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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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Biological relevance of identified proteins for carbon and ethanol metabolism. The biological relevance of differentially expressed proteins in glucose versus ethanol yeast was evaluated. The GPS assay was based on six CIMS antibodies. A, all identified proteins by GPS for biosample 1 (ethanol (K6, R10) - glucose) plotted as overall fold change as a function of protein intensity in the MS. Protein ratios are color-coded according to their ratio significance (significance B, as described in (29)). B, all identified proteins by SCX for biosample 1(ethanol (K6, R10) - glucose) plotted as overall fold change as a function of protein intensity in the MS. Protein ratios are color-coded according to their ratio significance (significance B, as described in (29)). C, GPS and SCX analysis of the central carbon metabolism. Protein expression values generated by GPS and SCX were mapped to the central carbon metabolism pathway(s) displayed in a condensed version according to (34). D, Protein-protein interaction maps of GPS identified up- and down-regulated proteins for biosample 1. Thirty-one up-regulated proteins (limited to proteins reported/annotated with a single Swiss-Prot ID, MaxQuant significance B value of p < 0.05 resulting in proteins with ≥5.5-fold up-regulation). Twenty-three down-regulated proteins (limited to proteins reported/annotated with a single Swiss-Prot ID, MaxQuant significance B value of p < 0.1 resulting in proteins with ≥twofold down-regulation).
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Figure 6: Biological relevance of identified proteins for carbon and ethanol metabolism. The biological relevance of differentially expressed proteins in glucose versus ethanol yeast was evaluated. The GPS assay was based on six CIMS antibodies. A, all identified proteins by GPS for biosample 1 (ethanol (K6, R10) - glucose) plotted as overall fold change as a function of protein intensity in the MS. Protein ratios are color-coded according to their ratio significance (significance B, as described in (29)). B, all identified proteins by SCX for biosample 1(ethanol (K6, R10) - glucose) plotted as overall fold change as a function of protein intensity in the MS. Protein ratios are color-coded according to their ratio significance (significance B, as described in (29)). C, GPS and SCX analysis of the central carbon metabolism. Protein expression values generated by GPS and SCX were mapped to the central carbon metabolism pathway(s) displayed in a condensed version according to (34). D, Protein-protein interaction maps of GPS identified up- and down-regulated proteins for biosample 1. Thirty-one up-regulated proteins (limited to proteins reported/annotated with a single Swiss-Prot ID, MaxQuant significance B value of p < 0.05 resulting in proteins with ≥5.5-fold up-regulation). Twenty-three down-regulated proteins (limited to proteins reported/annotated with a single Swiss-Prot ID, MaxQuant significance B value of p < 0.1 resulting in proteins with ≥twofold down-regulation).

Mentions: Finally, the biological relevance of proteins pin-pointed as differentially expressed in glucose versus ethanol cultivated yeast, using GPS and SCX was examined (Fig. 6 and supplemental Fig. S6). The results showed that 27 and 50 differentially expressed proteins (p < 0.01, biosample 1) were identified using GPS (Fig. 6A) and SCX (Fig. 6B), respectively. Focusing on the central carbon metabolism pathways, including the glycolysis, tricarboxylic acid (TCA) cycle, and glyoxylate cycle, several key differentially expressed proteins could be mapped (Fig. 6C). Noteworthy, the GPS set-up and the SCX approach showed coherent profiles.


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)

Biological relevance of identified proteins for carbon and ethanol metabolism. The biological relevance of differentially expressed proteins in glucose versus ethanol yeast was evaluated. The GPS assay was based on six CIMS antibodies. A, all identified proteins by GPS for biosample 1 (ethanol (K6, R10) - glucose) plotted as overall fold change as a function of protein intensity in the MS. Protein ratios are color-coded according to their ratio significance (significance B, as described in (29)). B, all identified proteins by SCX for biosample 1(ethanol (K6, R10) - glucose) plotted as overall fold change as a function of protein intensity in the MS. Protein ratios are color-coded according to their ratio significance (significance B, as described in (29)). C, GPS and SCX analysis of the central carbon metabolism. Protein expression values generated by GPS and SCX were mapped to the central carbon metabolism pathway(s) displayed in a condensed version according to (34). D, Protein-protein interaction maps of GPS identified up- and down-regulated proteins for biosample 1. Thirty-one up-regulated proteins (limited to proteins reported/annotated with a single Swiss-Prot ID, MaxQuant significance B value of p < 0.05 resulting in proteins with ≥5.5-fold up-regulation). Twenty-three down-regulated proteins (limited to proteins reported/annotated with a single Swiss-Prot ID, MaxQuant significance B value of p < 0.1 resulting in proteins with ≥twofold down-regulation).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3412966&req=5

Figure 6: Biological relevance of identified proteins for carbon and ethanol metabolism. The biological relevance of differentially expressed proteins in glucose versus ethanol yeast was evaluated. The GPS assay was based on six CIMS antibodies. A, all identified proteins by GPS for biosample 1 (ethanol (K6, R10) - glucose) plotted as overall fold change as a function of protein intensity in the MS. Protein ratios are color-coded according to their ratio significance (significance B, as described in (29)). B, all identified proteins by SCX for biosample 1(ethanol (K6, R10) - glucose) plotted as overall fold change as a function of protein intensity in the MS. Protein ratios are color-coded according to their ratio significance (significance B, as described in (29)). C, GPS and SCX analysis of the central carbon metabolism. Protein expression values generated by GPS and SCX were mapped to the central carbon metabolism pathway(s) displayed in a condensed version according to (34). D, Protein-protein interaction maps of GPS identified up- and down-regulated proteins for biosample 1. Thirty-one up-regulated proteins (limited to proteins reported/annotated with a single Swiss-Prot ID, MaxQuant significance B value of p < 0.05 resulting in proteins with ≥5.5-fold up-regulation). Twenty-three down-regulated proteins (limited to proteins reported/annotated with a single Swiss-Prot ID, MaxQuant significance B value of p < 0.1 resulting in proteins with ≥twofold down-regulation).
Mentions: Finally, the biological relevance of proteins pin-pointed as differentially expressed in glucose versus ethanol cultivated yeast, using GPS and SCX was examined (Fig. 6 and supplemental Fig. S6). The results showed that 27 and 50 differentially expressed proteins (p < 0.01, biosample 1) were identified using GPS (Fig. 6A) and SCX (Fig. 6B), respectively. Focusing on the central carbon metabolism pathways, including the glycolysis, tricarboxylic acid (TCA) cycle, and glyoxylate cycle, several key differentially expressed proteins could be mapped (Fig. 6C). Noteworthy, the GPS set-up and the SCX approach showed coherent profiles.

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