteome and provides an opportunity to identify multiple proteins in a 2D gel spot in an analysis of complex human tissue proteome and maximize the coverage of NFPA proteome.
Here we describe detailed protocols of 2DE-MALDI MS PMF, 2DE-MALDI MS/MS, and 2DE-LC-MS/MS which have been successfully used in the analysis of human NFPA proteome. The protocols include preparation of samples, first dimension (isoelectric focusing, IEF), second dimension (SDS-PAGE), visualization of proteins (silver staining and Coomassie blue staining), image analysis of 2D gel, in-gel trypsin digestion, purification of tryptic peptides, PMF, MS/MS, and database searing3,8,25,26. Moreover, this protocol easily translates for the analysis of other human tissue proteomes.

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			<td height="61" style="height:61px;width:207px;">Ettan IPGphor 3&#160;</td>
			<td style="width:264px;">GE Healthcare</td>
			<td style="width:76px;"></td>
			<td style="width:160px;">isoelectric focusing system.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Ettan DALTsix multigel caster</td>
			<td style="width:264px;">Amersham Pharmacia Biotech, Piscataway, NJ, USA</td>
			<td style="width:76px;"></td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="59">
			<td height="59" style="height:59px;width:207px;">Ettan DALT II System</td>
			<td style="width:264px;">Amersham Pharmacia Biotech, Piscataway, NJ, USA</td>
			<td style="width:76px;"></td>
			<td style="width:160px;">The vertical electrophoresis system</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Ettan IPGphor strip holder</td>
			<td style="width:264px;">Amersham Pharmacia Biotech, Piscataway, NJ, USA</td>
			<td style="width:76px;"></td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Ettan DALTsix multigel caster</td>
			<td style="width:264px;">Amersham Pharmacia Biotech, Piscataway, NJ, USA</td>
			<td style="width:76px;"></td>
			<td style="width:160px;">Multigel caster</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Voyager DE STR MALDI-TOF MS&#160;</td>
			<td style="width:264px;">ABI, Foster City,CA</td>
			<td style="width:76px;"></td>
			<td style="width:160px;">MALDI-MS PMF</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">MALDI-TOF-TOF</td>
			<td style="width:264px;">Autoflex III, Bruker</td>
			<td style="width:76px;"></td>
			<td style="width:160px;">MALDI-MS/MS mass spectrometer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">LTQ-OrbiTrap Velos Pro ETD</td>
			<td style="width:264px;">Thermo Scientific, Waltham, MA, USA</td>
			<td style="width:76px;"></td>
			<td style="width:160px;">ESI-MS/MS mass spectrometer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">EASY-nano LC system&#160;</td>
			<td style="width:264px;">Proxeon Biosystems, Odense, Denmark</td>
			<td style="width:76px;"></td>
			<td style="width:160px;">High performance liquid chromatography system</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">PepMap C18 trap column&#160;</td>
			<td style="width:264px;">300 &#956;m i.d. &#215; 5 mm length; Dionex Corp., Sunnyvale, CA, USA</td>
			<td style="width:76px;"></td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:207px;">RP C18 column</td>
			<td style="width:264px;">75 &#956;m i.d., 15 cm length; Dionex Corp., Sunnyvale, CA, USA</td>
			<td style="width:76px;"></td>
			<td style="width:160px;"></td>
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			<td height="47" style="height:47px;width:207px;">KimWipe</td>
			<td style="width:264px;">Kimvipe&#160;</td>
			<td style="width:76px;"></td>
			<td style="width:160px;">Insoluble paper towel</td>
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		<tr height="47">
			<td height="47" style="height:47px;width:207px;">Watter</td>
			<td style="width:264px;">Made by PURELAB flex instrument</td>
			<td style="width:76px;"></td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:207px;">Polytron Model P710/35 homogenizer</td>
			<td style="width:264px;">Brinkmann Instruments, Westbury, NY</td>
			<td style="width:76px;"></td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:207px;">PDQuest</td>
			<td style="width:264px;">Bio-Rad,&#160; Hercules, CA</td>
			<td style="width:76px;"></td>
			<td style="width:160px;">2D gel image analysis software</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:207px;">SEQUEST&#160;</td>
			<td style="width:264px;">Thermo Proteome Discoverer 1.3 (version No. 1.3.0.339)</td>
