Automated Phosphoproteome Analysis for Cultured Cancer Cells by Two-Dimensional NanoLC-MS Using a Calcined Titania/C18 Biphasic Column
Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0017, Japan. Analytical Sciences
(Impact Factor: 1.39).
02/2008; 24(1):161-6. DOI: 10.2116/analsci.24.161
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.
Available from: Yu-Qi Feng
- "For example, most of metal oxides show preferential enrichment of mono-phosphopeptides  . Different metal oxides or the same one with different crystal structures or morphologies display distinct affinity for phosphopeptides , whereas the theoretical mechanism is ambiguous  . On the other hand, composite oxides containing at least two kinds of metal oxides or nonmetallic oxides possess more remarkable properties than single-component metal oxide and show great potential in the specific capture of phosphopeptides   . "
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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.
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Available from: Kyuya Harada
- "Fax: +81-45-787-2787 Abbreviations: MOAC, metal oxide affinity chromatography; MPPD, Medicago PhosphoProtein Database; FDR, false discovery rate perform genome database driven proteome analysis in this organism. Meanwhile, the development of high-performance MS and phosphopeptide-enrichment techniques such as IMAC and metal oxide affinity chromatography (MOAC)     have made it possible to comprehensively analyze phosphoproteins by MS. In this study, using the genome database and these analytical techniques, we profiled phosphoprotein expression in the cotyledons and hypocotyls of L. japonicus seeds after absorption of water. "
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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.
Available from: Jean Cook
- "Some isoforms decreased in abundance but new isoforms accumulated in the S phase samples (Figure 5C, compare lane 1 with lanes 2 and 3). Of note, the hnRNPA3 protein has been reported to be heavily phosphorylated, raising the possibility that the decrease observed by mass spectrometry was due to cell cycle regulated post-translational modifications , , , , , , , . Indeed, a number of hnRNPs, including hnRNPD0, were identified as Cyclin A/Cdk2 substrates . "
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ABSTRACT: Cell proliferation involves dramatic changes in DNA metabolism and cell division, and control of DNA replication, mitosis, and cytokinesis have received the greatest attention in the cell cycle field. To catalogue a wider range of cell cycle-regulated processes, we employed quantitative proteomics of synchronized HeLa cells. We quantified changes in protein abundance as cells actively progress from G1 to S phase and from S to G2 phase. We also describe a cohort of proteins whose abundance changes in response to pharmacological inhibition of the proteasome. Our analysis reveals not only the expected changes in proteins required for DNA replication and mitosis but also cell cycle-associated changes in proteins required for biological processes not known to be cell-cycle regulated. For example, many pre-mRNA alternative splicing proteins are down-regulated in S phase. Comparison of this dataset to several other proteomic datasets sheds light on global mechanisms of cell cycle phase transitions and underscores the importance of both phosphorylation and ubiquitination in cell cycle changes.
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