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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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Robotic magnetic automated phosphopeptide enrichment.(a) KingFisherFlex configured for automated phosphopeptide enrichment. (b) Automatedphosphopeptide enrichment steps (program has been deposited in PRIDE:PXD000892). (c) 100 μg and 500 μg of a common trypticdigest was phospho-enriched using TiO2 and Ti-IMAC hyper-porousmicrospheres via manual and automated methods and analyzed by LC-MS/MS.DDA runs, red dots, n = 24.
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fig1: Robotic magnetic automated phosphopeptide enrichment.(a) KingFisherFlex configured for automated phosphopeptide enrichment. (b) Automatedphosphopeptide enrichment steps (program has been deposited in PRIDE:PXD000892). (c) 100 μg and 500 μg of a common trypticdigest was phospho-enriched using TiO2 and Ti-IMAC hyper-porousmicrospheres via manual and automated methods and analyzed by LC-MS/MS.DDA runs, red dots, n = 24.

Mentions: All experimentswere performed with a KingFisher Flex (Thermo Scientific) magneticparticle-processing robot. The automated phosphopeptide enrichmentprogram was developed using BindIt Software 3.0 (Thermo Scientific).The program file has been uploaded alongside the MS/MS data (see below)with the identifier Automated_Phosphopeptide_Enrichment.msz. Thisprogram can be freely downloaded and run on any KingFisher Flex system.TiO2 (MR-TID010) and Ti-IMAC (MR-TIM010) hyper-porous magneticmicrospheres were purchased from ReSyn Biosciences. The KingFisherFlex was configured for automated phosphopeptide enrichment, as illustratedin Figure 1a. In brief, deep-well 96-well plates(VWR 733-3004) were assigned to each of the eight carousel positions.Individual positions were loaded with (1) 96-well tip heads (ThermoScientific); (2) hyper-porous magnetic microspheres (in 100% MeCN);(3) wash buffer 1 (80% MeCN, 5% TFA, + 1 M glycolic acid); (4) 100μg Lys-C/trypsin digested lysate (in 80% MeCN, 5% TFA, + 1 Mglycolic acid); (5) wash buffer 1; (6) wash buffer 2 (80% MeCN, 1%TFA); (7) wash buffer 3 (10% MeCN, 0.2% TFA); and (8) elution buffer(1–5% NH4OH). 500 μL of the relevant buffer was addedto each well, except for the sample binding and elution steps, whereonly 200 μL of sample and elution buffer were used. Unless statedotherwise, all experiments were performed with the buffers describedabove, 1 mg of magnetic microspheres, and three automated phosphopeptideenrichment cycles. (Technical note: The amount of microspheres cangreatly affect enrichment performance.20 For maximum sample-to-sample fidelity, vortex the microsphere slurrybefore aliquoting.)


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)

Robotic magnetic automated phosphopeptide enrichment.(a) KingFisherFlex configured for automated phosphopeptide enrichment. (b) Automatedphosphopeptide enrichment steps (program has been deposited in PRIDE:PXD000892). (c) 100 μg and 500 μg of a common trypticdigest was phospho-enriched using TiO2 and Ti-IMAC hyper-porousmicrospheres via manual and automated methods and analyzed by LC-MS/MS.DDA runs, red dots, n = 24.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4206527&req=5

fig1: Robotic magnetic automated phosphopeptide enrichment.(a) KingFisherFlex configured for automated phosphopeptide enrichment. (b) Automatedphosphopeptide enrichment steps (program has been deposited in PRIDE:PXD000892). (c) 100 μg and 500 μg of a common trypticdigest was phospho-enriched using TiO2 and Ti-IMAC hyper-porousmicrospheres via manual and automated methods and analyzed by LC-MS/MS.DDA runs, red dots, n = 24.
Mentions: All experimentswere performed with a KingFisher Flex (Thermo Scientific) magneticparticle-processing robot. The automated phosphopeptide enrichmentprogram was developed using BindIt Software 3.0 (Thermo Scientific).The program file has been uploaded alongside the MS/MS data (see below)with the identifier Automated_Phosphopeptide_Enrichment.msz. Thisprogram can be freely downloaded and run on any KingFisher Flex system.TiO2 (MR-TID010) and Ti-IMAC (MR-TIM010) hyper-porous magneticmicrospheres were purchased from ReSyn Biosciences. The KingFisherFlex was configured for automated phosphopeptide enrichment, as illustratedin Figure 1a. In brief, deep-well 96-well plates(VWR 733-3004) were assigned to each of the eight carousel positions.Individual positions were loaded with (1) 96-well tip heads (ThermoScientific); (2) hyper-porous magnetic microspheres (in 100% MeCN);(3) wash buffer 1 (80% MeCN, 5% TFA, + 1 M glycolic acid); (4) 100μg Lys-C/trypsin digested lysate (in 80% MeCN, 5% TFA, + 1 Mglycolic acid); (5) wash buffer 1; (6) wash buffer 2 (80% MeCN, 1%TFA); (7) wash buffer 3 (10% MeCN, 0.2% TFA); and (8) elution buffer(1–5% NH4OH). 500 μL of the relevant buffer was addedto each well, except for the sample binding and elution steps, whereonly 200 μL of sample and elution buffer were used. Unless statedotherwise, all experiments were performed with the buffers describedabove, 1 mg of magnetic microspheres, and three automated phosphopeptideenrichment cycles. (Technical note: The amount of microspheres cangreatly affect enrichment performance.20 For maximum sample-to-sample fidelity, vortex the microsphere slurrybefore aliquoting.)

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