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">
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			<td height="40" style="height:40px;width:207px;">SDS</td>
			<td style="width:264px;">AMRESCO</td>
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			<td height="40" style="height:40px;width:207px;">CHAPS</td>
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			<td height="40" style="height:40px;width:207px;">Ammonium Persulfate</td>
			<td style="width:264px;">AMRESCO</td>
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			<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>
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			<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>
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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.

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