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Identification of a major phosphopeptide in human tristetraprolin by phosphopeptide mapping and mass spectrometry.

ABSTRACT: Tristetraprolin/zinc finger protein 36 (TTP/ZFP36) binds and destabilizes some pro-inflammatory cytokine mRNAs. TTP-deficient mice develop a profound inflammatory syndrome due to excessive production of pro-inflammatory cytokines. TTP expression is induced by various factors including insulin and extracts from cinnamon and green tea. TTP is highly phosphorylated in vivo and is a substrate for several protein kinases. Multiple phosphorylation sites are identified in human TTP, but it is difficult to assign major vs. minor phosphorylation sites. This study aimed to generate additional information on TTP phosphorylation using phosphopeptide mapping and mass spectrometry (MS). Wild-type and site-directed mutant TTP proteins were expressed in transfected human cells followed by in vivo radiolabeling with [32P]-orthophosphate. Histidine-tagged TTP proteins were purified with Ni-NTA affinity beads and digested with trypsin and lysyl endopeptidase. The digested peptides were separated by C18 column with high performance liquid chromatography. Wild-type and all mutant TTP proteins were localized in the cytosol, phosphorylated extensively in vivo and capable of binding to ARE-containing RNA probes. Mutant TTP with S90 and S93 mutations resulted in the disappearance of a major phosphopeptide peak. Mutant TTP with an S197 mutation resulted in another major phosphopeptide peak being eluted earlier than the wild-type. Additional mutations at S186, S296 and T271 exhibited little effect on phosphopeptide profiles. MS analysis identified the peptide that was missing in the S90 and S93 mutant protein as LGPELSPSPTSPTATSTTPSR (corresponding to amino acid residues 83-103 of human TTP). MS also identified a major phosphopeptide associated with the first zinc-finger region. These analyses suggest that the tryptic peptide containing S90 and S93 is a major phosphopeptide in human TTP.

SUBMITTER: Cao H

PROVIDER: S-EPMC4091943 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Identification of a major phosphopeptide in human tristetraprolin by phosphopeptide mapping and mass spectrometry.

Cao Heping H Deterding Leesa J LJ Blackshear Perry J PJ

PloS one 20140710 7

Tristetraprolin/zinc finger protein 36 (TTP/ZFP36) binds and destabilizes some pro-inflammatory cytokine mRNAs. TTP-deficient mice develop a profound inflammatory syndrome due to excessive production of pro-inflammatory cytokines. TTP expression is induced by various factors including insulin and extracts from cinnamon and green tea. TTP is highly phosphorylated in vivo and is a substrate for several protein kinases. Multiple phosphorylation sites are identified in human TTP, but it is difficult ...[more]

PMID: 25010646

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Project description:MotivationComplex patterns of protein phosphorylation mediate many cellular processes. Tandem mass spectrometry (MS/MS) is a powerful tool for identifying these post-translational modifications. In high-throughput experiments, mass spectrometry database search engines, such as MASCOT provide a ranked list of peptide identifications based on hundreds of thousands of MS/MS spectra obtained in a mass spectrometry experiment. These search results are not in themselves sufficient for confident assignment of phosphorylation sites as identification of characteristic mass differences requires time-consuming manual assessment of the spectra by an experienced analyst. The time required for manual assessment has previously rendered high-throughput confident assignment of phosphorylation sites challenging.ResultsWe have developed a knowledge base of criteria, which replicate expert assessment, allowing more than half of cases to be automatically validated and site assignments verified with a high degree of confidence. This was assessed by comparing automated spectral interpretation with careful manual examination of the assignments for 501 peptides above the 1% false discovery rate (FDR) threshold corresponding to 259 putative phosphorylation sites in 74 proteins of the Trypanosoma brucei proteome. Despite this stringent approach, we are able to validate 80 of the 91 phosphorylation sites (88%) positively identified by manual examination of the spectra used for the MASCOT searches with a FDR < 15%.ConclusionsHigh-throughput computational analysis can provide a viable second stage validation of primary mass spectrometry database search results. Such validation gives rapid access to a systems level overview of protein phosphorylation in the experiment under investigation.AvailabilityA GPL licensed software implementation in Perl for analysis and spectrum annotation is available in the supplementary material and a web server can be assessed online at http://www.compbio.dundee.ac.uk/prophossi.

Project description:Antibody mediated strategies for protein biomarker detection are common, but may limit discovery. We hypothesized that the use of antibody-free proteomics is feasible for detecting protein biomarkers in plasma of patients sustaining major trauma. A subset of subjects with major trauma from a prospective observational trial were analyzed. Patients were assigned to one of four groups based on their presenting Abbreviated Injury Severity Score (AIS). Sensitive, antibody-free selective reaction monitoring (SRM) mass spectrometry (MS), with spiked-in isotopically labeled synthetic peptides, was used for targeted protein quantification of a panel of 10 prospective targets. An overall tiered sensitivity analytical approach was used for peptide detection and quantification based upon plasma immunoaffinity depletion and PRISM fractionation. Forty-four patients were included in the analysis, of which 82% were men with a mean age of 50 (±19) years. Half had isolated head injury (n = 22), with the remaining patients experiencing multiple injuries or polytrauma (n = 14), isolated body injury (n = 2), or minor injury (n = 6). Peptides from 3 proteins (vascular adhesion molecule 1 [VCAM1], intercellular adhesion molecule 1 [ICAM1], and matrix metalloproteinase 9 [MMP9]) were detected and quantified in non-depleted processed plasma. Peptides from 2 proteins (angiopoietin 2 [Ang2] and plasminogen activator inhibitor-1 [PAI1]) were detected and quantification in depleted plasma, whereas the remaining 5 of the 10 prospective targets were undetected. VCAM1 (p = 0.02) and MMP9 (p = 0.03) were significantly upregulated in in the major trauma groups (1-3) versus mild injury group (4), whereas the others were not. There were no differences in protein expression between patients with traumatic brain injury (TBI; groups 1 and 2) versus those without TBI (groups 3 and 4). We detected non-specific upregulation of proteins reflecting blood-brain barrier breakdown in severely injured patients, indicating label-free MS techniques are feasible and may be informative.

Dataset Information

Identification of a major phosphopeptide in human tristetraprolin by phosphopeptide mapping and mass spectrometry.

Publications

Identification of a major phosphopeptide in human tristetraprolin by phosphopeptide mapping and mass spectrometry.

Similar Datasets

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