Sequential Elution from IMAC (SIMAC): An Efficient Method for Enrichment and Separation of Mono- and Multi-phosphorylated Peptides.
(2016) In Methods in Molecular Biology 1355. p.147-160- Abstract
- Phosphoproteomics relies on methods for efficient purification and sequencing of phosphopeptides from highly complex biological systems, especially when using low amounts of starting material. Current methods for phosphopeptide enrichment, e.g., Immobilized Metal ion Affinity Chromatography and titanium dioxide chromatography provide varying degrees of selectivity and specificity for phosphopeptide enrichment. The number of multi-phosphorylated peptides identified in most published studies is rather low. Here we describe a protocol for a strategy that separates mono-phosphorylated peptides from multiply phosphorylated peptides using Sequential elution from Immobilized Metal ion Affinity Chromatography. The method relies on the initial... (More)
- Phosphoproteomics relies on methods for efficient purification and sequencing of phosphopeptides from highly complex biological systems, especially when using low amounts of starting material. Current methods for phosphopeptide enrichment, e.g., Immobilized Metal ion Affinity Chromatography and titanium dioxide chromatography provide varying degrees of selectivity and specificity for phosphopeptide enrichment. The number of multi-phosphorylated peptides identified in most published studies is rather low. Here we describe a protocol for a strategy that separates mono-phosphorylated peptides from multiply phosphorylated peptides using Sequential elution from Immobilized Metal ion Affinity Chromatography. The method relies on the initial enrichment and separation of mono- and multi-phosphorylated peptides using Immobilized Metal ion Affinity Chromatography and a subsequent enrichment of the mono-phosphorylated peptides using titanium dioxide chromatography. The two separate phosphopeptide fractions are then subsequently analyzed by mass spectrometric methods optimized for mono-phosphorylated and multi-phosphorylated peptides, respectively, resulting in improved identification of especially multi-phosphorylated peptides from a minimum amount of starting material. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/8235104
- author
- Thingholm, Tine LU and Larsen, Martin R
- organization
- publishing date
- 2016
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Methods in Molecular Biology
- volume
- 1355
- pages
- 147 - 160
- publisher
- Springer
- external identifiers
-
- pmid:26584924
- scopus:84947998541
- pmid:26584924
- ISSN
- 1940-6029
- DOI
- 10.1007/978-1-4939-3049-4_10
- language
- English
- LU publication?
- yes
- id
- 9c64ab14-795e-41bc-9409-0a0e31d98fe0 (old id 8235104)
- alternative location
- http://www.ncbi.nlm.nih.gov/pubmed/26584924?dopt=Abstract
- date added to LUP
- 2016-04-04 08:55:46
- date last changed
- 2022-03-23 03:25:17
@article{9c64ab14-795e-41bc-9409-0a0e31d98fe0,
abstract = {{Phosphoproteomics relies on methods for efficient purification and sequencing of phosphopeptides from highly complex biological systems, especially when using low amounts of starting material. Current methods for phosphopeptide enrichment, e.g., Immobilized Metal ion Affinity Chromatography and titanium dioxide chromatography provide varying degrees of selectivity and specificity for phosphopeptide enrichment. The number of multi-phosphorylated peptides identified in most published studies is rather low. Here we describe a protocol for a strategy that separates mono-phosphorylated peptides from multiply phosphorylated peptides using Sequential elution from Immobilized Metal ion Affinity Chromatography. The method relies on the initial enrichment and separation of mono- and multi-phosphorylated peptides using Immobilized Metal ion Affinity Chromatography and a subsequent enrichment of the mono-phosphorylated peptides using titanium dioxide chromatography. The two separate phosphopeptide fractions are then subsequently analyzed by mass spectrometric methods optimized for mono-phosphorylated and multi-phosphorylated peptides, respectively, resulting in improved identification of especially multi-phosphorylated peptides from a minimum amount of starting material.}},
author = {{Thingholm, Tine and Larsen, Martin R}},
issn = {{1940-6029}},
language = {{eng}},
pages = {{147--160}},
publisher = {{Springer}},
series = {{Methods in Molecular Biology}},
title = {{Sequential Elution from IMAC (SIMAC): An Efficient Method for Enrichment and Separation of Mono- and Multi-phosphorylated Peptides.}},
url = {{http://dx.doi.org/10.1007/978-1-4939-3049-4_10}},
doi = {{10.1007/978-1-4939-3049-4_10}},
volume = {{1355}},
year = {{2016}},
}