Enhancing the identification of phosphopeptides from putative basophilic kinase substrates using Ti (IV) based IMAC enrichment

Houjiang Zhou, Low Teck Yew, Marco L. Hennrich, Henk Der Van Toorn, Thomas Schwend, Hanfa Zou, Shabaz Mohammed, Albert J.R. Heck

Research output: Contribution to journalArticle

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Abstract

Metal and metal oxide chelating-based phosphopeptide enrichment technologies provide powerful tools for the in-depth profiling of phosphoproteomes. One weakness inherent to current enrichment strategies is poor binding of phosphopeptides containing multiple basic residues. The problem is exacerbated when strong cation exchange (SCX) is used for pre-fractionation, as under low pH SCX conditions phosphorylated peptides with multiple basic residues elute with the bulk of the tryptic digest and therefore require more stringent enrichment. Here, we report a systematic evaluation of the characteristics of a novel phosphopeptide enrichment approach based on a combination of low pH SCX and Ti 4+-immobilized metal ion affinity chromatography (IMAC) comparing it one-to-one with the well established low pH SCX-TiO 2 enrichment method. We also examined the effect of 1,1,1,3,3,3- hexafluoroisopropanol (HFP), trifluoroacetic acid (TFA), or 2,5-dihydroxybenzoic acid (DHB) in the loading buffer, as it has been hypothesized that high levels of TFA and the perfluorinated solvent HFP improve the enrichment of phosphopeptides containing multiple basic residues. We found that Ti 4+-IMAC in combination with TFA in the loading buffer, outperformed all other methods tested, enabling the identification of around 5000 unique phosphopeptides containing multiple basic residues from 400 μg of a HeLa cell lysate digest. In comparison, ∼2000 unique phosphopeptides could be identified by Ti 4+-IMAC with HFP and close to 3000 by TiO 2. We confirmed, by motif analysis, the basic phosphopeptides enrich the number of putative basophilic kinases substrates. In addition, we performed an experiment using the SCX/Ti 4+-IMAC methodology alongside the use of collision-induced dissociation (CID), higher energy collision induced dissociation (HCD) and electron transfer dissociation with supplementary activation (ETD) on considerably more complex sample, consisting of a total of 400 μg of triple dimethyl labeled MCF-7 digest. This analysis led to the identification of over 9,000 unique phosphorylation sites. The use of three peptide activation methods confirmed that ETD is best capable of sequencing multiply charged peptides. Collectively, our data show that the combination of SCX and Ti 4+-IMAC is particularly advantageous for phosphopeptides with multiple basic residues.

Original languageEnglish
JournalMolecular and Cellular Proteomics
Volume10
Issue number10
DOIs
Publication statusPublished - 1 Oct 2011
Externally publishedYes

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Affinity chromatography
Ion chromatography
Phosphopeptides
Affinity Chromatography
Metal ions
Phosphotransferases
Metals
Ions
Cations
Substrates
Ion exchange
Trifluoroacetic Acid
Peptides
Buffers
Chemical activation
Phosphorylation
Depth profiling
Fractionation
Chelation
HeLa Cells

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Molecular Biology

Cite this

Enhancing the identification of phosphopeptides from putative basophilic kinase substrates using Ti (IV) based IMAC enrichment. / Zhou, Houjiang; Teck Yew, Low; Hennrich, Marco L.; Van Toorn, Henk Der; Schwend, Thomas; Zou, Hanfa; Mohammed, Shabaz; Heck, Albert J.R.

In: Molecular and Cellular Proteomics, Vol. 10, No. 10, 01.10.2011.

Research output: Contribution to journalArticle

Zhou, Houjiang ; Teck Yew, Low ; Hennrich, Marco L. ; Van Toorn, Henk Der ; Schwend, Thomas ; Zou, Hanfa ; Mohammed, Shabaz ; Heck, Albert J.R. / Enhancing the identification of phosphopeptides from putative basophilic kinase substrates using Ti (IV) based IMAC enrichment. In: Molecular and Cellular Proteomics. 2011 ; Vol. 10, No. 10.
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