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Extensive quantitative remodeling of the proteome between normal colon tissue and adenocarcinoma.

Wiśniewski JR, Ostasiewicz P, Duś K, Zielińska DF, Gnad F, Mann M - Mol. Syst. Biol. (2012)

Bottom Line: Functionally similar changes in the proteome were observed comparing rapidly growing and differentiated CaCo-2 cells.In contrast, there was minimal proteomic remodeling between primary cancer and metastases, suggesting that no drastic proteome changes are necessary for the tumor to propagate in a different tissue context.Our proteomic data set furthermore allows mapping quantitative changes of functional protein classes, enabling novel insights into the biology of colon cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Proteomics and Signal Transduction, Max-Planck-Institute of Biochemistry, Martinsried, Germany. jwisniew@biochem.mpg.de

ABSTRACT
We report a proteomic analysis of microdissected material from formalin-fixed and paraffin-embedded colorectal cancer, quantifying > 7500 proteins between patient matched normal mucosa, primary carcinoma, and nodal metastases. Expression levels of 1808 proteins changed significantly between normal and cancer tissues, a much larger fraction than that reported in transcript-based studies. Tumor cells exhibit extensive alterations in the cell-surface and nuclear proteomes. Functionally similar changes in the proteome were observed comparing rapidly growing and differentiated CaCo-2 cells. In contrast, there was minimal proteomic remodeling between primary cancer and metastases, suggesting that no drastic proteome changes are necessary for the tumor to propagate in a different tissue context. Additionally, we introduce a new way to determine protein copy numbers per cell without protein standards. Copy numbers estimated in enterocytes and cancer cells are in good agreement with CaCo-2 and HeLa cells and with the literature data. Our proteomic data set furthermore allows mapping quantitative changes of functional protein classes, enabling novel insights into the biology of colon cancer.

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Validation of total protein approach (TPA) for estimation of absolute copy numbers. (A) TPA calculation applied to analysis of a mixture of standard proteins (UPS2; Sigma). Protein standards were solubilized in SDS containing buffer and processed with the FASP method using trypsin. The digest was analyzed by LC-MS/MS using 4 h acetonitrile gradient. The protein concentrations were calculated either using directly protein intensities or iBAQ values. (B) Comparison of protein copy numbers in HeLa cells determined using isotope-labeled standards (SILAC-PrEST) with values obtained by label-free protein intensity-based TPA calculation. HeLa cell lysates were analyzed using the MED FASP-SAX method (Wisniewski and Mann, 2012). The SILAC-PrEST values are from Zeiler et al (2012). (C) Comparison of the protein copy numbers calculated using data from the current analysis (B) and a previously published analysis of HeLa cells (Wisniewski et al, 2009b).
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f7: Validation of total protein approach (TPA) for estimation of absolute copy numbers. (A) TPA calculation applied to analysis of a mixture of standard proteins (UPS2; Sigma). Protein standards were solubilized in SDS containing buffer and processed with the FASP method using trypsin. The digest was analyzed by LC-MS/MS using 4 h acetonitrile gradient. The protein concentrations were calculated either using directly protein intensities or iBAQ values. (B) Comparison of protein copy numbers in HeLa cells determined using isotope-labeled standards (SILAC-PrEST) with values obtained by label-free protein intensity-based TPA calculation. HeLa cell lysates were analyzed using the MED FASP-SAX method (Wisniewski and Mann, 2012). The SILAC-PrEST values are from Zeiler et al (2012). (C) Comparison of the protein copy numbers calculated using data from the current analysis (B) and a previously published analysis of HeLa cells (Wisniewski et al, 2009b).

Mentions: Recent large-scale proteomic analyses estimated protein copy numbers per cell by extrapolating from added standards (Beck et al, 2011; Schwanhausser et al, 2011). Here, we determine copy numbers per cell from our data simply on the basis of individual LFQ intensities compared with the total MS signal of the measured proteome. We named this method as total protein approach (TPA). By dividing these values by the molecular weight and multiplying by the Avogadro constant and by the protein content of a single cell, they can be converted into copy number values. To validate this approach, we first used a mixture of proteins with known protein concentrations. We found a linear response of the calculated protein amount with the amount of protein digested and measured by LC-MS/MS (Figure 7A). Calculations using summed peptide intensities or the iBAQ algorithm resulted in similar values. Next, we applied our calculation method to an LC-MS/MS analysis of a HeLa cell lysate. We compared the copy numbers of 23 proteins for which such values have recently been measured using stable isotope-labeled PrEST standards (Zeiler et al, 2012). We found that the values obtained by the TPA and SILAC-PrEST approaches were similar (r2=0.85; Figure 7B). This is even more remarkable considering that the SILAC-PrEST study used standard tryptic digestion and was performed on an Orbitrap Velos instrument whereas the TPA analysis employed two enzyme digestion and SAX fractionation (MED-FASP-SAX) followed by peptide analysis on the Q Exactive mass spectrometer. Next, we employed the TPA method to calculate protein copy numbers from a HeLa data set generated using FASP, OFFGEL peptide separation, and Orbitrap Velos analysis (Wisniewski et al, 2009b) and compared the values with the copy numbers obtained in this study. Figure 7C shows that the copy number values are similar in both experiments (r2=0.87). This evaluation demonstrates that the TPA-based copy number estimation is applicable to diverse large-scale proteomic data set generated in the past.