			<td style="width:76px;"></td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:207px;">DataExplore (ver. 4.0.0.0) software</td>
			<td style="width:264px;"></td>
			<td style="width:76px;"></td>
			<td style="width:160px;">MS spectrum-processing software</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:207px;">Mascot software</td>
			<td style="width:264px;"></td>
			<td style="width:76px;"></td>
			<td style="width:160px;">PMF-based protein searching software&#160;&#160;</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:207px;">Mascot software</td>
			<td style="width:264px;"></td>
			<td style="width:76px;"></td>
			<td style="width:160px;">MS/MS-based protein searching software</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:207px;">Proteome Discoverer software v.1.3 beta</td>
			<td style="width:264px;">Thermo Scientific</td>
			<td style="width:76px;"></td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:207px;">Xcalibur software v.2.1</td>
			<td style="width:264px;"></td>
			<td style="width:76px;"></td>
			<td style="width:160px;">MS/MS data-acquired management software&#160;</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:207px;">Uniprot version 201410.1_HUMAN.fasta</td>
			<td style="width:264px;"></td>
			<td style="width:76px;"></td>
			<td style="width:160px;">Human protein database</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:207px;">SEQUEST (version No. 1.3.0.339)&#160;</td>
			<td style="width:264px;"></td>
			<td style="width:76px;"></td>
			<td style="width:160px;">MS/MS-based protein searching software I</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:207px;">MASCOT (version 2.3.02)&#160;</td>
			<td style="width:264px;"></td>
			<td style="width:76px;"></td>
			<td style="width:160px;">MS/MS-based protein searching software II</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:207px;">C18 ZipTip microcolumn</td>
			<td style="width:264px;">Millipore</td>
			<td style="width:76px;"></td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:207px;">PeptideMass Standard kit&#160;</td>
			<td style="width:264px;">Perspective Biosystems</td>
			<td style="width:76px;"></td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Pierce BCA Protein Assay Kit&#160;</td>
			<td style="width:264px;">Thermo&#160;Fisher Scientific</td>
			<td style="width:76px;">23227</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">2-D Quant Kit</td>
			<td style="width:264px;">GE Healthcare</td>
			<td style="width:76px;">80-6483-56</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">BIS-ACRYLAMIDE</td>
			<td style="width:264px;">AMRESCO</td>
			<td style="width:76px;">0172</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">ACRYLAMIDE</td>
			<td style="width:264px;">AMRESCO</td>
			<td style="width:76px;">0341</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">DTT</td>
			<td style="width:264px;">Sigma-Aldrich</td>
			<td style="width:76px;">D0632</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Thiourea</td>
			<td style="width:264px;">Sigma-Aldrich</td>
			<td style="width:76px;">T8656</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Urea</td>
			<td style="width:264px;">VETEC</td>
			<td style="width:76px;">V900119</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">SDS</td>
			<td style="width:264px;">AMRESCO</td>
			<td style="width:76px;">0227</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">CHAPS</td>
			<td style="width:264px;">AMRESCO</td>
			<td style="width:76px;">0465</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">TEMED</td>
			<td style="width:264px;">AMRESCO</td>
			<td style="width:76px;">M146</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Ammonium Persulfate</td>
			<td style="width:264px;">AMRESCO</td>
			<td style="width:76px;">M133</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Trypsin</td>
			<td style="width:264px;">Promega, Madison, WI, USA</td>
			<td style="width:76px;">V5111</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">IPG buffer pH 3-10, NL</td>
			<td style="width:264px;">GE Healthcare</td>
			<td style="width:76px;">17-6000-87</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Immobiline Dry Strip pH 3-10NL,18cm</td>
			<td style="width:264px;">GE Healthcare</td>
			<td style="width:76px;">17-1235-01</td>
			<td style="width:160px;"></td>
		</tr>
	</tbody>
</table>

two-dimensional gel electrophoresis coupled with mass spectrometry methods for an analysis of human pituitary adenoma tissue proteome

