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.

<ol>
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Rev. 24 (6), 783-813 (2005).</li><li>Wang, X., Guo, T., Peng, F., Long, Y., Mu, Y., Yang, H., Ye, N., Li, X., 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=Proteomic+and+functional+profiles+of+a+follicle-stimulating+hormone-positive+human+nonfunctional+pituitary+adenoma.">Proteomic and functional profiles of a follicle-stimulating hormone-positive human nonfunctional pituitary adenoma.</a> Electrophoresis. 36 (11-12), 1289-1304 (2015).</li><li>Karppinen, A., Kivipelto, L., Vehkavaara, S., Ritvonen, E., Tikkanen, E., Kivisaari, R., Hernesniemi, J., Set&#228;l&#228;, K., Schalin-J&#228;ntti, C., Niemel&#228;, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transition+from+microscopic+to+endoscopic+transsphenoidal+surgery+for+nonfunctional+pituitary+adenomas.">Transition from microscopic to endoscopic transsphenoidal surgery for nonfunctional pituitary adenomas.</a> World Neurosurg. 84 (1), 48-57 (2015).</li><li>Liu, X., Ma, S., Dai, C., Cai, F., Yao, Y., Yang, Y., Feng, M., Deng, K., Li, G., Ma, W., Xin, B., Lian, W., Xiang, G., Zhang, B., Wang, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiproliferative,+antiinvasive,+and+proapoptotic+activity+of+folate+receptor+&#945;-targeted+liposomal+doxorubicin+in+nonfunctional+pituitary+adenoma+cells.">Antiproliferative, antiinvasive, and proapoptotic activity of folate receptor &#945;-targeted liposomal doxorubicin in nonfunctional pituitary adenoma cells.</a> Endocrinol. 154 (4), 1414-1423 (2013).</li><li>Zhan, X., Wang, X., Desiderio, D. 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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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The authors declare that there is no conflict of interests.

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.

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

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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 ...

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

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Cancer Research

13.4K Views.  Central South University. Hier presenteren we een twee-dimensionale gelelektroforese (2DE) dat is gekoppeld aan massaspectrometrie (MS) te scheiden en te identificeren menselijke hypofyse adenoom weefsel Proteoom, waarin een goede en reproduceerbare 2DE patroon. Veel eiwitten worden bij elke 2DE plek waargenomen bij het analyseren van complexe kanker Proteoom met het gebruik van hoog-gevoeligheids MS.

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

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Biochemistry

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 ...

Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or pathological conditions. Basically, this technique is based on the separation of proteins according to their isoelectric point in a first step, and secondly according to their molecular weights by SDS polyacrylamide gel electrophoresis (SDS-PAGE). In this report an optimized sample preparation protocol for little amount of human post-mortem and mouse brain tissue is described. This method enables to perform both two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mini 2DE immunoblotting. The combination of these approaches allows one to not only find new proteins and/or protein modifications in their expression thanks to its compatibility with mass spectrometry detection, but also a new insight into markers validation. Thus, mini-2DE coupled to western blotting permits to identify and validate post-translational modifications, proteins catabolism and provides a qualitative comparison among different conditions and/or treatments. Herein, we provide a method to study components of protein aggregates found in AD and Lewy body dementia such as the amyloid-beta peptide and the alpha-synuclein. Our method can thus be adapted for the analysis of the proteome and insoluble proteins extract from human brain tissue and mice models too. In parallel, it may provide useful information for the study of molecular and cellular pathways involved in neurodegenerative diseases as well as potential novel biomarkers and therapeutic targets.

A common protein extraction protocol using urea/thiourea/SDS buffer for human and mice brain tissue allows indentification of proteins by 2D-DIGE and their subsequent characterization by mini 2DE immunoblotting. This method enables one to obtain more reproducible and reliable results from human biopsies and experimental models.

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or ...

Neuroscience

From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a consequence, for the clinical management of patients. In the present work we will describe an optimized proteomic approach for the identification of molecules involved in the progression of Chronic Lymphocytic Leukemia (CLL). In detail, leukemic cell lysates are resolved by 2-dimensional Electrophoresis (2DE) and visualized as &#8220;spots&#8221; on the 2DE gels. Comparative analysis of proteomic maps allows the identification of differentially expressed proteins (in terms of abundance and post-translational modifications) that are picked, isolated and identified by Mass Spectrometry (MS). The biological function of the identified candidates can be tested by different assays (i.e. migration, adhesion and F-actin polymerization), that we have optimized for primary leukemic cells.

Here we describe a protocol that couples two proteomic techniques, namely 2-dimensional Electrophoresis (2DE) and Mass Spectrometry (MS), to identify differentially expressed/post-translational modified proteins among two or more groups of primary samples. This approach, together with functional experiments, allows the identification and characterization of prognostic markers/therapeutic targets.

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a ...

Medicine

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.

In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry.

