| Chapter title |
Improving the Phosphoproteome Coverage for Limited Sample Amounts Using TiO2-SIMAC-HILIC (TiSH) Phosphopeptide Enrichment and Fractionation.
|
|---|---|
| Chapter number | 11 |
| Book title |
Phospho-Proteomics
|
| Published in |
Methods in molecular biology, January 2016
|
| DOI | 10.1007/978-1-4939-3049-4_11 |
| Pubmed ID | |
| Book ISBNs |
978-1-4939-3048-7, 978-1-4939-3049-4
|
| Authors |
Kasper Engholm-Keller, Martin R. Larsen |
| Editors |
Louise von Stechow |
| Abstract |
Obtaining high phosphoproteome coverage requires specific enrichment of phosphorylated peptides from the often extremely complex peptide mixtures generated by proteolytic digestion of biological samples, as well as extensive chromatographic fractionation prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Due to the sample loss resulting from fractionation, this procedure is mainly performed when large quantities of sample are available. To make large-scale phosphoproteomics applicable to smaller amounts of protein we have recently combined highly specific TiO2-based phosphopeptide enrichment with sequential elution from immobilized metal affinity chromatography (SIMAC) for fractionation of mono- and multi-phosphorylated peptides prior to capillary scale hydrophilic interaction liquid chromatography (HILIC) based fractionation of monophosphorylated peptides. In the following protocol we describe the procedure step by step to allow for comprehensive coverage of the phosphoproteome utilizing only a few hundred micrograms of protein. |
Mendeley readers
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| France | 1 | 4% |
| Unknown | 27 | 96% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Student > Ph. D. Student | 5 | 18% |
| Professor > Associate Professor | 3 | 11% |
| Student > Master | 2 | 7% |
| Professor | 2 | 7% |
| Student > Doctoral Student | 1 | 4% |
| Other | 3 | 11% |
| Unknown | 12 | 43% |
| Readers by discipline | Count | As % |
|---|---|---|
| Biochemistry, Genetics and Molecular Biology | 4 | 14% |
| Agricultural and Biological Sciences | 4 | 14% |
| Chemistry | 3 | 11% |
| Medicine and Dentistry | 2 | 7% |
| Computer Science | 1 | 4% |
| Other | 2 | 7% |
| Unknown | 12 | 43% |