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Reproducible automated phosphopeptide enrichment using magnetic TiO2 and Ti-IMAC.

Tape CJ, Worboys JD, Sinclair J, Gourlay R, Vogt J, McMahon KM, Trost M, Lauffenburger DA, Lamont DJ, Jørgensen C - Anal. Chem. (2014)

Bottom Line: As a result, automated phosphopeptide enrichment enables statistical analysis of label-free phosphoproteomic samples in a high-throughput manner.This technique uses commercially available, off-the-shelf components and can be easily adopted by any laboratory interested in phosphoproteomic analysis.We provide a free downloadable automated phosphopeptide enrichment program to facilitate uniform interlaboratory collaboration and exchange of phosphoproteomic data sets.

View Article: PubMed Central - PubMed

Affiliation: The Institute of Cancer Research , 237 Fulham Road, London SW3 6JB, United Kingdom.

ABSTRACT
Reproducible, comprehensive phosphopeptide enrichment is essential for studying phosphorylation-regulated processes. Here, we describe the application of hyper-porous magnetic TiO2 and Ti-IMAC microspheres for uniform automated phosphopeptide enrichment. Combining magnetic microspheres with a magnetic particle-handling robot enables rapid (45 min), reproducible (r2 ≥ 0.80) and high-fidelity (>90% purity) phosphopeptide purification in a 96-well format. Automated phosphopeptide enrichment demonstrates reproducible synthetic phosphopeptide recovery across 2 orders of magnitude, "well-to-well" quantitative reproducibility indistinguishable to internal SILAC standards, and robust "plate-to-plate" reproducibility across 5 days of independent enrichments. As a result, automated phosphopeptide enrichment enables statistical analysis of label-free phosphoproteomic samples in a high-throughput manner. This technique uses commercially available, off-the-shelf components and can be easily adopted by any laboratory interested in phosphoproteomic analysis. We provide a free downloadable automated phosphopeptide enrichment program to facilitate uniform interlaboratory collaboration and exchange of phosphoproteomic data sets.

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Automated phosphopeptide enrichment intra-platereproducibility.(a) Light KPC cells were treated with 100 ng/mL EGF for 5 min andSILAC heavy (K +8 Da; R +10 Da) KPC cells were left untreated. EachSILAC population was digested separately and mixed either before (premixed,green) or after (postmixed, blue) automated magnetic TiO2 phosphopeptide enrichment. Phosphopeptides were then analyzed byDDA LC-MS/MS. (b) Pre- and postmixed samples were randomly dividedinto two groups (A and B; n = 3/group). (c–e)The light/heavy SILAC ratios of each population were assessed by Pearsoncorrelation (two-tailed P < 0.0001). LC-MS/MSDDA runs n = 12.
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fig3: Automated phosphopeptide enrichment intra-platereproducibility.(a) Light KPC cells were treated with 100 ng/mL EGF for 5 min andSILAC heavy (K +8 Da; R +10 Da) KPC cells were left untreated. EachSILAC population was digested separately and mixed either before (premixed,green) or after (postmixed, blue) automated magnetic TiO2 phosphopeptide enrichment. Phosphopeptides were then analyzed byDDA LC-MS/MS. (b) Pre- and postmixed samples were randomly dividedinto two groups (A and B; n = 3/group). (c–e)The light/heavy SILAC ratios of each population were assessed by Pearsoncorrelation (two-tailed P < 0.0001). LC-MS/MSDDA runs n = 12.

Mentions: As the automated phosphopeptideenrichment platform subjects eachwell to identical conditions (e.g., uniform incubation times, agitationfrequencies, cycles, etc.), we hypothesized this technique enricheswell-to-well phosphopeptides in a highly reproducible manner. To investigateintraplate fidelity, we used the automated phosphopeptide enrichmentplatform to compare a “gold standard” standard SILACexperiment (samples mixed prior to enrichment)30 with a “split” SILAC experiment (samplescombined postenrichment). To this end, we compared the relative “light”/“heavy”ratios of two distinct SILAC populations mixed either before (premixed)or after (postmixed) enrichment (Figure 3a)(n = 3/group). To simulate a typical biological experimentand evaluate the dynamic range of the workflow, light labeled cellswere treated with 100 ng/mL EGF for 5 min, and heavy labeled cellswere left untreated. In agreement with earlier experiments, this analysisconfirmed intraplate technical replicates enrich approximately equalnumbers of unique phosphopeptides at high purity (>90% phosphorylatedpeptides) (Figure 3b). Crucially, correlatingthe light/heavy ratios of premixed and postmixed phosphopeptide samplesshowed no discernible difference in phosphopeptide enrichment fidelitybetween replicates (r2 = 0.80) (Figure 3c–e). As a result, we propose automated magnetic particlehandling as a highly reproducible platform for uniform intraplatephosphopeptide enrichment.