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may be classified as either clinically functional or nonfunctional PA (FPA and NFPA). NFPA is difficult for early stage diagnosis and therapy due to barely elevating hormones in the blood compared to FPA. Our long-term goal is to use proteomics methods to discover reliable biomarkers for clarification of PA molecular mechanisms and recognition of effective diagnostic, prognostic markers and therapeutic targets. Effective two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) methods were presented here to analyze human PA proteomes, including preparation of samples, 2D gel electrophoresis, protein visualization, image analysis, in-gel trypsin digestion, peptide mass fingerprint (PMF), and tandem mass spectrometry (MS/MS). 2-Dimensional gel electrophoresis matrix-assisted laser desorption/ionization mass spectrometry PMF (2DE-MALDI MS PMF), 2DE-MALDI MS/MS, and 2DE-liquid chromatography (LC) MS/MS procedures have been successfully applied in an analysis of NFPA proteome. With the use of a high-sensitivity mass spectrometer, many proteins were identified with the 2DE-LC-MS/MS method in each 2D gel spot in an analysis of complex PA tissue to maximize the coverage of human PA proteome.

1. 2DE-MALDI MS PMF: With the experimental procedure described above, a total of 150 &#181;g proteins were extracted from FSH-expressed NFPA tissues (female; 50 years old, ACTH (-), GH (-), PRL (-), LH (-), FSH (+), and TSH (-)) and arrayed with an 18 cm IPG strip (pH 3-10 NL) and a large-format SDS-PAGE gel, then visualized with silver staining. We obtained a reproducible and good 2DE gel pattern of NFPA tissue proteome (Figure 1</st...

Watch this Scientific Journal Video about Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome at JoVE.com

Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome

Here, we present a two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) to separate and identify human pituitary adenoma tissue proteome, which presents a good and reproducible 2DE pattern. Many proteins are observed in each 2DE spot when analyzing complex cancer proteome with the use of high-sensitivity MS.

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may ...

