Name: from a 2de-gel spot to protein function: lesson learned from hs1 in chronic lymphocytic leukemia
Uploaded: 2014-10-19T16:00:00
Description: Watch this Scientific Journal Video about From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia at JoVE.com

We thank the Lymphoid Malignancies Unit, Elisa ten Hacken, Lydia Scarf&#242;,&#160;Massimo Alessio, Antonio Conti, Angela Bachi, and Angela Cattaneo.
This project was supported by: Associazione Italiana per la Ricerca sul Cancro AIRC (Investigator Grant and Special Program Molecular Clinical Oncology &#8211; 5 per mille #9965)
C.S. and B.A designed the study, performed the experiments, analyzed the data and wrote the manuscript. M.T.S.B., U.R., F.B. P.R. assisted in writing the manuscript. P.G. and F.C.C. designed the study provided patients&#8217; samples and clinical data.

NOTE: All tissue samples were obtained with approval of the institutional review board of San Raffaele Hospital (Milan, Italy).
1. Human Tissue Samples and Cell Purification (Figure 1)
NOTE: Leukemic lymphocytes were obtained from the Peripheral Blood (PB) of CLL patients, diagnosed according to Mulligan et al. 9
1.1) Leukemic Cell Separation from PB
<ol>
	<li>Collect PB in vacutainer blood collection tubes with Sodium ...

<ol>
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The aim of this manuscript is to describe an optimized protocol for the identification of molecules involved in the pathogenesis of CLL, even if this approach can be extended to other diseases and/or other sample types16-18. By comparing the proteomic profiles of 2 subsets of CLL patients, good vs bad prognosis19, we demonstrated that they differ in the phosphorylation status of HS1 and that its activation affects the migration and adhesion capacity of leukemic cells.
<p class="jove_con...

Chronic Lymphocytic Leukemia (CLL), the most common adult leukemia in the Western countries, derives from the accumulation of monoclonal neoplastic CD5+ B lymphocytes and is clinically very heterogeneous. It may be present as a pre-leukemic form, defined as monoclonal B-cell lymphocytosis (MBL) 1,2,3. In other cases the disease can appear either with an indolent clinical course, that can remain stable for years before needing treatment, or as a progressive chemorefractory disease with dismal prognosis despite therapy. Finally, it may progress into a frequently lethal high grade lymphoma (Richter&#8217;s Syndrome-RS) 2,3,4. Patients are usually classified in two main subsets: good and bad prognosis, based on a set of prognostic factors that provides complementary information on predictors of disease outcome and survival. Clinical heterogeneity likely reflects biological heterogeneity. A number of genetic, microenvironmental and cellular factors have been shown to concur to disease pathogenesis though no unifying mechanisms have been found 5. In detail, several studies have demonstrated that differences in the clinical course of the disease can be partially explained by the presence (or the absence) of some biological markers that have a prognostic value 6,7,8. These data demonstrated that a better understanding of the disease biology could provide additional hints for the clinical management of the pathology, by the identification of both prognostic markers and therapeutic targets.
The goal of the present paper is to show how a combination of different proteomic techniques can be used for the identification of proteins involved in CLL onset and progression. Our approach demonstrates that it is possible to join together basic proteomic and clinical data5.
In the present workflow, primary leukemic cells from selected patients (good vs bad prognosis) are isolated and cell lysates are then used to obtain proteomic maps. 2DE allows the characterization of the proteomic profile of a cell population, including post-translational modifications, thus giving indirect information on the biological activity of each protein. Whole cell lysates are resolved by a first dimension run based on the isoelectric point, followed by a second dimension run on a polyacrylamide gel that resolves proteins based on their molecular weight. Comparative analysis of proteomic maps allows the identification of differentially expressed proteins (both in terms of abundance and post-translational modifications) as spots on the gel that can be cut and analyzed by Mass Spectrometry. The role of each candidate can be then exploited by different assays.
This approach, restricted to CLL in the current manuscript, can be easily expanded to other diseases/samples, thus providing information about the proteomic/biological differences between two groups (i.e. pathologic vs normal, stimulated vs unstimulated, wild-type vs knockdown).