Reproducible automated phosphopeptide enrichment using magnetic TiO2 and Ti-IMAC.

Tape CJ, Worboys JD, Sinclair J, Gourlay R, Vogt J, McMahon KM, Trost M, Lauffenburger DA, Lamont DJ, Jørgensen C - Anal. Chem. (2014)

Automated phosphopeptide enrichment intra-platereproducibility.(a) Light KPC cells were treated with 100 ng/mL EGF for 5 min andSILAC heavy (K +8 Da; R +10 Da) KPC cells were left untreated. EachSILAC population was digested separately and mixed either before (premixed,green) or after (postmixed, blue) automated magnetic TiO2 phosphopeptide enrichment. Phosphopeptides were then analyzed byDDA LC-MS/MS. (b) Pre- and postmixed samples were randomly dividedinto two groups (A and B; n = 3/group). (c–e)The light/heavy SILAC ratios of each population were assessed by Pearsoncorrelation (two-tailed P < 0.0001). LC-MS/MSDDA runs n = 12.
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Related In: Results  -  Collection

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fig3: Automated phosphopeptide enrichment intra-platereproducibility.(a) Light KPC cells were treated with 100 ng/mL EGF for 5 min andSILAC heavy (K +8 Da; R +10 Da) KPC cells were left untreated. EachSILAC population was digested separately and mixed either before (premixed,green) or after (postmixed, blue) automated magnetic TiO2 phosphopeptide enrichment. Phosphopeptides were then analyzed byDDA LC-MS/MS. (b) Pre- and postmixed samples were randomly dividedinto two groups (A and B; n = 3/group). (c–e)The light/heavy SILAC ratios of each population were assessed by Pearsoncorrelation (two-tailed P < 0.0001). LC-MS/MSDDA runs n = 12.
Mentions: As the automated phosphopeptideenrichment platform subjects eachwell to identical conditions (e.g., uniform incubation times, agitationfrequencies, cycles, etc.), we hypothesized this technique enricheswell-to-well phosphopeptides in a highly reproducible manner. To investigateintraplate fidelity, we used the automated phosphopeptide enrichmentplatform to compare a “gold standard” standard SILACexperiment (samples mixed prior to enrichment)30 with a “split” SILAC experiment (samplescombined postenrichment). To this end, we compared the relative “light”/“heavy”ratios of two distinct SILAC populations mixed either before (premixed)or after (postmixed) enrichment (Figure 3a)(n = 3/group). To simulate a typical biological experimentand evaluate the dynamic range of the workflow, light labeled cellswere treated with 100 ng/mL EGF for 5 min, and heavy labeled cellswere left untreated. In agreement with earlier experiments, this analysisconfirmed intraplate technical replicates enrich approximately equalnumbers of unique phosphopeptides at high purity (>90% phosphorylatedpeptides) (Figure 3b). Crucially, correlatingthe light/heavy ratios of premixed and postmixed phosphopeptide samplesshowed no discernible difference in phosphopeptide enrichment fidelitybetween replicates (r2 = 0.80) (Figure 3c–e). As a result, we propose automated magnetic particlehandling as a highly reproducible platform for uniform intraplatephosphopeptide enrichment.

Bottom Line: As a result, automated phosphopeptide enrichment enables statistical analysis of label-free phosphoproteomic samples in a high-throughput manner.This technique uses commercially available, off-the-shelf components and can be easily adopted by any laboratory interested in phosphoproteomic analysis.We provide a free downloadable automated phosphopeptide enrichment program to facilitate uniform interlaboratory collaboration and exchange of phosphoproteomic data sets.

View Article: PubMed Central - PubMed

Affiliation: The Institute of Cancer Research , 237 Fulham Road, London SW3 6JB, United Kingdom.

ABSTRACT
Reproducible, comprehensive phosphopeptide enrichment is essential for studying phosphorylation-regulated processes. Here, we describe the application of hyper-porous magnetic TiO2 and Ti-IMAC microspheres for uniform automated phosphopeptide enrichment. Combining magnetic microspheres with a magnetic particle-handling robot enables rapid (45 min), reproducible (r2 ≥ 0.80) and high-fidelity (>90% purity) phosphopeptide purification in a 96-well format. Automated phosphopeptide enrichment demonstrates reproducible synthetic phosphopeptide recovery across 2 orders of magnitude, "well-to-well" quantitative reproducibility indistinguishable to internal SILAC standards, and robust "plate-to-plate" reproducibility across 5 days of independent enrichments. As a result, automated phosphopeptide enrichment enables statistical analysis of label-free phosphoproteomic samples in a high-throughput manner. This technique uses commercially available, off-the-shelf components and can be easily adopted by any laboratory interested in phosphoproteomic analysis. We provide a free downloadable automated phosphopeptide enrichment program to facilitate uniform interlaboratory collaboration and exchange of phosphoproteomic data sets.

Show MeSH