The overall goal of this two-dimensional gel electrophoresis coupled with mass spectrometry procedure is to separate and identify human pituitary adenoma tissue proteome. This method can help answer key questions in the proteomes field. Such as high of proteome.And discovery of protein situation, protein four, and protein spacious. The main advantage of this technique is that two dimensional gel electrophoresis is easily coupled with high sensitivity mass spectrometry to identify proteome, especially low abundance proteins. Protein situation, protein four, and protein spacious.The implementations of this technique is tend toward PQ itery of animal therapy. Because of key protein for protein spacious or protein pattern is useful in patients diagnosis. We show a demonstration of this method is critical as the two dimensional gel electrophoresis steps are difficult to learn because it takes time consuming labor intensive and there are several experimental tricks.To begin this procedure add 250 microliters of a previously prepared protein sample solution to each slot of a 13 centimeter IPG strip holder. Place an 18 centimeter IPG strip, gel side down, onto each protein sample solution taking care to avoid bubbles. Then add three to four milliliters of mineral oil to cover each IPG strip.Next, assemble each IPG strip holder into an isoelectric focusing system with the pointed end on the back plate and the square end on the front plate. After rehydrating the IPG strips for 18 hours at room temperature, set the IEF parameters on the isoelectric focusing system with a total run time of 7.5 hours and 36, 875 volts hours. After IEF, take out each IPG strip from the isoelectric focusing system and remove the excess mineral oil with an insoluble paper towel.Then wrap each strip in a sheet of plastic wrap and store at minus 80 degrees Celsius. To cast a 12%PAGE gel add double distilled water, 1.5 molar tris-hydrochloric buffer, 40%acrylamide, bis-acrylamide stock solution, 10%immodium persulfate, and timed to a beaker. Mix the gel solution gently, taking care to avoid bubbles.Gently pour the gel solution into a multi-casting chamber up to the expected gel height of 19 centimeters. Immediately add about three milliliters of double distilled water to the multi-casting chamber to cover each resolving gel and allow each gel to polymerize for about one hour. This step is very important to prepare a high quality SDS-PAGE gel and the water must be immediately added on the top of each gel.Prepare two liters of electrophoresis buffer then add the buffer to an electrophoresis separation unit buffer tank. After removing the focused IPG strips from the freezer, place them in a plastic vessel containing one milliliter of reducing equilibrium buffer and a trace of bromophenol blue and equilibrate them for 10 minutes with gentle rocking. After removing the reducing equilibrium buffer, add four milliliters of excalation equilibrium buffer and a trace of bromophenol blue to the IPG strips and equilibrate them for 10 minutes with gentle rocking.During gel equilibrium, disassemble the multi-casting chamber and remove the prepared resolving gel cassette. Rinse the gel cassette three times with double distilled water, then remove access water from the gel cassette with an insoluble paper towel and place it in a gel stander. Following this, rinse the equilibrated IPG strips with electrophoresis buffer and remove excess liquid on each IPG strip surface with an insoluble paper towel.Place an IPG strip onto the resolving SDS-PAGE gel, allowing the IPG strip's plastic side to contact the longer glass plate with the pointed end to the left. Now quickly add three to four milliliters of hot 1%agarose and SDS electrophoresis buffer to seal the IPG strip on the top of each SDS-PAGE gel and place the top side of the IPG strip down on the top of the shorter glass plate taking care to avoid bubbles. These steps are very important to seal the IPG strip to the SDS-PAGE gel.The hot agarose solutions must be immediately added and the IPG strip must be immediately placed into the top of the SDS gel. After polymerization, insert the assembled gel cassette vertically between the plastic gaskets in a vertical electrophoresis system. Place the top of the gel with the IPG strip next to the cathode.Next, adjust the level of electrophoresis buffer to immerse the gel cassette by adding the appropriate volume of buffer to the electrophoresis system. Connect and set the power supply in constant voltage mode and run at 200 volts for about 370 minutes while monitoring the dye. After the run is complete, take the gel cassette out of the electrophoresis system and gently remove the gel from the gel cassette, taking care to avoid tearing the gel.Carefully transfer the gel to a tray containing 250 milliliters of fixation solution and gently shake the gel for 20 minutes. Following this, discard the solution and add 250 milliliters of 50%methanol to the tray. Then gently shake the gel for 10 minutes.After shaking the gel in double distilled water, add 250 milliliters of sensitization buffer containing 0.02%of sodium sulfate. Gently shake the gel for one minute. After shaking the gel twice in double distilled water, add 250 milliliters of silver reaction solution and gently shake the gel for 20 minutes.After shaking the gel in double distilled water, add 250 milliliters of development solution and gently shake until the desired staining is obtained, which is typically one to three minutes. Now add 250 milliliters of 5%acidic acid to terminate staining and gently shake the gel for 10 minutes. After shaking the gel in double distilled water, add 250 milliliters of 8.8%glycerol and store the gel at four degrees Celsius until analysis.A reproducible and good two dimensional gel pattern of NFPA tissue proteome was obtained from gel electrophoresis with approximately 1, 200 protein spots detected in the two dimensional gel map that were mainly distributed within the area of pH four to eight and mass 15 to 150 kilodaltons. A total of 192 redundant proteins were identified from 141 gel spots out of the 337 analyzed gel spots in the MALDI-MS PMF analysis and no protein was identified in the remaining 196 spots. Staining with commassie blue yielded a reproducible and high quality two dimensional gel pattern of invasive NFPA tissue proteome and approximately 1, 100 protein spots detected in a two dimensional gel map that were mainly distributed within the range of pH four to eight and mass 15 to 150 kilodaltons.An average of one protein per spot was identified with MALDI tof tof MS MS analysis. Where as many proteins in each corresponding spot were identified with LC-ESI-MS/MS analysis. This data clearly demonstrates that each two dimensional gel spot contains many proteins in the complex human cancer tissue proteome.Once mastered this technique can be done in 32 hours for two dimensional gel electrophoresis if it is performed properly. While attempting this procedure, it is important to remember to follow the steps, especially according to the protocol. Following this procedure, other methods like stable labeling can be performed in order to answer additional questions like the codification of proteins in the proteome.After its development, this technique paved the way for researchers in the field of protein mix to explore larger scale low abundance protein post translational modifications, protein specialization, protein spacious, and protein four in model conditions, patients, and organ systems. After watching this video you should have a good understanding of how to perform two dimensional gel electrophoresis coupled with mass spectrometry achieving good and reproducible two dimensional gel electrophoresis pattern and identify many proteins in each spot. Don't forget that working with SDS achreomy and TEMED can be extremely hazardous and precautions such as wearing plastic gloves, should always be taken while performing this procedure.