<table border="0" cellpadding="0" cellspacing="0" width="796">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:359px;">Name of Material/ Equipment</td>
			<td style="width:160px;">Company</td>
			<td style="width:111px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:359px;">Acetonitril</td>
			<td style="width:160px;">MERCK</td>
			<td style="width:111px;">61830025001730&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Ammonium Bicarbonate</td>
			<td>SIGMA</td>
			<td style="width:111px;">A6141-500G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">1,4-Dithioerythritol</td>
			<td>SIGMA</td>
			<td style="width:111px;">D9680-5G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Iodoacetamide</td>
			<td>SIGMA</td>
			<td style="width:111px;">I6125-25G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Calcium chloride dihydrate</td>
			<td>SIGMA</td>
			<td style="width:111px;">223506-25G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Trypsin, Sequencing Grade</td>
			<td style="width:160px;">ROCHE</td>
			<td style="width:111px;">11418475001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#945;-cyano-4-hydroxycinnamic acid</td>
			<td>SIGMA</td>
			<td style="width:111px;">C2020-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Trifluoroacetic acid</td>
			<td>SIGMA</td>
			<td style="width:111px;">T6580</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:359px;">ZipTip&#181;-C18</td>
			<td>MILLIPORE</td>
			<td>ZTC18M096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">RosetteSep Human B Cell Enrichment Cocktail</td>
			<td>STEMCELL</td>
			<td>15064</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">PBS</td>
			<td>EUROCLONE</td>
			<td>ECB4004L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">FBS</td>
			<td>DOMINIQUE DUTSCHER</td>
			<td>S1810</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Lymphoprep</td>
			<td>SENTINEL DIAGNOSTICS</td>
			<td>1114547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Trypan Blue</td>
			<td>SIGMA</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">CD19</td>
			<td>BECKMAN COULTER</td>
			<td>082A07770</td>
			<td>ECD</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">CD5</td>
			<td>BECKMAN COULTER</td>
			<td>082A07754</td>
			<td>PC5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">CD3</td>
			<td>BECKMAN COULTER</td>
			<td>082A07746</td>
			<td>FITC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">CD14</td>
			<td>BD</td>
			<td>345785</td>
			<td>PE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">CD16</td>
			<td>BECKMAN COULTER</td>
			<td>082A07767</td>
			<td>PC5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">CD56</td>
			<td>BECKMAN COULTER</td>
			<td>082A07789</td>
			<td>PC5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Ettan IPGphor 3 Isoelectric Focusing Unit</td>
			<td style="width:160px;">GE-Healthcare</td>
			<td>11-0033-64&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Urea</td>
			<td>SIGMA</td>
			<td>U6504</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Tris-Hcl Buffer</td>
			<td>BIO-RAD</td>
			<td>161-0799</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">CHAPS</td>
			<td>SIGMA</td>
			<td>C2020-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">DTT</td>
			<td>SIGMA</td>
			<td>D9163-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">IPGstrips</td>
			<td>GE-Healthcare</td>
			<td>17-1233-01&#160;</td>
			<td>Linear pH 4-7 18cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">IPGbuffer</td>
			<td>GE-Healthcare</td>
			<td>17-6000-86</td>
			<td>pH 4-7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Glycerol</td>
			<td>SIGMA</td>
			<td>G6279</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">PROTEAN II XL Cell</td>
			<td>BIO-RAD</td>
			<td>165-3188</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">SDS</td>
			<td>INVITROGEN</td>
			<td>NP0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Acrilamide</td>
			<td>BIO-RAD</td>
			<td>161-0156</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Agarosio</td>
			<td>INVITROGEN</td>
			<td>16500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Methanol</td>
			<td>SIGMA</td>
			<td>32213</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Acetic Acid</td>
			<td>VWR</td>
			<td>631000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Formalin</td>
			<td>BIO-OPTICAL</td>
			<td>05-K01009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Ethanol</td>
			<td>VWR</td>
			<td>9832500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Sodium Thiosulfate</td>
			<td>FLUKA</td>
			<td>72049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Silver Nitrate</td>
			<td>FLUKA</td>
			<td>85228</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Molecular Dynamics Personal SI Laser Densitometer</td>
			<td>Amersham Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageMaster 2D Platinum 5.0</td>
			<td>Amersham Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Sodium Carbonate</td>
			<td>MERCK</td>
			<td>A0250292</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">MALDI-TOF Voyager-DE STR</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Data Explorer software (version 3.2)&#160;</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">transwell chambre 6.6 mm diameter</td>
			<td>CORNING</td>
			<td>3421</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">RPMI</td>
			<td>EUROCLONE</td>
			<td>ECB2000L</td>
			<td>WITH L-GLUTAMINE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Gentamicin</td>
			<td>SIGMA</td>
			<td>G1397</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">SDF-1</td>
			<td>PREPROTECH</td>
			<td>300-28A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Flat-bottom 96-well plate</td>
			<td>BECTON DICKINSON</td>
			<td>353072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">BSA</td>
			<td>SANTA CRUZ</td>
			<td>9048-46-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">ICAM-1</td>
			<td>R&#38;D Systems</td>
			<td>720-IC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">CMFDA</td>
			<td>Life Thechnology</td>
			<td>C7025</td>
			<td>Green</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">IgM</td>
			<td>SOUTHERNBIOTECH</td>
			<td>2020-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Paraformaldehyde</td>
			<td>SIGMA</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Saponine</td>
			<td>SIGMA</td>
			<td>S-7900</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Phalloidin&#160;</td>
			<td>Life Thechnology</td>
			<td>A12379</td>
			<td>Alexa fluor 488</td>
		</tr>
	</tbody>
</table>

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.

We isolated primary leukemic B cells form PB of CLL patients and we analyzed proteomic maps. Samples (n = 104) were grouped in two main subsets based on the clinical features of each patient (bad prognosis vs good prognosis) and comparative analysis of 2DE gels was performed.
The analysis allowed identification of spots differentially expressed between the two subsets (in term of presence/absence or shift on the gel, implying changes in post-translational modifications; n &#8776; 16)....

Watch this Scientific Journal Video about From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia at JoVE.com

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

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 ...

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Medicine

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