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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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Dynamic range and sensitivity. Glucose grown yeast SILAC-proteomes, mixed at three ratios of H and L, were profiled using two CIMS antibodies, A, CIMS-17-E02 and B, CIMS-33–3D-F06, and mapped to the known protein abundances generated by orthogonal methods (32). In total, the data was based on six capture trials (two for each isotopically labeled spike in proteome ratio mix) per CIMS-antibody (supplemental Tables S3–S5). For Swiss-Prot ID's matching two different synonymous yeast SGD ids and two different values in (32), the highest reported value was plotted. In cases with multiple Swiss-Prot ID's, they were checked in alphabetical order for presence in (32), and if no value was present the second Swiss-Prot id was checked and onwards. A number of identified peptides/proteins were reported to be present in less than 50 copies/cell or had no reported value at all and thereby could not be plotted and are listed separately in a box.
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Figure 2: Dynamic range and sensitivity. Glucose grown yeast SILAC-proteomes, mixed at three ratios of H and L, were profiled using two CIMS antibodies, A, CIMS-17-E02 and B, CIMS-33–3D-F06, and mapped to the known protein abundances generated by orthogonal methods (32). In total, the data was based on six capture trials (two for each isotopically labeled spike in proteome ratio mix) per CIMS-antibody (supplemental Tables S3–S5). For Swiss-Prot ID's matching two different synonymous yeast SGD ids and two different values in (32), the highest reported value was plotted. In cases with multiple Swiss-Prot ID's, they were checked in alphabetical order for presence in (32), and if no value was present the second Swiss-Prot id was checked and onwards. A number of identified peptides/proteins were reported to be present in less than 50 copies/cell or had no reported value at all and thereby could not be plotted and are listed separately in a box.

Mentions: In order to evaluate the dynamic range (and sensitivity) of the GPS platform, glucose grown yeast SILAC-proteome were profiled using two CIMS antibodies, and the identified yeast proteins were then mapped to the known absolute protein abundances generated by orthogonal methods (32) (Fig. 2 and supplemental Tables S5A and S5B). For both CIMS antibodies, the results showed that yeast proteins could be detected in a wide range of abundances, spanning over a million and reaching down to less than 50 copies per cell (Fig. 2). Noteworthy, a significant set of the identified peptides (19 and 27%, respectively) had not previously been reported in PeptideAtlas (supplemental Tables S3B and S4B). Hence, the GPS set-up was shown to display a dynamic range of at least three orders of magnitude, high sensitivity, and a complementary peptide coverage.


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)

Dynamic range and sensitivity. Glucose grown yeast SILAC-proteomes, mixed at three ratios of H and L, were profiled using two CIMS antibodies, A, CIMS-17-E02 and B, CIMS-33–3D-F06, and mapped to the known protein abundances generated by orthogonal methods (32). In total, the data was based on six capture trials (two for each isotopically labeled spike in proteome ratio mix) per CIMS-antibody (supplemental Tables S3–S5). For Swiss-Prot ID's matching two different synonymous yeast SGD ids and two different values in (32), the highest reported value was plotted. In cases with multiple Swiss-Prot ID's, they were checked in alphabetical order for presence in (32), and if no value was present the second Swiss-Prot id was checked and onwards. A number of identified peptides/proteins were reported to be present in less than 50 copies/cell or had no reported value at all and thereby could not be plotted and are listed separately in a box.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Dynamic range and sensitivity. Glucose grown yeast SILAC-proteomes, mixed at three ratios of H and L, were profiled using two CIMS antibodies, A, CIMS-17-E02 and B, CIMS-33–3D-F06, and mapped to the known protein abundances generated by orthogonal methods (32). In total, the data was based on six capture trials (two for each isotopically labeled spike in proteome ratio mix) per CIMS-antibody (supplemental Tables S3–S5). For Swiss-Prot ID's matching two different synonymous yeast SGD ids and two different values in (32), the highest reported value was plotted. In cases with multiple Swiss-Prot ID's, they were checked in alphabetical order for presence in (32), and if no value was present the second Swiss-Prot id was checked and onwards. A number of identified peptides/proteins were reported to be present in less than 50 copies/cell or had no reported value at all and thereby could not be plotted and are listed separately in a box.
Mentions: In order to evaluate the dynamic range (and sensitivity) of the GPS platform, glucose grown yeast SILAC-proteome were profiled using two CIMS antibodies, and the identified yeast proteins were then mapped to the known absolute protein abundances generated by orthogonal methods (32) (Fig. 2 and supplemental Tables S5A and S5B). For both CIMS antibodies, the results showed that yeast proteins could be detected in a wide range of abundances, spanning over a million and reaching down to less than 50 copies per cell (Fig. 2). Noteworthy, a significant set of the identified peptides (19 and 27%, respectively) had not previously been reported in PeptideAtlas (supplemental Tables S3B and S4B). Hence, the GPS set-up was shown to display a dynamic range of at least three orders of magnitude, high sensitivity, and a complementary peptide coverage.

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