two-dimensional-gel-electrophoresis-coupled-with-mass-spectrometry

Research

JoVE Journal

Cancer Research

Modified Terminal Restriction Fragment Analysis for Quantifying Telomere Length Using In-gel Hybridization

There are several different techniques for measuring telomere length, each with their own advantages and disadvantages. The traditional approach, Telomere Restriction Fragment (TRF) analysis, utilizes a DNA hybridization technique whereby genomic DNA samples are digested with restriction enzymes, leaving behind telomere DNA repeats and some sub-telomeric DNA. These are separated by agarose gel electrophoresis, transferred to a filter membrane and hybridized to oligonucleotide probes tagged with either chemiluminescence or radioactivity to visualize telomere restriction fragments. This approach, while requiring a larger quantity of DNA than other techniques such as PCR, can measure the telomere length distribution of a population of cells and allows measurement expressed in absolute kilobases. This manuscript demonstrates a modified DNA hybridization procedure for determining telomere length. Genomic DNA is first digested with restriction enzymes (that do not cut telomeres) and separated by agarose gel electrophoresis. The gel is then dried and the DNA is denatured and hybridized in situ to a radiolabeled oligonucleotide probe. This in situ hybridization avoids loss of telomere DNA and improves signal intensity. Following hybridization, the gels are imaged utilizing phosphor screens and the telomere length is quantified using a graphing program. This procedure was developed by the laboratories of Drs. Woodring Wright and Jerry Shay at the University of Texas Southwestern1,2. Here, we present a detailed description of this procedure, with some modifications.

In this article, a detailed protocol for quantifying telomere length using a modified terminal restriction fragment analysis is discussed that provides fast and efficient direct measurement of telomere length. This technique can be applied to a variety of cell sources of DNA for quantifying telomere length.

There are several different techniques for measuring telomere length, each with their own advantages and disadvantages. The traditional approach, Telomere ...

Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction pathways and is tightly regulated by kinases and phosphatases. Therefore, characterizing the phosphoproteome may provide insights into identifying novel targets and biomarkers for oncologic therapy. Mass spectrometry provides a way to globally detect and quantify thousands of unique phosphorylation events. However, phosphopeptides are much less abundant than non-phosphopeptides, making biochemical analysis more challenging. To overcome this limitation, methods to enrich phosphopeptides prior to the mass spectrometry analysis are required. We describe a procedure to extract and digest proteins from tissue to yield peptides, followed by an enrichment for phosphotyrosine (pY) and phosphoserine/threonine (pST) peptides using an antibody-based and/or titanium dioxide (TiO2)-based enrichment method. After the sample preparation and mass spectrometry, we subsequently identify and quantify phosphopeptides using liquid chromatography-mass spectrometry and analysis software.

This protocol describes a procedure to extract and enrich phosphopeptides from prostate cancer cell lines or tissues for an analysis of the phosphoproteome via mass spectrometry-based proteomics.

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction ...

Capturing Small Molecule Communication Between Tissues and Cells Using Imaging Mass Spectrometry

Imaging mass spectrometry (IMS) has routinely been applied to three types of samples: tissue sections, spheroids, and microbial colonies. These sample types have been analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to visualize the distribution of proteins, lipids, and metabolites across the biological sample of interest. We have developed a novel sample preparation method that combines the strengths of the three previous applications to address an underexplored approach for identifying chemical communication in cancer, by seeding mammalian cell cultures into agarose in coculture with healthy tissues followed by desiccation of the sample. Mammalian tissue and cells are cocultured in close proximity allowing chemical communication via diffusion between the tissue and cells. At specific time points, the agarose-based sample is dried in the same manner as microbial colonies prepared for IMS analysis. Our method was developed to model the communication between high grade serous ovarian cancer derived from the fallopian tube as it interacts with the ovary during metastasis. Optimization of the sample preparation resulted in the identification of norepinephrine as a key chemical component in the ovarian microenvironment. This newly developed method can be applied to other biological systems that require an understanding of chemical communication between adjacent cells or tissues.

