Organic-inorganic hybrid silica monolith based immobilized titanium ion affinity chromatography column for analysis of mitochondrial phosphoproteome.
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 microm 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 Ti(4+)-IMAC column, 15 phosphopeptides were effectively isolated from the digest mixture of alpha-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 Ti(4+)-IMAC monolithic column were measured to be 1.4 micromol/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 Ti(4+)-IMAC column might provide a promising tool for large-scale phosphopeptide enrichment, facilitating the in-depth understanding of the biological functions of phosphoproteomes.
Hou
Chunyan
C
Key Laboratory of Separation Science for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China.
Ma
Junfeng
J
Tao
Dingyin
D
Shan
Yichu
Y
Liang
Zhen
Z
Zhang
Lihua
L
Zhang
Yukui
Y
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D008099:Liver / Q000737:chemistry*; D024101:Mitochondrial Proteins / Q000032:analysis*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010455:Peptides / Q000032:analysis* / Q000235:genetics; D010750:Phosphoproteins / Q000032:analysis*; D011437:Propylamines; D040901:Proteomics / Q000379:methods*; D051381:Rats; D012821:Silanes; D012822:Silicon Dioxide; D053719:Tandem Mass Spectrometry; D014025:Titanium
D024101:Mitochondrial Proteins; D010455:Peptides; D010750:Phosphoproteins; D011437:Propylamines; D012821:Silanes; D012822:Silicon Dioxide; D014025:Titanium; C477625:amino-propyl-triethoxysilane