Nanodiamond/TiO2 core/shell nanocomposite as a promising material for analysis of phosphopeptides
Date Issued
2010
Date
2010
Author(s)
Hung, Wan-Yu
Abstract
Protein phosphorylation is an important post-translational control of protein activity in cells. However, low abundances, low stoichiometry, and poor ionization of phosphopeptides make the isolation and concentration steps indispensable prior to MS analysis. In this study, we utilized a new probe of high affinity for phosphopeptide enrichment with titanium dioxide-coated nanodiamonds (TiO2-coated NDs). Nanodiamond (ND) holds several unique properties such as small particle size, large specific surface area, wide optical transparency range, and facile surface functionalizability, making it a promising solid-phase substrate for affinity purification mass spectrometry. The enrichment conditions were optimized using tryptic digests of β-casein, and the high specificity of the TiO2-coateed NDs was demonstrated by selectively enriching phosphopeptides from the tryptic digests of protein mixture of β-casein and bovine serum albumin (BSA) with a molar ratio of 1: 5000, followed by MALDI-TOF MS characterization. The new protocol was also coupled with nano-LC-MS/MS system without difficulty. Analysis of tryptic digests from cytoplasmic fraction of HeLa cells yielded numbers of phosphopeptide identifications comparable to that obtained using commercial phosphopeptide isolation tool (Phos-trapTM 96 Enrichment Kit) and almost 59.3% recovery of phosphopeptides, presumable due to the presence of a large numbers of sites available on TiO2-coated NDs for binding or incomplete removal of nonspecific bound peptides. In 120 μg of equivalent of HeLa cell lysates, we identified 696 unique phosphopeptides and 925 phosphorylation sites, indicating the excellent performance of the TiO2-coated NDs.
Subjects
phosphorylation
trypsin
sol-gel
nanodiamond
β-casein
bovine serum albumin
HeLa cell lysate
Type
thesis
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