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Cited by 891

The DOI matches its Crossref record; 12 of the 12 references it cites were found in a registry or an index.

Selective Isolation at the Femtomole Level of Phosphopeptides from Proteolytic Digests Using 2D-NanoLC-ESI-MS/MS and Titanium Oxide Precolumns

Pinkse, Martijn W. H., Uitto, Pauliina M., Hilhorst, Martijn J., Ooms, Bert, Heck, Albert J. R.·2004·Analytical Chemistry
DOI 10.1021/ac0498617
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Abstract

Selective detection of phosphopeptides from proteolytic digests is a challenging and highly relevant task in many proteomics applications. Often phosphopeptides are present in small amounts and need selective isolation or enrichment before identification. Here we report a novel automated method for the enrichment of phosphopeptides from complex mixtures. The method employs a two-dimensional column setup, with titanium oxide-based solid-phase material (Titansphere) as the first dimension and reversed-phase material as the second dimension. Phosphopeptides are separated from nonphosphorylated peptides by trapping them under acidic conditions on a TiO(2) precolumn. Nonphosphorylated peptides break through and are trapped on a reversed-phase precolumn after which they are analyzed by nanoflow LC-ESI-MS/MS. Subsequently, phosphopeptides are desorbed from the TiO(2) column under alkaline conditions, reconcentrated onto the reversed-phase precolumn, and analyzed by nanoflow LC-ESI-MS/MS. The selectivity and practicality of using TiO(2) precolumns for trapping phosphopeptides are demonstrated via the analysis of a model peptide RKISASEF, in a 1:1 mixture of a non- and a monophosphorylated form. A sample of 125 fmol of the phosphorylated peptide could easily be isolated from the nonphosphorylated peptide with a recovery above 90%. In addition, proteolytic digests of three different autophosphorylation forms of the 153-kDa homodimeric cGMP-dependent protein kinase are analyzed. From proteolytic digests of the fully autophosphorylated protein at least eight phosphorylation sites are identified, including two previously uncharacterized sites, namely, Ser-26 and Ser-44. Ser-26 is characterized as a minor phosphorylation site in purified PKG samples, while Ser-44 is identified as a novel in vitro autophosphorylation target. These results clearly show that TiO(2) has strong affinity for phosphorylated peptides, and thus, we conclude that this material has a high potential in the field of phosphoproteomics.

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Metadata

Authors
Pinkse, M. W. H., Uitto, P. M., Hilhorst, M. J., Ooms, B., & Heck, A. J. R.
Venue
Analytical Chemistry
Publisher
American Chemical Society (ACS)
Published
2004-06-15
Volume
76
Issue
14
Pages
3935-3943
ISSN
  • 0003-2700 (ISSN-L)
  • 1520-6882
DOI
10.1021/ac0498617
OpenAlex
W2033889320
Cited by
891
References
12
FWCI
18.39
Type
journal article
Language
en
Open access
closed

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