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A Double-Barrel Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) System to Quantify 96 Interactomes per Day.

Hosp F, Scheltema RA, Eberl HC, Kulak NA, Keilhauer EC, Mayr K, Mann M - Mol. Cell Proteomics (2015)

Bottom Line: The modified LC platform eliminates idle time between measurements, and the high sequencing speed of the Q Exactive HF reduces required measurement time.Thus, sample throughput, sensitivity and LC/MS-MS duty cycle are improved severalfold compared with established workflows.The pipeline can be extended to different types of interaction studies and to other medium complexity proteomes.

View Article: PubMed Central - PubMed

Affiliation: From the ‡Department of Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.

No MeSH data available.


Related in: MedlinePlus

Double-barrel chromatography with 14 min gradients on three pull-downs. (A) Base-peak chromatogram of a biological triplicate RSC8 pull-down run on the double-barrel LC-MS/MS setup. Chromatography in all cases is very reproducible. (B) Comparison of RSC8, SPT7 and SWI3 pull-downs; all measured in triplicates. The matrix of 36 correlation plots reveals high correlations between MaxLFQ intensities within triplicates. (C) Zoom into SPT7_02 versus the SWI3_01 correlation plot. While most proteins were detected with very similar MaxLFQ intensities, the two outlier populations marked in orange (SPT7) and blue (SWI3) represent the different complex members of the distinct protein complexes.
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Figure 4: Double-barrel chromatography with 14 min gradients on three pull-downs. (A) Base-peak chromatogram of a biological triplicate RSC8 pull-down run on the double-barrel LC-MS/MS setup. Chromatography in all cases is very reproducible. (B) Comparison of RSC8, SPT7 and SWI3 pull-downs; all measured in triplicates. The matrix of 36 correlation plots reveals high correlations between MaxLFQ intensities within triplicates. (C) Zoom into SPT7_02 versus the SWI3_01 correlation plot. While most proteins were detected with very similar MaxLFQ intensities, the two outlier populations marked in orange (SPT7) and blue (SWI3) represent the different complex members of the distinct protein complexes.

Mentions: To investigate the reproducibility of protein quantification between different measurements, we acquired PPI data for the yeast chromatin remodelers RSC8, SPT7, and SWI3 with our workflow. Visual inspection of the chromatograms for the RSC8 pull-down, measured in triplicates, already shows a high degree of technical reproducibility for the double barrel system with back pressure matched analytical columns (Fig. 4A). In modern PPI experiments, the number of background binders can be in the thousands as opposed to only a few true interactors. We take advantage of these unspecific binders to estimate reproducibility by calculating the correlation between each pair of the measurements where only the generally small number of true interactors degrade the correlation (21). Most of the detected unspecific binders were indeed reproducibly quantified in all three samples. There was one exception with a slightly reduced Pearson correlation coefficient for the RSC8 pull-down (Fig. 4B), for which we concluded based on the large number of imputed values that the enrichment was not completely successful. A small outlier population observed for each bait protein indeed represented the expected interaction partners (Fig. 4C and Supplemental Fig. S4). Collectively, these results indicate that our double-barrel setup can be operated with very low MS idling time between two independent measurements and achieves high reproducibility at the same time.


A Double-Barrel Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) System to Quantify 96 Interactomes per Day.

Hosp F, Scheltema RA, Eberl HC, Kulak NA, Keilhauer EC, Mayr K, Mann M - Mol. Cell Proteomics (2015)

Double-barrel chromatography with 14 min gradients on three pull-downs. (A) Base-peak chromatogram of a biological triplicate RSC8 pull-down run on the double-barrel LC-MS/MS setup. Chromatography in all cases is very reproducible. (B) Comparison of RSC8, SPT7 and SWI3 pull-downs; all measured in triplicates. The matrix of 36 correlation plots reveals high correlations between MaxLFQ intensities within triplicates. (C) Zoom into SPT7_02 versus the SWI3_01 correlation plot. While most proteins were detected with very similar MaxLFQ intensities, the two outlier populations marked in orange (SPT7) and blue (SWI3) represent the different complex members of the distinct protein complexes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4587330&req=5

Figure 4: Double-barrel chromatography with 14 min gradients on three pull-downs. (A) Base-peak chromatogram of a biological triplicate RSC8 pull-down run on the double-barrel LC-MS/MS setup. Chromatography in all cases is very reproducible. (B) Comparison of RSC8, SPT7 and SWI3 pull-downs; all measured in triplicates. The matrix of 36 correlation plots reveals high correlations between MaxLFQ intensities within triplicates. (C) Zoom into SPT7_02 versus the SWI3_01 correlation plot. While most proteins were detected with very similar MaxLFQ intensities, the two outlier populations marked in orange (SPT7) and blue (SWI3) represent the different complex members of the distinct protein complexes.
Mentions: To investigate the reproducibility of protein quantification between different measurements, we acquired PPI data for the yeast chromatin remodelers RSC8, SPT7, and SWI3 with our workflow. Visual inspection of the chromatograms for the RSC8 pull-down, measured in triplicates, already shows a high degree of technical reproducibility for the double barrel system with back pressure matched analytical columns (Fig. 4A). In modern PPI experiments, the number of background binders can be in the thousands as opposed to only a few true interactors. We take advantage of these unspecific binders to estimate reproducibility by calculating the correlation between each pair of the measurements where only the generally small number of true interactors degrade the correlation (21). Most of the detected unspecific binders were indeed reproducibly quantified in all three samples. There was one exception with a slightly reduced Pearson correlation coefficient for the RSC8 pull-down (Fig. 4B), for which we concluded based on the large number of imputed values that the enrichment was not completely successful. A small outlier population observed for each bait protein indeed represented the expected interaction partners (Fig. 4C and Supplemental Fig. S4). Collectively, these results indicate that our double-barrel setup can be operated with very low MS idling time between two independent measurements and achieves high reproducibility at the same time.

Bottom Line: The modified LC platform eliminates idle time between measurements, and the high sequencing speed of the Q Exactive HF reduces required measurement time.Thus, sample throughput, sensitivity and LC/MS-MS duty cycle are improved severalfold compared with established workflows.The pipeline can be extended to different types of interaction studies and to other medium complexity proteomes.

View Article: PubMed Central - PubMed

Affiliation: From the ‡Department of Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.

No MeSH data available.


Related in: MedlinePlus