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...

A Streamlined Approach for Mass Spectrometry-Based Proteomics Using Selected Tissue Regions

Mass spectrometry (MS)-based proteomics enables comprehensive proteome analysis across a wide range of biological samples, including cells, tissues, and body fluids. Formalin-fixed, paraffin-embedded (FFPE) tissue sections, commonly used for long-term archiving, have emerged as valuable resources for proteomic studies. Beyond their storage benefits, researchers can isolate regions of interest (ROIs) from normal tissue regions through collaborative efforts with pathologists. Despite this potential, a streamlined approach for proteomic experiments encompassing ROI isolation, proteomic sample preparation, and MS analysis remains lacking. In this protocol, an integrated workflow that combines macrodissection of ROIs, suspension trapping-based sample preparation, and high-throughput MS analysis is presented. Through this approach, the ROIs of patients' FFPE tissues, consisting of benign serous cystic neoplasms (SCN) and precancerous intraductal papillary mucinous neoplasms (IPMN) diagnosed by pathologists, were macrodissected, collected, and analyzed, resulting in high proteome coverage. Furthermore, molecular differences between the two distinct pancreatic cystic neoplasms were successfully identified, thus demonstrating the applicability of this approach for advancing proteomic research with FFPE tissues.

Here, we establish a mass spectrometry-based proteomic method using isolated regions of interest in formalin-fixed, paraffin-embedded tissue sections. This protocol is used to analyze proteome from specific tissue areas in archived formalin-fixed, paraffin-embedded tissue sections.

Mass spectrometry (MS)-based proteomics enables comprehensive proteome analysis across a wide range of biological samples, including cells, tissues, and ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

Biology

High-throughput and Deep-proteome Profiling by 16-plex Tandem Mass Tag Labeling Coupled with Two-dimensional Chromatography and Mass Spectrometry

Isobaric tandem mass tag (TMT) labeling is widely used in proteomics because of its high multiplexing capacity and deep proteome coverage. Recently, an expanded 16-plex TMT method has been introduced, which further increases the throughput of proteomic studies. In this manuscript, we present an optimized protocol for 16-plex TMT-based deep-proteome profiling, including protein sample preparation, enzymatic digestion, TMT labeling reaction, two-dimensional reverse-phase liquid chromatography (LC/LC) fractionation, tandem mass spectrometry (MS/MS), and computational data processing. The crucial quality control steps and improvements in the process specific for the 16-plex TMT analysis are highlighted. This multiplexed process offers a powerful tool for profiling a variety of complex samples such as cells, tissues, and clinical specimens. More than 10,000 proteins and posttranslational modifications such as phosphorylation, methylation, acetylation, and ubiquitination in highly complex biological samples from up to 16 different samples can be quantified in a single experiment, providing a potent tool for basic and clinical research.

Presented here is an optimized high-throughput protocol developed with 16-plex tandem mass tag reagents, enabling quantitative proteome profiling of biological samples. Extensive basic pH fractionation and high-resolution LC-MS/MS mitigate ratio compression and provide deep proteome coverage.

Isobaric tandem mass tag (TMT) labeling is widely used in proteomics because of its high multiplexing capacity and deep proteome coverage. Recently, an ...

Quantitative Proteomics Workflow using Multiple Reaction Monitoring Based Detection of Proteins from Human Brain Tissue

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related disorders continue to be a major contributor of deaths around the world, necessitating the need for even greater understanding of their pathobiology. Traditional antibody-based techniques like western blotting or immunohistochemistry suffer from being low-throughput besides being labor-intensive and qualitative or semi-quantitative. Even conventional mass spectrometry-based shotgun approaches fail to provide conclusive evidence to support a certain hypothesis. Targeted proteomics approaches are largely hypothesis driven and differ from the conventional shotgun proteomics approaches that have been long in use. Multiple reaction monitoring is one such targeted approach that requires the use of a special mass spectrometer called the tandem quadrupole mass spectrometer or triple quadrupole mass spectrometer. In the current study, we have systematically highlighted the major steps involved in performing a successful tandem quadrupole mass spectrometry-based proteomics workflow using human brain tissue with an aim to introduce this workflow to a broader research community.

The protocol aims to introduce the use of a triple quadrupole mass spectrometer for Multiple Reaction Monitoring (MRM) of proteins from clinical samples. We have provided a systematic workflow starting from sample preparation to data analysis for clinical samples with all the necessary precautions to be taken.

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related ...

