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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

Coverage—comparison of GPS and SCX. Yeast SILAC-proteomes, cultivated in ethanol or glucose, were profiled using GPS and SCX. A, Venn diagram showing the number of peptides identified by GPS (based on 6 CIMS antibodies) and SCX; a total number of 4126 peptides were identified of which 417 were detected in both. B, Venn diagram showing the number of proteins identified by GPS and SCX; a total number of 987 proteins were identified of which 382 were detected in both. C, Statistics of all the identified peptides and proteins. D, peptide length plotted for all identified peptides using GPS and SCX. E, peptide length plotted for peptides identified in both biological replicates with a ratio count of ≥2 using GPS and SCX.
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Figure 3: Coverage—comparison of GPS and SCX. Yeast SILAC-proteomes, cultivated in ethanol or glucose, were profiled using GPS and SCX. A, Venn diagram showing the number of peptides identified by GPS (based on 6 CIMS antibodies) and SCX; a total number of 4126 peptides were identified of which 417 were detected in both. B, Venn diagram showing the number of proteins identified by GPS and SCX; a total number of 987 proteins were identified of which 382 were detected in both. C, Statistics of all the identified peptides and proteins. D, peptide length plotted for all identified peptides using GPS and SCX. E, peptide length plotted for peptides identified in both biological replicates with a ratio count of ≥2 using GPS and SCX.

Mentions: To further evaluate the quantitative capability of GPS, the data was compared with that obtained for the same samples analyzed in parallel experiments using one of the main orthogonal approaches within conventional quantitative proteomics, namely SCX LC-MS/MS (Fig. 3 and supplemental Tables S6A–S6C). To this end, SILAC-proteomes generated from S. cerevisiae, cultivated in glucose or ethanol, were profiled using six CIMS antibodies and any differentially expressed analytes were identified and quantified. Although two biological replicates and two technical replicates were applied in both cases, the GPS set-up relied on six CIMS antibodies (in total 24 runs) and the SCX set-up on eight fractions (in total 32 runs).


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)

Coverage—comparison of GPS and SCX. Yeast SILAC-proteomes, cultivated in ethanol or glucose, were profiled using GPS and SCX. A, Venn diagram showing the number of peptides identified by GPS (based on 6 CIMS antibodies) and SCX; a total number of 4126 peptides were identified of which 417 were detected in both. B, Venn diagram showing the number of proteins identified by GPS and SCX; a total number of 987 proteins were identified of which 382 were detected in both. C, Statistics of all the identified peptides and proteins. D, peptide length plotted for all identified peptides using GPS and SCX. E, peptide length plotted for peptides identified in both biological replicates with a ratio count of ≥2 using GPS and SCX.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Coverage—comparison of GPS and SCX. Yeast SILAC-proteomes, cultivated in ethanol or glucose, were profiled using GPS and SCX. A, Venn diagram showing the number of peptides identified by GPS (based on 6 CIMS antibodies) and SCX; a total number of 4126 peptides were identified of which 417 were detected in both. B, Venn diagram showing the number of proteins identified by GPS and SCX; a total number of 987 proteins were identified of which 382 were detected in both. C, Statistics of all the identified peptides and proteins. D, peptide length plotted for all identified peptides using GPS and SCX. E, peptide length plotted for peptides identified in both biological replicates with a ratio count of ≥2 using GPS and SCX.
Mentions: To further evaluate the quantitative capability of GPS, the data was compared with that obtained for the same samples analyzed in parallel experiments using one of the main orthogonal approaches within conventional quantitative proteomics, namely SCX LC-MS/MS (Fig. 3 and supplemental Tables S6A–S6C). To this end, SILAC-proteomes generated from S. cerevisiae, cultivated in glucose or ethanol, were profiled using six CIMS antibodies and any differentially expressed analytes were identified and quantified. Although two biological replicates and two technical replicates were applied in both cases, the GPS set-up relied on six CIMS antibodies (in total 24 runs) and the SCX set-up on eight fractions (in total 32 runs).

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