Sample prep for proteomics of breast cancer: proteomics and gene ontology reveal dramatic differences in protein solubilization preferences of radioimmunoprecipitation assay and urea lysis buffers

doi:10.1186/1477-5956-6-30

Proteome Science

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Sample prep for proteomics of breast cancer: proteomics and gene ontology reveal dramatic differences in protein solubilization preferences of radioimmunoprecipitation assay and urea lysis buffers

Lambert CM Ngoka¹^,2

¹Department of Chemistry, Virginia Commonwealth University, 1001 West Main Street, P. O. Box 842006, Richmond, VA 23284-2006, USA

²Department of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, MSB-1, 5001 El Paso Drive, El Paso, TX 79905, USA

author email corresponding author email

Proteome Science 2008, 6:30doi:10.1186/1477-5956-6-30


Published:	24 October 2008

Additional files

Additional file 1:

BLAST Table for discoidin domain receptor 1 (DDR1). BLAST Table for discoidin domain receptor 1 (gi|68533097|dbj|BAE06103.1|/894). For each protein input query, the BLASTmachine generates a BLAST Table [34]. The table shows parameters of the BLAST search, including Sequences producing significant alignments, Gene Name, Accession number, e-Value, align-length, positives, similarity %, hsp and mapping (Ontologies found), for each of the 40 hits requested.

Format: XLS Size: 96KB Download file

This file can be viewed with: Microsoft Excel Viewer

Additional file 2:

MudPIT Mass Spectra of the breast tumor T2-018 TUMOR. The set of 12 MudPIT mass spectra of the RIPA-soluble fraction are shown at left, whereas those for the urea-soluble fraction are shown at right. A typical MudPIT experiment consists of a 12-cycle run in which a 60-minute nano-LC gradient is run for each of: 1. 1D_2 μL sample; 2. 2D_10 μL sample; 3. 2D_0 mM NH₄COO^-; 4. 2D_25 mM NH₄COO^-; 5. 2D_50 mM NH₄COO^-; 6. 2D_75 mM NH₄COO^-; 7. 2D_100 mM NH₄COO^-; 8. 2D_150 mM NH₄COO^-; 9. 2D_200 mM NH₄COO^-; 10. 2D_250 mM NH₄COO^-; 11. 2D_300 mM NH₄COO^-, and 12. 2D_500 mM NH₄COO^-. NH₄COO^-is ammonium formate.

Format: TIFF Size: 8MB Download file

Additional file 3:

MudPIT Mass Spectra of the breast tumor T2-048 TUMOR. MudPIT Mass Spectra of the breast tumor T2-048 TUMOR. Spectra of RIPA-soluble fraction are shown at left, whereas those for the urea-soluble fraction are shown at right.

Format: TIFF Size: 7.4MB Download file

Additional file 4:

MudPIT Mass Spectra of the matched normal breast tissue T2-048 NORMAL. The set of 12 MudPIT mass spectra of the RIPA-soluble fraction are shown at left, whereas those for the urea-soluble fraction are shown at right.

Format: TIFF Size: 7.2MB Download file

Additional file 5:

MudPIT Mass Spectra of the bilateral breast tumor T2-029 TUMOR. The set of 12 MudPIT mass spectra of the RIPA-soluble fraction are shown at left, whereas those for the urea-soluble fraction are shown at right.

Format: TIFF Size: 6.1MB Download file

Additional file 6:

Expanded view of the extracellular region of the Cellular Component DAG for the bilateral proteome T2-029T (UREA). The node filter was reduced to 0 to obtain this complete display. In contrast, lowering the DAG node filter for the RIPA DAG counterpart did not produce appreciable change in the number of nodes displayed within the extracellular region.

Format: PNG Size: 98KB Download file

Additional file 7:

Cellular Component DAG for the proteome T2-048T (RIPA). Extracellular matrix proteins are not seen, even when the node filter is lowered to 10.

Format: PNG Size: 635KB Download file

Additional file 8:

Cellular Component DAG for the proteome T2-048T (UREA). At a node filter setting of 16, there are 31 extracellular matrix proteins. Thus, extracellular matrix proteins are soluble almost exclusively in urea buffer. None is seen in the RIPA buffer fraction of this proteome (Additional File 7).

Format: PNG Size: 1.4MB Download file

Additional file 9:

Cellular Component DAG of the matched normal proteome T2-048N (RIPA). Extracellular matrix proteins are not seen, even at a node filter setting of 14.

Format: PNG Size: 669KB Download file

Additional file 10:

Cellular Component DAG of the matched normal proteome T2-048N (UREA). Twenty-nine extracellular matrix proteins are present in this urea buffer fraction of T2-048N, even when the DAG is displayed with a node filter setting of just 5 (cf. Node filter is 14 in Additional File 9 above); no extracellular matrix proteins are present in the RIPA buffer fraction of this proteome, shown in Additional File 9 above.

Format: PNG Size: 772KB Download file

Additional file 11:

Cellular Component DAG of the bilateral Adenocarcinoma proteome T2-029T (RIPA). Extracellular matrix proteins are not seen, even at a node filter setting of 12.

Format: PNG Size: 771KB Download file

Additional file 12:

Cellular Component DAG of the bilateral Adenocarcinoma proteome T2-029T (UREA). Thirty-seven extracellular matrix proteins are observed, at a node filter setting of 16. No extracellular matrix proteins are observed in the RIPA buffer fraction of this proteome, which is shown in Additional File 11 above.

Format: PNG Size: 861KB Download file

Additional file 13:

Molecular Function DAG for the proteome T2-048T (RIPA). Extracellular matrix structural constituents are not seen, even when the node filter is set at 12.

Format: PNG Size: 643KB Download file

Additional file 14:

Molecular Function DAG of the proteome T2-048T (UREA). The Structural Molecule Activity (SMA) of the urea proteome contains 20 extracellular matrix structural constituents, none of which is observed in the RIPA buffer fraction DAG of Additional File 13 shown above. Thus, extracellular matrix proteins are soluble primarily in urea buffer.

Format: PNG Size: 590KB Download file

Additional file 15:

Molecular Function DAG of the matched normal proteome in RIPA buffer T2-048N (RIPA). Extracellular matrix structural constituents are not seen, even when the node filter is set at 17.

Format: PNG Size: 354KB Download file

Additional file 16:

Molecular Function DAG for the proteome T2-048N (UREA). The Structural Molecule Activity of the urea proteome contains 13 extracellular matrix structural constituents. None is observed in the RIPA buffer fraction DAG of Additional File 15 shown above. Thus, extracellular matrix proteins appear to be soluble primarily in urea buffer.

Format: PNG Size: 338KB Download file

Additional file 17:

Molecular Function DAG of the matched normal proteome T2-029T (RIPA). Extracellular matrix structural constituents are not observed, even at a node filter setting of 19. Thus, extracellular matrix proteins do not appear to be soluble in RIPA buffer.

Format: PNG Size: 477KB Download file

Additional file 18:

Molecular Function DAG for the proteome T2-029T (UREA). The Structural Molecule Activity of the urea proteome contains 25 extracellular matrix structural constituents. None of these constituents is observed in the RIPA buffer fraction DAG of Additional File 17 shown above. Thus, extracellular matrix proteins are soluble primarily in urea buffer.

Format: PNG Size: 377KB Download file