Two-Dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis (2DGE) is a technique that can resolve thousands of biomolecules from a mixture. This technique involves two distinct separation methods that have been coupled together: isoelectric focusing (IEF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This physically separates compounds across two axes of a gel by their isoelectric points (an electrochemical property) and their molecular weights.
The procedure in this video covers the main concepts of 2DGE and a general procedure for characterizing the composition of a complex protein solution. Three examples of this technique are shown in the applications section, including biomarker detection for disease initiation and progress, monitoring treatment in patients, and the study of proteins following posttranslational modification (PTM).
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Two-dimensional, or 2D, gel electrophoresis is a technique utilizing two distinct separation methods which can separate thousands of proteins from a single mixture. One of the techniques, SDS-PAGE or sodium dodecyl sulfate polyacrylamide gel electrophoresis, cannot fully separate complex mixtures alone. 2D gel electrophoresis couples the SDS-PAGE to a second method, isoelectric focusing or IEF, which separates based on isoelectric points, allowing for the resolution of potentially all proteins in a cell lysate. This video will show the principles of 2D gel electrophoresis, a general procedure, and some of its biomedical applications.
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2D gel electrophoresis begins with IEF as the first dimension. Every protein has a pH value, called the isoelectric point or pI, where the net charge is zero. When a protein is subjected to an electric field, it will move toward the electrode with opposite charge. Samples of interest are loaded onto immobilized pH gradient, or IPG, strips which have embedded ampholytes, molecules containing both acidic and basic groups. An electric field is then applied to the pH gradient strip, causing the proteins to migrate until they reach the pH value matching their pI, where they lose their net charge.
Prior to running the second-dimension, the embedded proteins are treated with SDS, denaturing them and providing a uniform negative charge. Once completed, the IPG strips are placed onto a polyacrylamide gel. An applied electric field draws the proteins toward the anode, with larger proteins moving more slowly through the gel.
Once the protein mixture has been separated according to pI and molecular weight, the proteome map is visualized using stains, and proteins of interest are identified.
Now that we have discussed the principles of 2D gel electrophoresis, let&#39;s go over a typical laboratory procedure.
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Before the experiment can be performed, the proteins must be solubilized into media. Solubilization of the sample is achieved by de-aggregating proteins with a combination of chaotropic agents for disrupting hydrogen-bonding interactions, nonionic detergents to prevent altering of the proteins&#39; charge, reducing agents to break disulfide bonds, and buffers. To remove interfering abundant proteins and other molecules, the material is sequentially extracted by centrifugation, and collection of the resulting pellet; followed by treatment with endonuclease, an enzyme used to consume any DNA that would interfere with the experiment.
Once the proteins have been solubilized, IPG strips are prepared by rinsing with a strip-cleaning solution and left upside-down to dry. Each strip is then assigned a strip holder number. Once ready, the cellular extract is loaded onto the strips in a slow, sliding motion from the negative to the positive end. For the purpose of rehydration, damp blotting paper is placed on top of the electrode and under the gel strips; the IPG strips are then lined onto the IEF instrument. A high electric current is applied, and the proteins begin to migrate.
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Following completion of the first dimension, the gel for SDS-PAGE is prepared in a casting apparatus. The IPG strips are treated by placing them face down in SDS-containing equilibration buffer. The electrophoresis unit is readied with the addition of electrophoresis buffer. The treated IPG strips are collected using tweezers, placed on top of the gel plates, and sealed with agarose-sealing solution. A voltage source then applies an electric field, which is held until the fastest-moving proteins are 1 cm from the bottom of the gel.
After completion of the electrophoresis, the proteins must be visualized. Traditionally this is performed by staining with Coomassie blue or silver nitrate. Proteins of interest may be transferred from the gel, and analyzed by Western blot analysis.
A second identification approach involves excision of the proteins from the gel, digesting them, than analyzing them by mass spectrometry.
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Now that we&#39;ve reviewed a procedure, let&#39;s look at some of the uses for 2D gel electrophoresis.
One of the most common uses for this technique is the identification of molecules involved in disease initiation and progression. 2D gel electrophoresis, coupled with mass spectrometry, can detect the up- or down-regulation of specific proteins in diseased areas in comparison to healthy ones.
Additionally, 2D gel electrophoresis is useful in following the progress of patients&#39; response to a potential therapeutic drug. Specimens may be taken from patients at various timepoints following administration of the treatment. In this way, 2D gel electrophoresis coupled with Western blot or mass spectrometry analysis, can detect proteins associated with negative responses such as inflammation; or the absence of proteins in an alleviated state.
Another use for 2D gel electrophoresis is in the study of protein structure and function following posttranslational modification, or PTM, which are additions to proteins following their translation from mRNA. PTM&#39;s can regulate a variety of functions, including protein signaling, gene expression, or cause oxidative damage. 2D gel electrophoresis is sensitive to modifications such as methylation or acetylation, which can cause a shift in pI as well as the molecular weight.
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You&#39;ve just watched JoVE&#39;s video on 2D gel electrophoresis. This video described the principles of the technique, a typical experimental procedure, and several of its applications in the field of biomedicine.
Thanks for watching!
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Two-dimensional gel electrophoresis (2DGE) is a technique that can resolve thousands of biomolecules from a mixture. This technique involves two distinct ...

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

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