A novel method of sample preparation was developed to accommodate cell and tissue coculture to detect small molecule exchange using imaging mass spectrometry.

Imaging mass spectrometry (IMS) has routinely been applied to three types of samples: tissue sections, spheroids, and microbial colonies. These sample ...

Extraction of Aqueous Metabolites from Cultured Adherent Cells for Metabolomic Analysis by Capillary Electrophoresis-Mass Spectrometry

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but also to identify biomarkers for early-stage cancer detection and prediction of chemotherapy response in cancer patients. Preparation of uniform samples for metabolomic analysis is a critical issue that remains to be addressed. Here, we present an easy and reliable protocol for extracting aqueous metabolites from cultured adherent cells for metabolomic analysis using capillary electrophoresis-mass spectrometry (CE-MS). Aqueous metabolites from cultured cells are analyzed by culturing and washing cells, treating cells with methanol, extracting metabolites, and removing proteins and macromolecules with spin columns for CE-MS analysis. Representative results using lung cancer cell lines treated with diamide, an oxidative reagent, illustrate the clearly observable metabolic shift of cells under oxidative stress. This article would be especially valuable to students and investigators involved in metabolomics research, who are new to harvesting metabolites from cell lines for analysis by CE-MS.

The purpose of this article is to describe a protocol for extraction of aqueous metabolites from cultured adherent cells for metabolomic analysis, particularly, capillary electrophoresis-mass spectrometry.

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but ...

Measuring Real-time Drug Response in Organotypic Tumor Tissue Slices

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu constitutes the tumor microenvironment (TME) and can modulate response to therapy in vivo or drug response ex vivo. Conventional cancer drug discovery screens are carried out on cells cultured in a monolayer, a system critically lacking the influence of TME. Thus, experimental systems that integrate sensitive and high-throughput assays with physiological TME will strengthen the preclinical drug discovery process. Here, we introduce ex vivo tumor tissue slice culture as a platform for medium-high-throughput drug screening. Organotypic tissue slice culture constitutes precisely-cut, thin tumor sections that are maintained with the support of a porous membrane in a liquid-air interface. In this protocol, we describe the preparation and maintenance of tissue slices prepared from mouse tumors and tumors from patient-derived xenograft (PDX) models. To assess changes in tissue viability in response to drug treatment, we leveraged a biocompatible luminescence-based viability assay that enables real-time, rapid, and sensitive measurement of viable cells in the tissue. Using this platform, we evaluated dose-dependent responses of tissue slices to the multi-kinase inhibitor, staurosporine, and cytotoxic agent, doxorubicin. Further, we demonstrate the application of tissue slices for ex vivo pharmacology by screening 17 clinical and preclinical drugs on tissue slices prepared from a single PDX tumor. Our physiologically-relevant, highly-sensitive, and robust ex vivo screening platform will greatly strengthen preclinical oncology drug discovery and treatment decision making.&#160;

We introduce a protocol for measuring real-time drug response in organotypic tumor tissue slices. The experimental strategy outlined here provides a platform to carry out medium-high throughput drug screens on tissue slices derived from clinical or mouse tumors in ex vivo conditions.

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu ...

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using supernatants from Lactobacillus fermentum cell culture, considered as probiotics cultures. The use of 3D cultures to test Lactobacillus cell-free supernatant (LCFS) are a better option than testing in 2D monolayers, especially as L. fermentum can produce anti-cancer effects within the gut. L. fermentum supernatant was identified to possess increased anti-proliferative effects against several colorectal cancer (CRC) cells in 3D culture conditions. Interestingly, these effects were strongly related to the culture model, demonstrating the notable ability of L. fermentum to induce cancer cell death. Stable spheroids were generated from diverse CRCs (colorectal cancer cells) using the protocol presented below. This protocol of generating 3D spheroid is time saving and cost effective. This system was developed to easily investigate the anti-cancer effects of LCFS in multiple types of CRC spheroids. As expected, CRC spheroids treated with LCFS strongly induced cell death during the experiment and expressed specific apoptosis molecular markers as analyzed by qRT-PCR, western blotting, and FACS analysis. Therefore, this method is valuable for exploring cell viability and evaluating the efficacy of anti-cancer drugs.

