PROTEOMIC CHANGES DURING B CELL MATURATION: 2D-DIGE APPROACH





Introduction

2D DIGE image overlays enable easy visual comparison between two protein samples labeled with Cy3 and Cy5 and presented in green and red colors. The more mature B cell type is presented in red color. Triple image overlays enable comparison between the two samples in relative to internal standard sample (Stnd) labeled with Cy2 and presented in blue color.

Explanation for colors in double image overlays:
Yellow: Proteins shared between the two samples.
Green and red: Unique proteins in the sample. In Ramos samples (Gels 5-7) red indicates up-regulation and green down-regulation in response to anti-IgM stimulation.

Triple image overlays:
White: Proteins shared between the three samples (no difference in protein abundance).
Yellow: Proteins shared between the two samples. Protein abundance is high in the two samples in relative to Stnd.
Blue: Protein abundance is negligible in relative to Stnd.
Green and red: Unique proteins in the sample. Protein abundance is high in relative to Stnd and to the other sample.
Turquoise and purple: Protein abundance is higher/lower in relative to the other sample.

Highlighted

Proteins highlighted in red are identified either by PMF or by combining MS and MS/MS spectral data for database searches. Proteins highlighted in blue are identified by comparing their positions on 2D DIGE gel to known proteins in other lymphocyte 2DE databases. Move the mouse pointer over a spot to see the protein name and number referring to Table 1 and Supplementary Tables 2-3. Click on the spot to get UniProt information about the protein.

Original view

Interactive 2D DIGE map. B cell protein samples labeled with Cy3 and Cy5 and separated in nonlinear pH gradient 3-10. Internal standard is labeled with Cy2 and run on the same gel.


 © 2012

 University of Tampere
 IBT Bioinformatics
 Finland

 Lund University
 Department of Experimental Medical Science
 Sweden

 

PROTEOMIC CHANGES DURING B CELL MATURATION: 2D-DIGE APPROACH

Johanna Salonen1,2,3, Gunilla Rönnholm4, Nisse Kalkkinen4 and Mauno Vihinen1,2,3,5

1Institute of Biomedical Technology, University of Tampere, Finland
2BioMediTech, Tampere, Finland
3Research Unit, Tampere University Hospital, Tampere, Finland
4Institute of Biotechnology, University of Helsinki, Finland
5Department of Experimental Medical Science, Lund University, Sweden

INTRODUCTION:

Two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) based proteomics was used to study differential protein abundance in various B cell types during B cell maturation (cell lines are listed in Supplementary Table 1). Further, B cell antigen receptor (BCR) activation induced proteomic changes were studied in anti-IgM stimulated Ramos B cells in a long time series. To view the results, click on 2D-DIGE analysis of B cell maturation. To view our previous results from two-dimensional gel electrophoresis (2D-GE) and MALDI-TOF-MS based study in anti-IgM stimulated Ramos B cells click on Proteome analysis of B cell maturation (Salonen, J. M., Valmu, L., Rönnholm, G., Kalkkinen, N., Vihinen, M., Proteome analysis of B-cell maturation. Proteomics 2006, 6, 5152-5168). At each maturation stage, expression of proteins affects the commitment to move to the next stage. Defects in genes and proteins important for B cell maturation block the maturation on certain stage and cause primary immunodeficiencies (IDbase).

Here you can view 2D-DIGE images representing B cell proteomes at different maturation stages. In 2D-DIGE method three protein samples differentially labeled with Cy3-, Cy5- and Cy2-dyes are separated in the same gel. Double image overlays highlight the differences between two samples labeled with Cy3 and Cy5. Triple image overlays enable visual comparison between two samples (labeled with Cy3 and Cy5) in relative to internal standard (Stnd) labeled with Cy2 and run in the same gel. The Stnd is a pooled reference sample prepared by combining protein extracts from all the samples included in the experiment. A total of 2063 protein spots across eight human B cell lines were clustered on the basis of the relative protein abundance (Supplementary Figure 1 and S2). Bioinformatic analysis of the proteomes revealed shared Gene Ontology (GO) terms and signaling pathways within the clusters of co-expressed proteins (Tables 1-3 and S4. To view all identified proteins (listed in Supplementary Table S2) click on Gel 3 and choose Stnd (Cy2) image.

