Automated Phosphoproteome Analysis for Cultured Cancer Cells by Two-Dimensional NanoLC-MS Using a Calcined Titania/C18 Biphasic Column

ArticleinAnalytical Sciences 24(1):161-6 · February 2008with4 Reads
DOI: 10.2116/analsci.24.161 · Source: PubMed
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.
    • "The variation in selectivity is seen not only in different metal oxides but also in the same metal oxide synthesized by different methods. Rutile-form titania exhibits greater selectivity in phosphopeptide enrichment than commercial anatase titania beads, although the inherent reason is still unclear [36] . The inhouse-prepared nanocast TiO2 sphere also exhibited properties complementary to commercial TiO2 material [37]. "
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    • "Previous studies have shown that TG2 is phosphorylated by PKA at Ser 215 and Ser 216 [52] and at an unknown site(s) by PTEN-induced putative kinase 1 (PINK1; [53]). Phosphoproteomic based studies have identified numerous phosphorylation sites on Ser, Thr and Tyr residues in human and rat TG2 [54][55][56][57][58][59][60]. It would therefore be of value to identify the specific site(s) of TG2-associated serine and threonine phosphorylation triggered by the A 1 adenosine receptor. "
    [Show abstract] [Hide abstract] ABSTRACT: The regulation of tissue transglutaminase (TG2) activity by the GPCR family is poorly understood. In this study, we investigated the modulation of TG2 activity by the A1 adenosine receptor in cardiomyocyte-like H9c2 cells.H9c2 cells were lysed following stimulation with the A1 adenosine receptor agonist N(6)-cyclopentyladenosine (CPA). Transglutaminase activity was determined using an amine incorporating and a protein cross linking assay. TG2 phosphorylation was assessed via immunoprecipitation and Western blotting. The role of TG2 in A1 adenosine receptor-induced cytoprotection was investigated by monitoring hypoxia-induced cell death. CPA induced time and concentration-dependent increases in amine incorporating and protein crosslinking activity of TG2. CPA-induced increases in TG2 activity were attenuated by the TG2 inhibitors Z-DON and R283. Responses to CPA were blocked by PKC (Ro 31-8220), MEK1/2 (PD 98059), p38 MAPK (SB 203580) and JNK1/2 (SP 600125) inhibitors and by removal of extracellular Ca(2+). CPA triggered robust increases in the levels of TG2-associated phosphoserine and phosphothreonine, which were attenuated by PKC, MEK1/2 and JNK1/2 inhibitors. Fluorescence microscopy revealed TG2-mediated biotin-X-cadaverine incorporation into proteins and proteomic analysis identified known (Histone H4) and novel (Hexokinase 1) protein substrates for TG2. CPA pre-treatment reversed hypoxia-induced LDH release and decreases in MTT reduction. TG2 inhibitors R283 and Z-DON attenuated A1 adenosine receptor-induced cytoprotection. TG2 activity was stimulated by the A1 adenosine receptor in H9c2 cells via a multi protein kinase dependent pathway. These results suggest a role for TG2 in A1 adenosine receptor-induced cytoprotection.
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    • "Generally, less than 1% of total ions from the ion source are utilized for separation. One method to compensate for this low duty cycle is summing the drift time and flight time datasets with multiple runs at the expense of time, especially for HPLC experiments [24]. Another approach to improve the duty cycle is the use of an ion trap as the ion gate [38, 39]. "
    [Show abstract] [Hide abstract] ABSTRACT: A high performance liquid chromatograph (HPLC)was interfaced to an atmospheric drift tube ion mobility time of flight mass spectrometry. The power of multidimensional separation was demonstrated using chili pepper extracts. The ambient pressure drift tube ion mobility provided high resolving powers up to 166 for the HPLC eluent. With implementation of Hadamard transform (HT), the duty cycle for the ion mobility drift tube was increased from less than 1% to 50%, and the ion transmission efficiency was improved by over 200 times compared with pulsed mode, improving signal to noise ratio 10 times. HT ion mobility and TOF mass spectrometry provide an additional dimension of separation for complex samples without increasing the analysis time compared with conventional HPLC. Graphical Abstract ᅟ
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