Advanced

Phosphopeptide Enrichment by Immobilized Metal Affinity Chromatography.

Thingholm, Tine LU and Larsen, Martin R (2016) In Methods in Molecular Biology 1355. p.123-133
Abstract
Immobilized metal affinity chromatography (IMAC) has been the method of choice for phosphopeptide enrichment prior to mass spectrometric analysis for many years and it is still used extensively in many laboratories. Using the affinity of negatively charged phosphate groups towards positively charged metal ions such as Fe(3+), Ga(3+), Al(3+), Zr(4+), and Ti(4+) has made it possible to enrich phosphorylated peptides from peptide samples. However, the selectivity of most of the metal ions is limited, when working with highly complex samples, e.g., whole-cell extracts, resulting in contamination from nonspecific binding of non-phosphorylated peptides. This problem is mainly caused by highly acidic peptides that also share high binding affinity... (More)
Immobilized metal affinity chromatography (IMAC) has been the method of choice for phosphopeptide enrichment prior to mass spectrometric analysis for many years and it is still used extensively in many laboratories. Using the affinity of negatively charged phosphate groups towards positively charged metal ions such as Fe(3+), Ga(3+), Al(3+), Zr(4+), and Ti(4+) has made it possible to enrich phosphorylated peptides from peptide samples. However, the selectivity of most of the metal ions is limited, when working with highly complex samples, e.g., whole-cell extracts, resulting in contamination from nonspecific binding of non-phosphorylated peptides. This problem is mainly caused by highly acidic peptides that also share high binding affinity towards these metal ions. By lowering the pH of the loading buffer nonspecific binding can be reduced significantly, however with the risk of reducing specific binding capacity. After binding, the enriched phosphopeptides are released from the metal ions using alkaline buffers of pH 10-11, EDTA, or phosphate-containing buffers.Here we describe a protocol for IMAC using Fe(3+) for phosphopeptide enrichment. The principles are illustrated on a semi-complex peptide mixture. (Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Methods in Molecular Biology
volume
1355
pages
123 - 133
publisher
Springer
external identifiers
  • pmid:26584922
  • scopus:84947996475
ISSN
1940-6029
DOI
10.1007/978-1-4939-3049-4_8
language
English
LU publication?
yes
id
20caced5-ab28-4e0a-9fe4-b45036acb473 (old id 8235114)
alternative location
http://www.ncbi.nlm.nih.gov/pubmed/26584922?dopt=Abstract
date added to LUP
2015-12-01 13:50:51
date last changed
2017-08-20 04:38:21
@article{20caced5-ab28-4e0a-9fe4-b45036acb473,
  abstract     = {Immobilized metal affinity chromatography (IMAC) has been the method of choice for phosphopeptide enrichment prior to mass spectrometric analysis for many years and it is still used extensively in many laboratories. Using the affinity of negatively charged phosphate groups towards positively charged metal ions such as Fe(3+), Ga(3+), Al(3+), Zr(4+), and Ti(4+) has made it possible to enrich phosphorylated peptides from peptide samples. However, the selectivity of most of the metal ions is limited, when working with highly complex samples, e.g., whole-cell extracts, resulting in contamination from nonspecific binding of non-phosphorylated peptides. This problem is mainly caused by highly acidic peptides that also share high binding affinity towards these metal ions. By lowering the pH of the loading buffer nonspecific binding can be reduced significantly, however with the risk of reducing specific binding capacity. After binding, the enriched phosphopeptides are released from the metal ions using alkaline buffers of pH 10-11, EDTA, or phosphate-containing buffers.Here we describe a protocol for IMAC using Fe(3+) for phosphopeptide enrichment. The principles are illustrated on a semi-complex peptide mixture.},
  author       = {Thingholm, Tine and Larsen, Martin R},
  issn         = {1940-6029},
  language     = {eng},
  pages        = {123--133},
  publisher    = {Springer},
  series       = {Methods in Molecular Biology},
  title        = {Phosphopeptide Enrichment by Immobilized Metal Affinity Chromatography.},
  url          = {http://dx.doi.org/10.1007/978-1-4939-3049-4_8},
  volume       = {1355},
  year         = {2016},
}