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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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Quantitative accuracy and reproducibility.A, Experimentally refined binding motif amino acid sequences as compiled from all MS-MS detected captured peptides (excluding background peptides) in the spike-in proteome experiments (glucose grown SILAC-labeled yeast proteomes mixed at three ratios of H and L) for two CIMS antibodies, clones CIMS17-E02 (97 peptides) and CIMS-33–3D-F06 (142 peptides). The frequency of each individual residue in the last six C-terminal positions is indicated. B, Peptide intensity/ratio distribution for peptides enriched, from predefined proteome mixtures, by the two CIMS antibodies. Data limited to YXR motif-containing peptides for CIMS-17-E02 and DXR motif-containing peptides for CIMS-33–3D-F06. (The two replicate captures for each condition were analyzed as one experiment in MaxQuant. Hence, raw (non-normalized) ratios, as calculated from associated redundant peptide ratios by means of median of the two replicates, are displayed. Furthermore, data limited to peptides with MaxQuant reported ratios in all conditions and replicate capture trials resulting in 39 different peptides for CIMS-17-E02 and 64 different peptides for CIMS-33–3D-F06). Noteworthy, the peptides were ordered based on total intensity, resulting in that peptide number 39 and 64 were the two peptides observed with the highest total intensity. C, The same data as in panel B, but now compensated (normalized) against the corresponding 50/50 ratio measurement set to be 0. D, Reproducibility between replicate capture experiments for CIMS-17-E02 (limited to YXR motif-containing peptides) and CIMS-33–3D-F06 (limited to DXR motif-containing peptides).
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Figure 1: Quantitative accuracy and reproducibility.A, Experimentally refined binding motif amino acid sequences as compiled from all MS-MS detected captured peptides (excluding background peptides) in the spike-in proteome experiments (glucose grown SILAC-labeled yeast proteomes mixed at three ratios of H and L) for two CIMS antibodies, clones CIMS17-E02 (97 peptides) and CIMS-33–3D-F06 (142 peptides). The frequency of each individual residue in the last six C-terminal positions is indicated. B, Peptide intensity/ratio distribution for peptides enriched, from predefined proteome mixtures, by the two CIMS antibodies. Data limited to YXR motif-containing peptides for CIMS-17-E02 and DXR motif-containing peptides for CIMS-33–3D-F06. (The two replicate captures for each condition were analyzed as one experiment in MaxQuant. Hence, raw (non-normalized) ratios, as calculated from associated redundant peptide ratios by means of median of the two replicates, are displayed. Furthermore, data limited to peptides with MaxQuant reported ratios in all conditions and replicate capture trials resulting in 39 different peptides for CIMS-17-E02 and 64 different peptides for CIMS-33–3D-F06). Noteworthy, the peptides were ordered based on total intensity, resulting in that peptide number 39 and 64 were the two peptides observed with the highest total intensity. C, The same data as in panel B, but now compensated (normalized) against the corresponding 50/50 ratio measurement set to be 0. D, Reproducibility between replicate capture experiments for CIMS-17-E02 (limited to YXR motif-containing peptides) and CIMS-33–3D-F06 (limited to DXR motif-containing peptides).

Mentions: To investigate the quantitative accuracy and reproducibility of the GPS measurements, two CIMS antibodies, clones CIMS-17-E02 and CIMS-33–3D-F06, were used to profile glucose grown yeast SILAC-proteomes mixed at three known ratios of H and L (80/20, 50/50, and 20/80) (Fig. 1 and supplemental Tables S3 and S4). In total, two captures per binder and proteome mix, i.e. 12 runs, were performed. First, the sequence of the enriched peptides were determined and matched to the motif specificity of the CIMS antibodies. The results showed that a total of 100 different peptides, including three background peptides, were enriched by CIMS-17-E02, whereas 145 different peptides (three background peptides) were captured by CIMS-33–3D-F06 (Fig. 1A).


