Identification and characterization of phosphorylated proteins in the human pituitary

Giorgianni, Francesco; Beranová-Giorgianni, Šárka; Desiderio, Dominic M.

doi:10.1002/pmic.200300584

Cited by 65 publications

(61 citation statements)

References 22 publications

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“…MS has been widely applied as a powerful tool to characterize protein modifications including phosphorylation due to its high sensitivity and capability of rapid sequencing by tandem mass spectrometric (MS n ) technique (3)(4)(5)(6). For the phosphoproteome analysis, satisfying results often cannot be obtained by direct mass spectrometric analysis of a protein digest.…”

Section: Molecular and Cellular Proteomics 6:1656 -1665 2007mentioning

confidence: 99%

Immobilized Zirconium Ion Affinity Chromatography for Specific Enrichment of Phosphopeptides in Phosphoproteome Analysis

Feng

Zhou

et al. 2007

Molecular & Cellular Proteomics

234

186

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Large scale characterization of phosphoproteins requires highly specific methods for purification of phosphopeptides because of the low abundance of phosphoproteins and substoichiometry of phosphorylation. Enrichment of phosphopeptides from complex peptide mixtures by IMAC is a popular way to perform phosphoproteome analysis. However, conventional IMAC adsorbents with iminodiacetic acid as the chelating group to immobilize Fe 3؉ lack enough specificity for efficient phosphoproteome analysis. Here we report a novel IMAC adsorbent through Zr 4؉ chelation to the phosphonate-modified poly(glycidyl methacrylate-co-ethylene dimethacrylate) polymer beads. The high specificity of Zr 4؉ -IMAC adsorbent was demonstrated by effectively enriching phosphopeptides from the digest mixture of phosphoprotein (␣-or ␤-casein) and bovine serum albumin with molar ratio at 1:100. Zr 4؉ -IMAC adsorbent was also successfully applied for the analysis of mouse liver phosphoproteome, resulting in the identification of 153 phosphopeptides (163 phosphorylation sites) from 133 proteins in mouse liver lysate. Significantly more phosphopeptides were identified than by the conventional Fe 3؉ -IMAC approach, indicating the excellent performance of the Zr 4؉ -IMAC approach. The high specificity of Zr 4؉ -IMAC adsorbent was found to mainly result from the strong interaction between chelating Zr 4؉ and phosphate group on phosphopeptides. Enrichment of phosphopeptides by Zr 4؉ -IMAC provides a powerful approach for large scale phosphoproteome analysis.

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Section: Molecular and Cellular Proteomics 6:1656 -1665 2007mentioning

confidence: 99%

Immobilized Zirconium Ion Affinity Chromatography for Specific Enrichment of Phosphopeptides in Phosphoproteome Analysis

Feng

Zhou

et al. 2007

Molecular & Cellular Proteomics

234

186

View full text Add to dashboard Cite

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“…39,40 Its biological function as potential precursor of active neuropeptide and disease-associated mechanism(s) remain uncharacterized. 41 Given that chromogranins A and B are present in human cerebrospinal fluid at higher concentration than in plasma, and have a role in the biochemistry of the synapses, they may serve as markers for large dense-core synaptic vesicle function and pathology. 39,42 Potential drug effects for the microarray screening of candidate genes were generally discussed previously.…”

Section: Synapse and Neurotransmissionmentioning

confidence: 99%

An integrated genomic analysis of gene-function correlation on schizophrenia susceptibility genes

Chu

Liu

2010

J Hum Genet

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Schizophrenia is a highly complex inheritable disease characterized by numerous genetic susceptibility elements, each contributing a modest increase in risk for the disease. Although numerous linkage or association studies have identified a large set of schizophrenia-associated loci, many are controversial. In addition, only a small portion of these loci overlaps with the large cumulative pool of genes that have shown changes of expression in schizophrenia. Here, we applied a genomic gene-function approach to identify susceptibility loci that show direct effect on gene expression, leading to functional abnormalities in schizophrenia. We carried out an integrated analysis by cross-examination of the literature-based susceptibility loci with the schizophrenia-associated expression gene list obtained from our previous microarray study (Journal of Human Genetics (2009) 54: 665-75) using bioinformatic tools, followed by confirmation of gene expression changes using qPCR. We found nine genes (CHGB, SLC18A2, SLC25A27, ESD, C4A/C4B, TCP1, CHL1 and CTNNA2) demonstrate gene-function correlation involving: synapse and neurotransmission; energy metabolism and defense mechanisms; and molecular chaperone and cytoskeleton. Our findings further support the roles of these genes in genetic influence and functional consequences on the development of schizophrenia. It is interesting to note that four of the nine genes are located on chromosome 6, suggesting a special chromosomal vulnerability in schizophrenia.

