Organic-Inorganic hybrid silica monolith based immobilized titanium ion affinity chromatography column for analysis of mitochondrial phosphoproteome

Chunyan Hou, Junfeng Ma, Dingyin Tao, Yichu Shan, Zhen Liang, Lihua Zhang, Yukui Zhang

Research output: Contribution to journalArticlepeer-review

54 Scopus citations


A novel kind of immobilized metal affinity chromatography (IMAC) column based on organic-inorganic hybrid silica monolith has been developed. The monolithic support was prepared in a 250 μm i.d. capillary by the sol-gel method with tetraethoxysilane (TEOS) and 3-aminopropyltriethoxysilane (APTES) as precursors. Subsequently, amine groups were functionalized by glutaraldehyde, and then activated with (aminomethyl) phosphonic acid, followed by Ti 4+ chelation. By such a hybrid silica monolithic Ti4+-IMAC column, 15 phosphopeptides were effectively isolated from the digest mixture of α-casein and BSA with the molar ratio as low as 1:200, illustrating its superior selectivity. With a synthetic phosphorylated peptide, YKVPQLEIVPNSpAEER, as the sample, the loading capacity and recovery of the Ti4+-IMAC monolithic column were measured to be 1.4 μmol/mL and 69%, respectively. Such an IMAC monolithic column was further applied to enrich phosphopeptides from rat liver mitochondria. In total, 224 unique phosphopeptides, corresponding to 148 phosphoprotein groups, were identified by duplicate nanoRPLC-LTQ MS/MS/MS runs with a false-positive rate of less than 1% at the peptide level. These results demonstrate that the hybrid silica monolith based Ti4+-IMAC column might provide a promising tool for large-scale phosphopeptide enrichment, facilitating the in-depth understanding of the biological functions of phosphoproteomes.

Original languageEnglish (US)
Pages (from-to)4093-4101
Number of pages9
JournalJournal of proteome research
Issue number8
StatePublished - Aug 6 2010
Externally publishedYes


  • immobilized metal ion affinity chromatography
  • mitochondrial phosphoproteome
  • organic-inorganic hybrid silica monolith
  • phosphopeptide enrichment
  • titanium ion

ASJC Scopus subject areas

  • Biochemistry
  • General Chemistry


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