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Quantitative proteomics targeting classes of motif-containing peptides using immunoaffinity-based mass spectrometry.

Olsson N, James P, Borrebaeck CA, Wingren C - Mol. Cell Proteomics (2012)

Bottom Line: On average, each of six different motif-specific antibodies was found to target about 75 different motif-containing proteins.It is worth noting that a significant set of peptides previously not reported in the PeptideAtlas database was among the profiled targets.Finally, several differentially expressed proteins, with both known and unknown functions, many relevant for the central carbon metabolism, could be detected in the glucose- versus ethanol-cultivated yeast.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunotechnology, Lund University, Lund, Sweden.

ABSTRACT
The development of high-performance technology platforms for generating detailed protein expression profiles, or protein atlases, is essential. Recently, we presented a novel platform that we termed global proteome survey, where we combined the best features of affinity proteomics and mass spectrometry, to probe any proteome in a species independent manner while still using a limited set of antibodies. We used so called context-independent-motif-specific antibodies, directed against short amino acid motifs. This enabled enrichment of motif-containing peptides from a digested proteome, which then were detected and identified by mass spectrometry. In this study, we have demonstrated the quantitative capability, reproducibility, sensitivity, and coverage of the global proteome survey technology by targeting stable isotope labeling with amino acids in cell culture-labeled yeast cultures cultivated in glucose or ethanol. The data showed that a wide range of motif-containing peptides (proteins) could be detected, identified, and quantified in a highly reproducible manner. On average, each of six different motif-specific antibodies was found to target about 75 different motif-containing proteins. Furthermore, peptides originating from proteins spanning in abundance from over a million down to less than 50 copies per cell, could be targeted. It is worth noting that a significant set of peptides previously not reported in the PeptideAtlas database was among the profiled targets. The quantitative data corroborated well with the corresponding data generated after conventional strong cation exchange fractionation of the same samples. Finally, several differentially expressed proteins, with both known and unknown functions, many relevant for the central carbon metabolism, could be detected in the glucose- versus ethanol-cultivated yeast. Taken together, the study demonstrated the potential of our immunoaffinity-based mass spectrometry platform for reproducible quantitative proteomics targeting classes of motif-containing peptides.

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Related in: MedlinePlus

Quantitative data—comparison of GPS and SCX. SILAC-labeled yeast proteomes, cultivated in ethanol or glucose, were profiled using GPS (based on six CIMS antibodies) and SCX. The correlation between the data sets was examined. A, all identified peptides for biosample 1 (isotopic amino acids present in ethanol condition). B, normalized data for all identified peptides in biosample 1 (isotopic amino acids present in ethanol condition). C, all identified proteins for biosample 1 (isotopic amino acids present in ethanol condition). D, normalized data for all identified proteins in biosample 1 (isotopic amino acids present in ethanol condition).
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Figure 4: Quantitative data—comparison of GPS and SCX. SILAC-labeled yeast proteomes, cultivated in ethanol or glucose, were profiled using GPS (based on six CIMS antibodies) and SCX. The correlation between the data sets was examined. A, all identified peptides for biosample 1 (isotopic amino acids present in ethanol condition). B, normalized data for all identified peptides in biosample 1 (isotopic amino acids present in ethanol condition). C, all identified proteins for biosample 1 (isotopic amino acids present in ethanol condition). D, normalized data for all identified proteins in biosample 1 (isotopic amino acids present in ethanol condition).

Mentions: Next, we determined the correlation between the quantitative data generated using GPS and SCX, again targeting SILAC-proteomes generated from S. cerevisiae, cultivated in glucose or ethanol (Fig. 4). The results showed that a high correlation between the quantitative data was obtained on both the peptide (r2-values of 0.86 and 0.82) and protein (r2-values of 0.91 and 0.89) levels covering a large concentration span of various analytes, demonstrating the quantitative capability of GPS.


Quantitative proteomics targeting classes of motif-containing peptides using immunoaffinity-based mass spectrometry.

Olsson N, James P, Borrebaeck CA, Wingren C - Mol. Cell Proteomics (2012)

Quantitative data—comparison of GPS and SCX. SILAC-labeled yeast proteomes, cultivated in ethanol or glucose, were profiled using GPS (based on six CIMS antibodies) and SCX. The correlation between the data sets was examined. A, all identified peptides for biosample 1 (isotopic amino acids present in ethanol condition). B, normalized data for all identified peptides in biosample 1 (isotopic amino acids present in ethanol condition). C, all identified proteins for biosample 1 (isotopic amino acids present in ethanol condition). D, normalized data for all identified proteins in biosample 1 (isotopic amino acids present in ethanol condition).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3412966&req=5

Figure 4: Quantitative data—comparison of GPS and SCX. SILAC-labeled yeast proteomes, cultivated in ethanol or glucose, were profiled using GPS (based on six CIMS antibodies) and SCX. The correlation between the data sets was examined. A, all identified peptides for biosample 1 (isotopic amino acids present in ethanol condition). B, normalized data for all identified peptides in biosample 1 (isotopic amino acids present in ethanol condition). C, all identified proteins for biosample 1 (isotopic amino acids present in ethanol condition). D, normalized data for all identified proteins in biosample 1 (isotopic amino acids present in ethanol condition).
Mentions: Next, we determined the correlation between the quantitative data generated using GPS and SCX, again targeting SILAC-proteomes generated from S. cerevisiae, cultivated in glucose or ethanol (Fig. 4). The results showed that a high correlation between the quantitative data was obtained on both the peptide (r2-values of 0.86 and 0.82) and protein (r2-values of 0.91 and 0.89) levels covering a large concentration span of various analytes, demonstrating the quantitative capability of GPS.

Bottom Line: On average, each of six different motif-specific antibodies was found to target about 75 different motif-containing proteins.It is worth noting that a significant set of peptides previously not reported in the PeptideAtlas database was among the profiled targets.Finally, several differentially expressed proteins, with both known and unknown functions, many relevant for the central carbon metabolism, could be detected in the glucose- versus ethanol-cultivated yeast.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunotechnology, Lund University, Lund, Sweden.

ABSTRACT
The development of high-performance technology platforms for generating detailed protein expression profiles, or protein atlases, is essential. Recently, we presented a novel platform that we termed global proteome survey, where we combined the best features of affinity proteomics and mass spectrometry, to probe any proteome in a species independent manner while still using a limited set of antibodies. We used so called context-independent-motif-specific antibodies, directed against short amino acid motifs. This enabled enrichment of motif-containing peptides from a digested proteome, which then were detected and identified by mass spectrometry. In this study, we have demonstrated the quantitative capability, reproducibility, sensitivity, and coverage of the global proteome survey technology by targeting stable isotope labeling with amino acids in cell culture-labeled yeast cultures cultivated in glucose or ethanol. The data showed that a wide range of motif-containing peptides (proteins) could be detected, identified, and quantified in a highly reproducible manner. On average, each of six different motif-specific antibodies was found to target about 75 different motif-containing proteins. Furthermore, peptides originating from proteins spanning in abundance from over a million down to less than 50 copies per cell, could be targeted. It is worth noting that a significant set of peptides previously not reported in the PeptideAtlas database was among the profiled targets. The quantitative data corroborated well with the corresponding data generated after conventional strong cation exchange fractionation of the same samples. Finally, several differentially expressed proteins, with both known and unknown functions, many relevant for the central carbon metabolism, could be detected in the glucose- versus ethanol-cultivated yeast. Taken together, the study demonstrated the potential of our immunoaffinity-based mass spectrometry platform for reproducible quantitative proteomics targeting classes of motif-containing peptides.

Show MeSH
Related in: MedlinePlus