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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

Identification repeatability—comparison of GPS and SCX. The reproducibility of the MS/MS identification was evaluated by determining the identification overlap for all replicate runs. A, percentage peptide ID overlap in replicate technical (capture + LC-MS/MS) and biological replicate GPS-assays (based on six CIMS antibodies) (biosample 1, ethanol (K6, R10) - glucose; biosample 2, ethanol - glucose (K6, R10)). It should be noted that the generated values were based on separate capture and LC-MS/MS steps. B, percentage peptide ID overlap for replicate runs (same SCX-fraction analyzed twice with LC-MS/MS) and biological replicate runs for the eight generated SCX fractions.
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Figure 5: Identification repeatability—comparison of GPS and SCX. The reproducibility of the MS/MS identification was evaluated by determining the identification overlap for all replicate runs. A, percentage peptide ID overlap in replicate technical (capture + LC-MS/MS) and biological replicate GPS-assays (based on six CIMS antibodies) (biosample 1, ethanol (K6, R10) - glucose; biosample 2, ethanol - glucose (K6, R10)). It should be noted that the generated values were based on separate capture and LC-MS/MS steps. B, percentage peptide ID overlap for replicate runs (same SCX-fraction analyzed twice with LC-MS/MS) and biological replicate runs for the eight generated SCX fractions.

Mentions: Next, the reproducibility of the MS/MS identification was evaluated for both GPS and SCX (Fig. 5). To this end, the identification overlap was determined for all replicate runs. The results showed that the median identification overlap was 68% for GPS (Fig. 5A), but only 43% for SCX (Fig. 5B). Furthermore, the identification overlap between biological samples was also found to be significantly higher for GPS than for SCX, 62% versus 31%, respectively (Fig. 5). It should be noted that the GPS data was based on two separate captures and LC-MS/MS runs, i.e. technical variation for the entire set-up, while the SCX data was based on the same eluate injected twice for LC-MS/MS analyses, i.e. technical variation only for the LC-MS/MS 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)

Identification repeatability—comparison of GPS and SCX. The reproducibility of the MS/MS identification was evaluated by determining the identification overlap for all replicate runs. A, percentage peptide ID overlap in replicate technical (capture + LC-MS/MS) and biological replicate GPS-assays (based on six CIMS antibodies) (biosample 1, ethanol (K6, R10) - glucose; biosample 2, ethanol - glucose (K6, R10)). It should be noted that the generated values were based on separate capture and LC-MS/MS steps. B, percentage peptide ID overlap for replicate runs (same SCX-fraction analyzed twice with LC-MS/MS) and biological replicate runs for the eight generated SCX fractions.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Identification repeatability—comparison of GPS and SCX. The reproducibility of the MS/MS identification was evaluated by determining the identification overlap for all replicate runs. A, percentage peptide ID overlap in replicate technical (capture + LC-MS/MS) and biological replicate GPS-assays (based on six CIMS antibodies) (biosample 1, ethanol (K6, R10) - glucose; biosample 2, ethanol - glucose (K6, R10)). It should be noted that the generated values were based on separate capture and LC-MS/MS steps. B, percentage peptide ID overlap for replicate runs (same SCX-fraction analyzed twice with LC-MS/MS) and biological replicate runs for the eight generated SCX fractions.
Mentions: Next, the reproducibility of the MS/MS identification was evaluated for both GPS and SCX (Fig. 5). To this end, the identification overlap was determined for all replicate runs. The results showed that the median identification overlap was 68% for GPS (Fig. 5A), but only 43% for SCX (Fig. 5B). Furthermore, the identification overlap between biological samples was also found to be significantly higher for GPS than for SCX, 62% versus 31%, respectively (Fig. 5). It should be noted that the GPS data was based on two separate captures and LC-MS/MS runs, i.e. technical variation for the entire set-up, while the SCX data was based on the same eluate injected twice for LC-MS/MS analyses, i.e. technical variation only for the LC-MS/MS 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