Extensive quantitative remodeling of the proteome between normal colon tissue and adenocarcinoma.

Wiśniewski JR, Ostasiewicz P, Duś K, Zielińska DF, Gnad F, Mann M - Mol. Syst. Biol. (2012)

Validation of total protein approach (TPA) for estimation of absolute copy numbers. (A) TPA calculation applied to analysis of a mixture of standard proteins (UPS2; Sigma). Protein standards were solubilized in SDS containing buffer and processed with the FASP method using trypsin. The digest was analyzed by LC-MS/MS using 4 h acetonitrile gradient. The protein concentrations were calculated either using directly protein intensities or iBAQ values. (B) Comparison of protein copy numbers in HeLa cells determined using isotope-labeled standards (SILAC-PrEST) with values obtained by label-free protein intensity-based TPA calculation. HeLa cell lysates were analyzed using the MED FASP-SAX method (Wisniewski and Mann, 2012). The SILAC-PrEST values are from Zeiler et al (2012). (C) Comparison of the protein copy numbers calculated using data from the current analysis (B) and a previously published analysis of HeLa cells (Wisniewski et al, 2009b).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3472694&req=5

f7: Validation of total protein approach (TPA) for estimation of absolute copy numbers. (A) TPA calculation applied to analysis of a mixture of standard proteins (UPS2; Sigma). Protein standards were solubilized in SDS containing buffer and processed with the FASP method using trypsin. The digest was analyzed by LC-MS/MS using 4 h acetonitrile gradient. The protein concentrations were calculated either using directly protein intensities or iBAQ values. (B) Comparison of protein copy numbers in HeLa cells determined using isotope-labeled standards (SILAC-PrEST) with values obtained by label-free protein intensity-based TPA calculation. HeLa cell lysates were analyzed using the MED FASP-SAX method (Wisniewski and Mann, 2012). The SILAC-PrEST values are from Zeiler et al (2012). (C) Comparison of the protein copy numbers calculated using data from the current analysis (B) and a previously published analysis of HeLa cells (Wisniewski et al, 2009b).
Mentions: Recent large-scale proteomic analyses estimated protein copy numbers per cell by extrapolating from added standards (Beck et al, 2011; Schwanhausser et al, 2011). Here, we determine copy numbers per cell from our data simply on the basis of individual LFQ intensities compared with the total MS signal of the measured proteome. We named this method as total protein approach (TPA). By dividing these values by the molecular weight and multiplying by the Avogadro constant and by the protein content of a single cell, they can be converted into copy number values. To validate this approach, we first used a mixture of proteins with known protein concentrations. We found a linear response of the calculated protein amount with the amount of protein digested and measured by LC-MS/MS (Figure 7A). Calculations using summed peptide intensities or the iBAQ algorithm resulted in similar values. Next, we applied our calculation method to an LC-MS/MS analysis of a HeLa cell lysate. We compared the copy numbers of 23 proteins for which such values have recently been measured using stable isotope-labeled PrEST standards (Zeiler et al, 2012). We found that the values obtained by the TPA and SILAC-PrEST approaches were similar (r2=0.85; Figure 7B). This is even more remarkable considering that the SILAC-PrEST study used standard tryptic digestion and was performed on an Orbitrap Velos instrument whereas the TPA analysis employed two enzyme digestion and SAX fractionation (MED-FASP-SAX) followed by peptide analysis on the Q Exactive mass spectrometer. Next, we employed the TPA method to calculate protein copy numbers from a HeLa data set generated using FASP, OFFGEL peptide separation, and Orbitrap Velos analysis (Wisniewski et al, 2009b) and compared the values with the copy numbers obtained in this study. Figure 7C shows that the copy number values are similar in both experiments (r2=0.87). This evaluation demonstrates that the TPA-based copy number estimation is applicable to diverse large-scale proteomic data set generated in the past.

Bottom Line: Functionally similar changes in the proteome were observed comparing rapidly growing and differentiated CaCo-2 cells.In contrast, there was minimal proteomic remodeling between primary cancer and metastases, suggesting that no drastic proteome changes are necessary for the tumor to propagate in a different tissue context.Our proteomic data set furthermore allows mapping quantitative changes of functional protein classes, enabling novel insights into the biology of colon cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Proteomics and Signal Transduction, Max-Planck-Institute of Biochemistry, Martinsried, Germany. jwisniew@biochem.mpg.de

ABSTRACT
We report a proteomic analysis of microdissected material from formalin-fixed and paraffin-embedded colorectal cancer, quantifying > 7500 proteins between patient matched normal mucosa, primary carcinoma, and nodal metastases. Expression levels of 1808 proteins changed significantly between normal and cancer tissues, a much larger fraction than that reported in transcript-based studies. Tumor cells exhibit extensive alterations in the cell-surface and nuclear proteomes. Functionally similar changes in the proteome were observed comparing rapidly growing and differentiated CaCo-2 cells. In contrast, there was minimal proteomic remodeling between primary cancer and metastases, suggesting that no drastic proteome changes are necessary for the tumor to propagate in a different tissue context. Additionally, we introduce a new way to determine protein copy numbers per cell without protein standards. Copy numbers estimated in enterocytes and cancer cells are in good agreement with CaCo-2 and HeLa cells and with the literature data. Our proteomic data set furthermore allows mapping quantitative changes of functional protein classes, enabling novel insights into the biology of colon cancer.

Show MeSH
Related in: MedlinePlus