Reproducible Automated Phosphopeptide Enrichment Using Magnetic TiO 2 and Ti-IMAC
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 magnet...
| Main Authors: | , , , , , , , , , |
|---|---|
| Other Authors: | |
| Format: | Article |
| Language: | en_US |
| Published: |
American Chemical Society (ACS)
2015
|
| Online Access: | http://hdl.handle.net/1721.1/96693 https://orcid.org/0000-0001-7004-8227 |
| _version_ | 1826201176833523712 |
|---|---|
| author | Tape, Christopher Worboys, Jonathan D. Sinclair, John Gourlay, Robert Vogt, Janis McMahon, Kelly M. Trost, Matthias Lauffenburger, Douglas A. Lamont, Douglas J. Jørgensen, Claus |
| author2 | Massachusetts Institute of Technology. Department of Biological Engineering |
| author_facet | Massachusetts Institute of Technology. Department of Biological Engineering Tape, Christopher Worboys, Jonathan D. Sinclair, John Gourlay, Robert Vogt, Janis McMahon, Kelly M. Trost, Matthias Lauffenburger, Douglas A. Lamont, Douglas J. Jørgensen, Claus |
| author_sort | Tape, Christopher |
| collection | MIT |
| description | 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. |
| first_indexed | 2024-09-23T11:47:44Z |
| format | Article |
| id | mit-1721.1/96693 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T11:47:44Z |
| publishDate | 2015 |
| publisher | American Chemical Society (ACS) |
| record_format | dspace |
| spelling | mit-1721.1/966932022-10-01T06:03:27Z Reproducible Automated Phosphopeptide Enrichment Using Magnetic TiO 2 and Ti-IMAC Tape, Christopher Worboys, Jonathan D. Sinclair, John Gourlay, Robert Vogt, Janis McMahon, Kelly M. Trost, Matthias Lauffenburger, Douglas A. Lamont, Douglas J. Jørgensen, Claus Massachusetts Institute of Technology. Department of Biological Engineering Tape, Christopher Lauffenburger, Douglas A. 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. Wellcome Trust (London, England) (Sir Henry Wellcome Postdoctoral Fellowship (098847/Z/12/Z)) Cancer Research UK (Career Establishment Award (C37293/A12905)) 2015-04-22T15:04:20Z 2015-04-22T15:04:20Z 2014-10 2014-07 Article http://purl.org/eprint/type/JournalArticle 0003-2700 1520-6882 http://hdl.handle.net/1721.1/96693 Tape, Christopher J., Jonathan D. Worboys, John Sinclair, Robert Gourlay, Janis Vogt, Kelly M. McMahon, Matthias Trost, Douglas A. Lauffenburger, Douglas J. Lamont, and Claus Jørgensen. “ Reproducible Automated Phosphopeptide Enrichment Using Magnetic TiO 2 and Ti-IMAC .” Analytical Chemistry 86, no. 20 (October 21, 2014): 10296–10302. © 2014 American Chemical Society. https://orcid.org/0000-0001-7004-8227 en_US http://dx.doi.org/10.1021/ac5025842 Analytical Chemistry Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf American Chemical Society (ACS) American Chemical Society |
| spellingShingle | Tape, Christopher Worboys, Jonathan D. Sinclair, John Gourlay, Robert Vogt, Janis McMahon, Kelly M. Trost, Matthias Lauffenburger, Douglas A. Lamont, Douglas J. Jørgensen, Claus Reproducible Automated Phosphopeptide Enrichment Using Magnetic TiO 2 and Ti-IMAC |
| title | Reproducible Automated Phosphopeptide Enrichment Using Magnetic TiO 2 and Ti-IMAC |
| title_full | Reproducible Automated Phosphopeptide Enrichment Using Magnetic TiO 2 and Ti-IMAC |
| title_fullStr | Reproducible Automated Phosphopeptide Enrichment Using Magnetic TiO 2 and Ti-IMAC |
| title_full_unstemmed | Reproducible Automated Phosphopeptide Enrichment Using Magnetic TiO 2 and Ti-IMAC |
| title_short | Reproducible Automated Phosphopeptide Enrichment Using Magnetic TiO 2 and Ti-IMAC |
| title_sort | reproducible automated phosphopeptide enrichment using magnetic tio 2 and ti imac |
| url | http://hdl.handle.net/1721.1/96693 https://orcid.org/0000-0001-7004-8227 |
| work_keys_str_mv | AT tapechristopher reproducibleautomatedphosphopeptideenrichmentusingmagnetictio2andtiimac AT worboysjonathand reproducibleautomatedphosphopeptideenrichmentusingmagnetictio2andtiimac AT sinclairjohn reproducibleautomatedphosphopeptideenrichmentusingmagnetictio2andtiimac AT gourlayrobert reproducibleautomatedphosphopeptideenrichmentusingmagnetictio2andtiimac AT vogtjanis reproducibleautomatedphosphopeptideenrichmentusingmagnetictio2andtiimac AT mcmahonkellym reproducibleautomatedphosphopeptideenrichmentusingmagnetictio2andtiimac AT trostmatthias reproducibleautomatedphosphopeptideenrichmentusingmagnetictio2andtiimac AT lauffenburgerdouglasa reproducibleautomatedphosphopeptideenrichmentusingmagnetictio2andtiimac AT lamontdouglasj reproducibleautomatedphosphopeptideenrichmentusingmagnetictio2andtiimac AT jørgensenclaus reproducibleautomatedphosphopeptideenrichmentusingmagnetictio2andtiimac |