Here methods are presented to understand anti-cancer effects of Lactobacillus cell-free supernatant (LCFS). Colorectal cancer cell lines show cell deaths when treated with LCFS in 3D cultures. The process of generating spheroids can be optimized depending on the scaffold and the analysis methods presented are useful for evaluating the involved signaling pathways.

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...

Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo

Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach in&#160;vaccine design. Antigen is delivered to DC via antibodies specific for endocytosis receptors such as DEC-205 that induce uptake, processing, and MHC class I- and II-presentation.
Efficient and reliable conjugation of the desired antigen to a suitable antibody is a critical step in DC targeting and among other factors depends on the format of the antigen. Chemical conjugation of&#160;full-length protein to purified antibodies is one possible strategy. In the past, we have successfully established cross-linking of the model antigen ovalbumin (OVA) and a DEC-205-specific IgG2a antibody (&#945;DEC-205)&#160;for in vivo DC targeting studies in mice. The first step of the protocol is the purification of the antibody from the supernatant of the NLDC (non-lymphoid dendritic cells)-145 hybridoma by affinity chromatography. The purified antibody is activated for chemical conjugation by sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate) while at the same time the sulfhydryl-groups of the OVA protein are exposed through incubation with TCEP-HCl (tris (2-carboxyethyl) phosphine hydrochloride). Excess TCEP-HCl and sulfo-SMCC are removed and the antigen is mixed with the activated antibody for overnight coupling. The resulting &#945;DEC-205/OVA conjugate is concentrated and freed from unbound OVA. Successful conjugation of OVA to &#945;DEC-205 is verified by western blot analysis and enzyme-linked immunosorbent assay (ELISA).
We have successfully used chemically crosslinked &#945;DEC-205/OVA to induce cytotoxic T cell responses in the liver and to compare different adjuvants for their potential in inducing humoral and cellular immunity following in vivo targeting of DEC-205+ DC. Beyond that, such chemically coupled antibody/antigen conjugates offer valuable tools for the efficient induction of vaccine responses to tumor antigens and have been proven to be superior to classical immunization approaches regarding the prevention and therapy of various types of tumors.

Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo

We describe a protocol for the chemical conjugation of the model antigen ovalbumin to an endocytosis receptor-specific antibody for in vivo dendritic cell targeting. The protocol includes purification of the antibody, chemical conjugation of the antigen, as well as purification of the conjugate and the verification of efficient conjugation.

Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach ...

Three Differential Expression Analysis Methods for RNA Sequencing: limma, EdgeR, DESeq2

RNA sequencing (RNA-seq) is one of the most widely used technologies in transcriptomics as it can reveal the relationship between the genetic alteration and complex biological processes and has great value in diagnostics, prognostics, and therapeutics of tumors. Differential analysis of RNA-seq data is crucial to identify aberrant transcriptions, and limma, EdgeR and DESeq2 are efficient tools for differential analysis. However, RNA-seq differential analysis requires certain skills with R language and the ability to choose an appropriate method, which is lacking in the curriculum of medical education.
Herein, we provide the detailed protocol to identify differentially expressed genes (DEGs) between cholangiocarcinoma (CHOL) and normal tissues through limma, DESeq2 and EdgeR, respectively, and the results are shown in volcano plots and Venn diagrams. The three protocols of limma, DESeq2 and EdgeR are similar but have different steps among the processes of the analysis. For example, a linear model is used for statistics in limma, while the negative binomial distribution is used in edgeR and DESeq2. Additionally, the normalized RNA-seq count data is necessary for EdgeR and limma but is not necessary for DESeq2.
Here, we provide a detailed protocol for three differential analysis methods: limma, EdgeR and DESeq2. The results of the three methods are partly overlapping. All three methods have their own advantages, and the choice of method only depends on the data.

A detailed protocol of differential expression analysis methods for RNA sequencing was provided: limma, EdgeR, DESeq2.

