Automated phosphoproteome analysis for cultured cancer cells by two-dimensional nanoLC-MS using a calcined titania/C18 biphasic column.
ABSTRACT We have developed an on-line automated system for phosphoproteome analysis using titania-based phosphopeptide enrichment followed by nanoLC-MS/MS. Titania beads were prepared by calcination of commercial chromatographic titania beads at 800 degrees C to convert the crystalline structure. The obtained rutile-form titania exhibited higher selectivity in phosphopeptide enrichment than commercial titania, even in the absence of a competitive chelating reagent for non-phosphopeptides. For phosphoproteome analysis of human cervical cancer HeLa cells, tryptic digests of the cell extracts were directly injected into this on-line system, and 696 non-redundant phosphopeptides with 671 unambiguously determined phosphorylation sites, derived from 512 phosphoproteins, were successfully identified. This is the first successful application of an on-line automated phosphoproteome analysis system to complex biological samples.
- Sensors and Actuators B Chemical 11/2013; 188:1073-1079. · 3.84 Impact Factor
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ABSTRACT: Selective and effective enrichment of phosphopeptides from complex samples is essential in phosphoproteome study by mass spectrometry (MS). In this work, we compared perovskites (MgTiO3, CaTiO3, SrTiO3, BaTiO3 and CaZrO3) with metal oxides (ZrO2 and TiO2) in their capability for the selective enrichment of phosphopeptides. It was found here that perovskites exhibited higher selectivity towards phosphopeptides than commonly used ZrO2 and TiO2, even though they all have high affinity to phosphopeptides. As for perovskites, CaTiO3 exhibited better selectivity for enrichment of phosphopeptides than SrTiO3, MgTiO3, BaTiO3 and CaZrO3, which might be ascribed to their crystal structures and electrophilic abilities. Moreover, to further confirm the performance of CaTiO3, CaTiO3 and TiO2 were applied to the enrichment of phosphopeptides from tryptic digest of proteins of human Jurkat-T cell lysate, respectively. The results showed CaTiO3 has much higher selectivity than TiO2 in the enrichment of phosphopeptides from the complex biological sample. Taken together, here we show that CaTiO3 is an excellent material for the highly selective enrichment of phosphopeptides and it could be potentially used in the large-scale phosphoproteome study. Copyright © 2014 Elsevier B.V. All rights reserved.Journal of Chromatography A 12/2014; · 4.26 Impact Factor
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ABSTRACT: We report the first dataset of phosphoproteins of the seeds of a reference plant, Lotus japonicus. This dataset might be useful in studying the regulatory mechanisms of seed germination in legume plants. By proteomic analysis of seeds following water absorption, we identified a total of 721 phosphopeptides derived from 343 phosphoproteins in cotyledons, and 931 phosphopeptides from 473 phosphoproteins in hypocotyls. Kinase-specific prediction analyses revealed that different kinases were activated in cotyledons and hypocotyls. In particular, many peptides containing ATM-kinase target motifs, X-X-pS/pT-Q-X-X, were detected in cotyledons. Moreover, by real-time RT-PCR analysis, we found that expression of a homolog of ATM kinase is upregulated specifically in cotyledons, suggesting that this ATM kinase homolog plays a significant role in cell proliferation in the cotyledons of L. japonicus seeds. The data have been deposited to the ProteomeXchange with identifier PXD000053. This article is protected by copyright. All rights reserved.Proteomics 11/2013; · 3.97 Impact Factor