Document Type

Article

Original Publication Date

2008

Journal/Book/Conference Title

Proteome Science

Volume

6

Issue

30

DOI of Original Publication

10.1186/1477-5956-6-30

Comments

Originally published at http://dx.doi.org/10.1186/1477-5956-6-30

Date of Submission

August 2014

Abstract

Background

An important step in the proteomics of solid tumors, including breast cancer, consists of efficiently extracting most of proteins in the tumor specimen. For this purpose, Radio-Immunoprecipitation Assay (RIPA) buffer is widely employed. RIPA buffer's rapid and highly efficient cell lysis and good solubilization of a wide range of proteins is further augmented by its compatibility with protease and phosphatase inhibitors, ability to minimize non-specific protein binding leading to a lower background in immunoprecipitation, and its suitability for protein quantitation.

Results

In this work, the insoluble matter left after RIPA buffer extraction of proteins from breast tumors are subjected to another extraction step, using a urea-based buffer. It is shown that RIPA and urea lysis buffers fractionate breast tissue proteins primarily on the basis of molecular weights. The average molecular weight of proteins that dissolve exclusively in urea buffer is up to 60% higher than in RIPA.

Gene Ontology (GO) and Directed Acyclic Graphs (DAG) are used to map the collective biological and biophysical attributes of the RIPA and urea proteomes. The Cellular Component and Molecular Function annotations reveal protein solubilization preferences of the buffers, especially the compartmentalization and functional distributions.

It is shown that nearly all extracellular matrix proteins (ECM) in the breast tumors and matched normal tissues are found, nearly exclusively, in the urea fraction, while they are mostly insoluble in RIPA buffer. Additionally, it is demonstrated that cytoskeletal and extracellular region proteins are more soluble in urea than in RIPA, whereas for nuclear, cytoplasmic and mitochondrial proteins, RIPA buffer is preferred.

Extracellular matrix proteins are highly implicated in cancer, including their proteinase-mediated degradation and remodelling, tumor development, progression, adhesion and metastasis. Thus, if they are not efficiently extracted by RIPA buffer, important information may be missed in cancer research.

Conclusion

For proteomics of solid tumors, a two-step extraction process is recommended. First, proteins in the tumor specimen should be extracted with RIPA buffer. Second, the RIPA-insoluble material should be extracted with the urea-based buffer employed in this work.

Rights

© 2008 Ngoka; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Is Part Of

VCU Chemistry Publications

1477-5956-6-30-s1.xls (95 kB)
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.

1477-5956-6-30-s2.tiff (8222 kB)
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.

1477-5956-6-30-s3.tiff (7547 kB)
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.

1477-5956-6-30-s4.tiff (7417 kB)
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.

1477-5956-6-30-s5.tiff (6207 kB)
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.

1477-5956-6-30-s6.png (97 kB)
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.

1477-5956-6-30-s7.png (634 kB)
Cellular Component DAG for the proteome T2-048T (RIPA). Extracellular matrix proteins are not seen, even when the node filter is lowered to 10.

1477-5956-6-30-s8.png (1439 kB)
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).

1477-5956-6-30-s9.png (668 kB)
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.

1477-5956-6-30-s10.png (771 kB)
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.

1477-5956-6-30-s11.png (770 kB)
Cellular Component DAG of the bilateral Adenocarcinoma proteome T2-029T (RIPA). 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.

1477-5956-6-30-s12.png (861 kB)
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.

1477-5956-6-30-s13.png (642 kB)
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.

1477-5956-6-30-s14.png (589 kB)
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.

1477-5956-6-30-s15.png (353 kB)
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.

1477-5956-6-30-s16.png (338 kB)
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.

1477-5956-6-30-s17.png (477 kB)
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.

1477-5956-6-30-s18.png (376 kB)
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.

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