RNA sequencing (RNA-seq) is one of the most widely used technologies in transcriptomics as it can reveal the relationship between the genetic alteration ...

Preparation of Human Tissues Embedded in Optimal Cutting Temperature Compound for Mass Spectrometry Analysis

Sphingolipids are cellular components that have well-established roles in human metabolism and disease. Mass spectrometry can be used to determine whether sphingolipids are altered in a disease and investigate whether sphingolipids can be targeted clinically. However, properly powered prospective studies that acquire tissues directly from the surgical suite can be time consuming, and technically, logistically, and administratively challenging. In contrast, retrospective studies can take advantage of cryopreserved human specimens already available, usually in large numbers, at tissue biorepositories. Other advantages of procuring tissues from biorepositories include access to information associated with the tissue specimens including histology, pathology, and in some instances clinicopathological variables, all of which can be used to examine correlations with lipidomics data. However, technical limitations related to the incompatibility of optimal cutting temperature compound (OCT) used in the cryopreservation and mass spectrometry is a technical barrier for the analysis of lipids. However, we have previously shown that OCT can be easily removed from human biorepository specimens through cycles of washes and centrifugation without altering their sphingolipid content. We have also previously established that sphingolipids in human tissues cryopreserved in OCT are stable for up to 16 years. In this report, we outline the steps and workflow to analyze sphingolipids in human tissue specimens that are embedded in OCT, including washing tissues, weighing tissues for data normalization, the extraction of lipids, preparation of samples for analysis by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), mass spectrometry data integration, data normalization, and data analysis.

Sphingolipids are bioactive metabolites with well-established roles in human disease. Characterizing alterations in tissues with mass-spectrometry can reveal roles in disease etiology or identify therapeutic targets. However, the OCT-compound used for cryopreservation in biorepositories interferes with mass-spectrometry. We outline methods to analyze sphingolipids in human tissues embedded in OCT with LC-ESI-MS/MS.

Sphingolipids are cellular components that have well-established roles in human metabolism and disease. Mass spectrometry can be used to determine whether ...

Expanding the Comprehension of the Tumor Microenvironment using Mass Spectrometry Imaging of Formalin-Fixed and Paraffin-Embedded Tissue Samples

Advances in immune-based therapies have revolutionized cancer treatment and research. This has triggered growing demand for the characterization of the tumor immune landscape. Although standard immunohistochemistry is suitable for studying tissue architecture, it is limited to the analysis of a small number of markers. Conversely, techniques such as flow cytometry can evaluate multiple markers simultaneously, although information about tissue morphology is lost. In recent years, multiplexed strategies that integrate phenotypic and spatial analysis have emerged as comprehensive approaches to the characterization of the tumor immune landscape. Herein, we discuss an innovative technology combining metal-labeled antibodies and secondary ion mass spectrometry focusing on the technical steps in assay development and optimization, tissue preparation, and image acquisition and processing. Before staining, a metal-labeled antibody panel must be developed and optimized. This hi-plex image system supports up to 40 metal-tagged antibodies in a single tissue section. Of note, the risk of signal interference increases with the number of markers included in the panel. After panel design, particular attention should be given to the metal isotope assignment to the antibody to minimize this interference. Preliminary panel testing is performed using a small subset of antibodies and subsequent testing of the entire panel in control tissues. Formalin-fixed, paraffin-embedded tissue sections are obtained and mounted on gold-coated slides and further stained. The staining takes 2 days and closely resembles standard immunohistochemical staining. Once samples are stained, they are placed in the image acquisition instrument. Fields of view are selected, and images are acquired, uploaded, and stored. The final stage is image preparation for the filtering and removal of interference using the system&#39;s image processing software. A disadvantage of this platform is the lack of analytical software. However, the images generated are supported by different computational pathology software.

In the era of cancer immunotherapy, interest in elucidating tumor microenvironment dynamics has increased strikingly. This protocol details a mass spectrometry imaging technique with respect to its staining and imaging steps, which allow for highly multiplexed spatial analysis.

Advances in immune-based therapies have revolutionized cancer treatment and research. This has triggered growing demand for the characterization of the ...