Tables:

Table 1: Biological process GO terms. Biological process GO terms and their p-values for co-expressed proteins
Table 2: Molecular function GO terms. Molecular function GO terms and their p-values for co-expressed proteins
Table 3: Cellular component GO terms. Cellular component GO terms and their p-values for co-expressed proteins

Supplementary Table S1: B cell lines. List of B cell lines with respective origin, differentiation stages and Ig chain expression.
Supplementary Table S2: Proteins in the B cell proteome database. List of identified proteins with respective UniProt accession numbers, experimental pI and Mr values, number of peptide matches, intensity and sequence coverage, as well as Mascot scores to show the reliability of the identifications (p<0.05).
Supplementary Table S3: Peptides identified from different proteins by fragment ion analysis. List of identified peptides with Mascot scores to show the reliability of the identifications (p<0.05)
Supplementary Table S4: Signalling pathways and identified proteins involved. List of Reactome, KEGG and BioCarta pathways and proteins involved with corresponding p-values.

2D DIGE images:

Gel 1

  • Nalm-1 (Cy3) early pre-B
  • REH (Cy5) pre-B
  • Nalm-1 (Cy3 green) + REH (Cy5 red). Overlay
  • Nalm-1 (Cy3 green) + REH (Cy5 red) + Stnd (Cy2 blue). Overlay

Gel 2

  • 697 (Cy3) pre-B
  • 697 (Cy3 green) + Ramos 2 h (Cy5 red). Overlay
  • 697 (Cy3 green) + Ramos 2 h (Cy5 red) + Stnd (Cy2 blue). Overlay

Gel 3

  • Stnd (Cy2) reference
  • U-266 (Cy3) plasma cell
  • U-266 (Cy3 red) + Ramos 96 h (Cy5 green). Overlay
  • U-266 (Cy3 red) + Ramos 96 h (Cy5 green) + Stnd (Cy2 blue). Overlay

Gel 4

  • 380 (Cy5) early pre-B
  • 380 (Cy5 green) + Ramos 120 h (Cy3 red). Overlay
  • 380 (Cy5 green) + Ramos 120 h (Cy3 red) + Stnd (Cy2 blue). Overlay

Gel 5

  • 2 h anti-IgM stimulated Ramos (Cy3)
  • 2 h anti-IgM stimulated Ramos (Cy3 red) + 2 h control Ramos (Cy5 green). Overlay
  • 2 h anti-IgM stimulated (Cy3 red) Ramos + 2 h control Ramos (Cy5 green) + Stnd (Cy2 blue). Overlay

Gel 6

  • 24 h anti-IgM stimulated Ramos cells (Cy5)
  • 24 h anti-IgM stimulated Ramos (Cy5 red) + control Ramos (Cy3 green). Overlay
  • 24 h anti-IgM stimulated Ramos (Cy5 red) + control Ramos (Cy3 green) + Stnd (Cy2 blue). Overlay

Gel 7

  • 72 h anti-IgM stimulated Ramos cells (Cy5)
  • 72 h anti-IgM stimulated Ramos (Cy5 red) + control Ramos (Cy3 green). Overlay
  • 72 h anti-IgM stimulated Ramos (Cy5 red) + control Ramos (Cy3 green) + Stnd (Cy2 blue). Overlay

Introduction
2D DIGE image overlays enable easy visual comparison between two protein samples labeled with Cy3 and Cy5 and presented in green and red colors. The more mature B cell type is presented in red color. Triple image overlays enable comparison between the two samples in relative to internal standard sample (Stnd) labeled with Cy2 and presented in blue color.
Explanation for colors in double image overlays:
Yellow: Proteins shared between the two samples.
Green and red: Unique proteins in the sample. In Ramos samples (Gels 5-7) red indicates up-regulation and green down-regulation in response to anti-IgM stimulation.
Triple image overlays:
White: Proteins shared between the three samples (no difference in protein abundance).
Yellow: Proteins shared between the two samples. Protein abundance is high in the two samples in relative to Stnd.
Blue: Protein abundance is negligible in relative to Stnd.
Green and red: Unique proteins in the sample. Protein abundance is high in relative to Stnd and to the other sample.
Turquoise and purple: Protein abundance is higher/lower in relative to the other sample.

Highlighted
Proteins highlighted in red are identified either by PMF or by combining MS and MS/MS spectral data for database searches. Proteins highlighted in blue are identified by comparing their positions on 2D DIGE gel to known proteins in other lymphocyte 2DE databases. Move the mouse pointer over a spot to see the protein name and number referring to Table 1 and Supplementary Tables 2-3. Click on the spot to get UniProt information about the protein.


Original view
 Interactive 2D DIGE map. B cell protein samples labeled with Cy3 and Cy5 and separated in nonlinear pH gradient 3-10. Internal standard is labeled with Cy2 and run on the same gel.