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Enrichment of Phosphopeptides by Fe 3+ -Immobilized Mesoporous Nanoparticles of MCM-41 for MALDI and Nano-LC−MS/MS Analysis

2006; American Chemical Society; Volume: 5; Issue: 11 Linguagem: Inglês

10.1021/pr0600125

1535-3907

Chensong Pan, Mingliang Ye, Yuge Liu, Shun Feng, Xiaogang Jiang, Guanghui Han, Jun‐Jie Zhu, Hanfa Zou,

Analytical chemistry methods development

Fe3+-immobilized mesoporous molecular sieves MCM-41 with particle size of ca. 600 nm and pore size of ca. 3 nm is synthesized and applied to selectively trap and separate phosphopeptides from tryptic digest of proteins. For the capture of phosphopeptides, typically 10 μL of tryptic digest solution was first diluted to 1 mL by solution of ACN/0.1% TFA (50:50, v/v) and incubated with 10 μL of 0.1% acetic acid dispersed Fe3+-immobilized MCM-41 for 1 h under vibration. Fe3+-immobilized MCM-41 with trapped phosphopeptides was separated by centrifugation. The deposition was first washed with a volume of 300 μL of solution containing 100 mM NaCl in ACN/0.1% TFA (50:50, v/v) and followed by a volume of 300 μL of solution of 0.1% acetic acid to remove nonspecifically bound peptides. The nanoparticles with trapped phosphopeptides are mixed with 2,5-dihydroxybenzoic acid (2,5-DHB) and deposited onto the target for analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI−MS). It was found that phosphopeptides from tryptic digest of α-casein and β-casein are effectively and specifically trapped on Fe3+-immobilized MCM-41 with few peptides nonspecifically adsorbed. After the extraction by Fe3+-immobilized MCM-41, the suppression to the detection of phosphopeptides caused by abundant nonphosphopeptides from tryptic digest is effectively eliminated, and the detection of phosphopeptides by MALDI is greatly enhanced with the value of signal-to-noise (S/N) increased by more than an order of magnitude. It is demonstrated that the mechanism of the adsorption of phosphopeptides on Fe3+-immobilized MCM-41 is based on the interaction between the Fe3+ and the phosphate group. Finally, Fe3+-immobilized MCM-41 is applied to extract phosphopeptides from tryptic digest of the lysate of mouse liver for phosphoproteome analysis by nano-LC−MS/MS. Keywords: phosphopeptides • MALDI • Fe3+-immobilized MCM-41 • nano-LC−MS/MS • enrichment