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“…Recent studies have introduced two new innovations, namely, immobilized metal affinity chromatography (IMAC) (1-3) and liquid chromatography-mass spectrometry (LC-MS) 3 neutral loss scanning (1)(2)(3)(4), to increase the efficiency of phosphopeptide identification. Quantification of phosphopeptides in this setting has been challenging and has been successful so far in cultured cells (5) and yeast (6) but not in native mammalian cells and tissues.…”

mentioning

confidence: 99%

Quantitative phosphoproteomics of vasopressin-sensitive renal cells: Regulation of aquaporin-2 phosphorylation at two sites

Hoffert

Pisitkun

Wang

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

327

325

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Protein phosphorylation plays a key role in vasopressin signaling in the renal-collecting duct. Large-scale identification and quantification of phosphorylation events triggered by vasopressin is desirable to gain a comprehensive systems-level understanding of this process. We carried out phosphoproteomic analysis of rat inner medullary collecting duct cells by using a combination of phosphopeptide enrichment by immobilized metal affinity chromatography and phosphorylation site identification by liquid chromatography-mass spectrometry n neutral loss scanning. A total of 714 phosphorylation sites on 223 unique phosphoproteins were identified from inner medullary collecting duct samples treated shortterm with either calyculin A or vasopressin. A number of proteins involved in cytoskeletal reorganization, vesicle trafficking, and transcriptional regulation were identified. Previously unidentified phosphorylation sites were found for membrane proteins essential to collecting duct physiology, including eight sites among aquaporin-2 (AQP2), aquaporin-4, and urea transporter isoforms A1 and A3. Through label-free quantification of phosphopeptides, we identified a number of proteins that significantly changed phosphorylation state in response to short-term vasopressin treatment: AQP2, Bclaf1, LRRC47, Rgl3, and SAFB2. In the presence of vasopressin, AQP2 monophosphorylated at S256 and diphosphorylated AQP2 (pS256͞261) increased in abundance, whereas AQP2 monophosphorylated at S261 decreased, raising the possibility that both sites are involved in vasopressin-dependent AQP2 trafficking. This study reveals the practicality of liquid chromotography-mass spectrometry n neutral loss scanning for large-scale identification and quantification of protein phosphorylation in the analysis of cell signaling in a native mammalian system. LC-MS͞MS ͉ collecting duct ͉ kidney ͉ Collecting Duct Phosphoprotein Database (CDPD) ͉ neutral loss E lucidation of cellular signaling networks requires methodologies for large-scale quantitative phosphoproteomic analysis that can be used to reveal dynamic system-wide changes in protein phosphorylation. Recent studies have introduced two new innovations, namely, immobilized metal affinity chromatography (IMAC) (1-3) and liquid chromatography-mass spectrometry (LC-MS) 3 neutral loss scanning (1-4), to increase the efficiency of phosphopeptide identification. Quantification of phosphopeptides in this setting has been challenging and has been successful so far in cultured cells (5) and yeast (6) but not in native mammalian cells and tissues. Here, we introduce a hybrid approach using IMAC for phosphopeptide enrichment, LC-MS multisequential (LC-MS n ) neutral loss scanning to identify phosphorylated residues in the peptides, and label-free quantification using numerical integration of pseudochromatograms constructed from MS peak heights.The method is used here for analysis of protein phosphorylation in vasopressin-sensitive inner medullary collecting duct (IMCD) cells freshly isolated from rat kidneys. ...

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Identification and characterization of phosphorylated proteins in the human pituitary

Cited by 65 publications

References 22 publications

Immobilized Zirconium Ion Affinity Chromatography for Specific Enrichment of Phosphopeptides in Phosphoproteome Analysis

Immobilized Zirconium Ion Affinity Chromatography for Specific Enrichment of Phosphopeptides in Phosphoproteome Analysis

An integrated genomic analysis of gene-function correlation on schizophrenia susceptibility genes

Quantitative phosphoproteomics of vasopressin-sensitive renal cells: Regulation of aquaporin-2 phosphorylation at two sites

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