Oxidized carbon nanotubes are tested as a matrix for analysis of small molecules by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). Compared with nonoxidized carbon nanotubes, oxidized carbon nanotubes facilitate sample preparation because of their higher solubility in water. The matrix layer of oxidized carbon nanotubes is much more homogeneous and compact than that of nonoxidized carbon nanotubes. The efficiency of desorption/ionization for analytes and the reproducibility of peak intensities within and between sample spots are greatly enhanced on the surface of oxidized carbon nanotubes. The advantage of the oxidized carbon nanotubes in comparison with ␣-cyano-4-hydroxycinnamic acid (CCA) and carbon nanotubes is demonstrated by MALDI-TOF-MS analysis of an amino acid mixture. The matrix is successfully used for analysis of synthetic hydroxypropyl -cyclodextrin, suggesting a great potential for monitoring reactions and for product quality control. Reliable quantitative analysis of jatrorrhizine and palmatine with a wide linear range (1-100 ng/mL) and good reproducibility of relative peak areas (RSD less than 10%) is achieved using this matrix. Concentrations of jatrorrhizine (8.65 mg/mL) and palmatine (10.4 mg/mL) in an extract of Coptis chinensis Franch are determined simultaneously using the matrix and a standard addition method. [27] developed a method for quantitative analysis of small molecules with desorption/ionization on porous silicon using electrospray deposition of analytes, and a quite good quantitative result with linear calibrations R 2 Ͼ 0.99 and values of RSD Ͻ 7% was achieved. It is believed that MALDI could be a powerful technique to provide both qualitative and quantitative determination of small compounds.Carbon nanotubes have been attracting wide interest [28,29] of scientists since they were initially discovered by Iijima et al. [30,31]. Recently, Carbon nanotubes have been developed as the matrix for MALDI-TOF-MS for analysis of small molecules [25], in which carbon nanotubes function both as the energy receptacle for laser radiation and as the energy transporter for desorption/ionization of analytes with the minimization of interference signals caused by matrix ion. The effectiveness of the method as matrix has been demonstrated by several compounds with low molecular weight. However, the low solubility of carbon nanotubes in water or organic solvent makes it hard to deposit carbon nanotubes onto the sample target and to form a homogeneous layer of matrix, leading to the relatively poor reproducibility and resolution of peaks for analytes.
Phosphorylation is one of the most important post-translational modifications of proteins, which modulates a wide range of biological functions and activity of proteins. The analysis of phosphopeptides is still one of the most challenging tasks in proteomics research by mass spectrometry. In this study, a novel phosphopeptide enrichment approach based on the strong interaction of zirconium phosphonate (ZrP) modified surface with phosphopeptides has been developed. ZrP modified porous silicon (ZrP-pSi) wafer was prepared to specifically capture the phosphopeptides from complex peptide mixtures, and then the captured phosphopeptides were analyzed by MALDI-TOF MS by directly placing the wafer on a MALDI target. The phosphopeptide enrichment and MALDI analysis were both performed on the ZrP-pSi wafer which significantly reduced the sample loss and simplified the analytical procedures. The prepared ZrP-pSi wafer has been successfully applied for the enrichment of phosphopeptides from the tryptic digest of standard phosphoproteins beta-casein and alpha-casein. The excellent selectivity of this approach was demonstrated by analyzing phosphopeptides in the digest mixture of beta-casein and bovine serum albumin with molar ratio of 1:100. High detection sensitivity has been achieved for the analysis of the phosphopeptides from tryptic digestion of 2 fmol beta-casein on the ZrP-pSi surface.
Large-scale characterization of phosphoproteins requires highly specific methods for the purification of phosphopeptides because of the low abundance of phosphoproteins and substoichiometry of phosphorylation. A phosphopeptide enrichment method using ZrO2 nanoparticles is presented. The high specificity of this approach was demonstrated by the isolation of phosphopeptides from the digests of model phosphoproteins. The strong affinity of ZrO2 nanoparticles to phosphopeptides enables the specific enrichment of phosphopeptides from a complex peptide mixture in which the abundance of phosphopeptides is two orders of magnitude lower than that of nonphosphopeptides. Superior selectivity of ZrO2 nanoparticles for the enrichment of phosphorylated peptides than that of conventional immobilized metal affinity chromatography was observed. Femtomole phosphopeptides from digestion products could be enriched by ZrO2 nanoparticles and can be well detected by MALDI mass spectrometric analysis. ZrO2 nanoparticles were further applied to selectively isolate phosphopeptides from the tryptic digestion of mouse liver lysate for phosphoproteome analysis by nanoliter LC MS/MS (nano-LC-MS/MS) and MS/MS/MS. A total of 248 defining phosphorylation sites and 140 phosphorylated peptides were identified by manual validation using a series of rigid criteria.
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