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 accuracy and reproducibility.A, Experimentally refined binding motif amino acid sequences as compiled from all MS-MS detected captured peptides (excluding background peptides) in the spike-in proteome experiments (glucose grown SILAC-labeled yeast proteomes mixed at three ratios of H and L) for two CIMS antibodies, clones CIMS17-E02 (97 peptides) and CIMS-33–3D-F06 (142 peptides). The frequency of each individual residue in the last six C-terminal positions is indicated. B, Peptide intensity/ratio distribution for peptides enriched, from predefined proteome mixtures, by the two CIMS antibodies. Data limited to YXR motif-containing peptides for CIMS-17-E02 and DXR motif-containing peptides for CIMS-33–3D-F06. (The two replicate captures for each condition were analyzed as one experiment in MaxQuant. Hence, raw (non-normalized) ratios, as calculated from associated redundant peptide ratios by means of median of the two replicates, are displayed. Furthermore, data limited to peptides with MaxQuant reported ratios in all conditions and replicate capture trials resulting in 39 different peptides for CIMS-17-E02 and 64 different peptides for CIMS-33–3D-F06). Noteworthy, the peptides were ordered based on total intensity, resulting in that peptide number 39 and 64 were the two peptides observed with the highest total intensity. C, The same data as in panel B, but now compensated (normalized) against the corresponding 50/50 ratio measurement set to be 0. D, Reproducibility between replicate capture experiments for CIMS-17-E02 (limited to YXR motif-containing peptides) and CIMS-33–3D-F06 (limited to DXR motif-containing peptides).
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Related In: Results  -  Collection

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Figure 1: Quantitative accuracy and reproducibility.A, Experimentally refined binding motif amino acid sequences as compiled from all MS-MS detected captured peptides (excluding background peptides) in the spike-in proteome experiments (glucose grown SILAC-labeled yeast proteomes mixed at three ratios of H and L) for two CIMS antibodies, clones CIMS17-E02 (97 peptides) and CIMS-33–3D-F06 (142 peptides). The frequency of each individual residue in the last six C-terminal positions is indicated. B, Peptide intensity/ratio distribution for peptides enriched, from predefined proteome mixtures, by the two CIMS antibodies. Data limited to YXR motif-containing peptides for CIMS-17-E02 and DXR motif-containing peptides for CIMS-33–3D-F06. (The two replicate captures for each condition were analyzed as one experiment in MaxQuant. Hence, raw (non-normalized) ratios, as calculated from associated redundant peptide ratios by means of median of the two replicates, are displayed. Furthermore, data limited to peptides with MaxQuant reported ratios in all conditions and replicate capture trials resulting in 39 different peptides for CIMS-17-E02 and 64 different peptides for CIMS-33–3D-F06). Noteworthy, the peptides were ordered based on total intensity, resulting in that peptide number 39 and 64 were the two peptides observed with the highest total intensity. C, The same data as in panel B, but now compensated (normalized) against the corresponding 50/50 ratio measurement set to be 0. D, Reproducibility between replicate capture experiments for CIMS-17-E02 (limited to YXR motif-containing peptides) and CIMS-33–3D-F06 (limited to DXR motif-containing peptides).
Mentions: To investigate the quantitative accuracy and reproducibility of the GPS measurements, two CIMS antibodies, clones CIMS-17-E02 and CIMS-33–3D-F06, were used to profile glucose grown yeast SILAC-proteomes mixed at three known ratios of H and L (80/20, 50/50, and 20/80) (Fig. 1 and supplemental Tables S3 and S4). In total, two captures per binder and proteome mix, i.e. 12 runs, were performed. First, the sequence of the enriched peptides were determined and matched to the motif specificity of the CIMS antibodies. The results showed that a total of 100 different peptides, including three background peptides, were enriched by CIMS-17-E02, whereas 145 different peptides (three background peptides) were captured by CIMS-33–3D-F06 (Fig. 1A).

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