Phosphorylation SitesPeptidesImmobilized metal ion affinity chromatographyEnrichment of phosphopeptidesQuantificationBottom-up proteomProteinsSyntheticIsolationIMACMixturesKinaseTrypticAssaysMassAnalysisGradientZinc fingerPhosphoproteinsAnalysesTissueSignificantlyComplexProtein IdentificationExperimentUniqueCutoffFractionBiomarkersMethodCancer
Phosphorylation Sites5
- Mass spectrometry analysis of phosphopeptides was used to determine two MPK phosphorylation sites in ZAT10. (springer.com)
- Stable isotope labeling of phosphopeptides for multiparallel kinase target analysis and identification of phosphorylation sites. (mpg.de)
- Recent developments in mass spectrometry-based phosphoproteomics have led to the identification of thousands of differentially phosphorylated proteins and their phosphorylation sites. (molcells.org)
- With this present work, we report an approach to identify a large number of phosphopeptides and their phosphorylation sites using TiO2-centered phosphopeptide enrichment followed by LC-MS/MS analysis using high level of sensitivity nano-electrospray and an LTQ-Orbitrap instrument. (scienza-under-18.org)
- Identification of in vitro 40 S ribosomal S6 phosphorylation sites. (concordia.ca)
Peptides10
- Custom Workflow for the Confident Identification of Sulfotyrosine-Containing Peptides and Their Discrimination from Phosphopeptides. (liverpool.ac.uk)
- Improved phosphopeptide analysis Ti-IMAC beads were added to tryptic peptides for enrichment. (thermofisher.com)
- IMAC can be used to selectively enrich phosphopeptides from complex peptide mixtures, but co-retention of acidic peptides together with the failure to retain some phosphopeptides restricts the general utility of the method. (reading.ac.uk)
- Immunocytochemical and molecular data guide peptide identification by mass spectrometry: Orcokinin and orcomyotropin-related peptides in the stomatogastric nervous system of several crustacean species. (mpg.de)
- Dr. Robin Park, Proteomics Bioinformatics Architect at Bruker, added: "Since our DL training model also incorporated phosphopeptide data, TIMScore enhanced the identification of phosphorylated PSMs and peptides by an average of 61% on our dataset. (przoom.com)
- Figure 1 schematically outlines workflows for identification and quantitation of phosphorylated proteins and peptides. (yale.edu)
- The ProteoExtract ® Phosphopeptide TiO 2 Enrichment Kit is a useful tool for enrichment of phosphorylated peptides from complex samples by a highly selective Titanium oxide solid phase. (emdmillipore.com)
- In the ERLIC mode the combination of electrostatic attraction and hydrophilic interaction does suffice to pull phosphopeptides away from the nonphosphorylated peptides in a tryptic digest. (polylc.com)
- We report that low percentages of dimethylsulfoxide (DMSO) in liquid chromatography solvents lead to a strong enhancement of electrospray ionization of peptides, improving the sensitivity of protein identification in bottom-up proteomics by up to tenfold. (hecklab.com)
- Overall, ~6000 proteins, ~9000 unique phosphopeptides and 221 acetylated peptides were accurately quantified across all tissues. (lu.se)
Immobilized metal ion affinity chromatography2
- Combining immunoprecipitation of tyrosine phosphopeptides with mass spec they identified 435 unique phosphopeptides, while enriching with immobilized metal ion affinity chromatography (IMAC) and using SILAC mass spec for quantification they identified 4,433 phosphopeptides. (genomeweb.com)
- Comprehensive and reproducible phosphopeptide enrichment using iron immobilized metal ion affinity chromatography (Fe-IMAC) columns. (hecklab.com)
Enrichment of phosphopeptides2
- The workflow is generally divided into two options: one for TiO 2 enrichment of phosphopeptides after the protein(s) of interest are purified (e.g. (yale.edu)
- Examples of analysis that can be performed at CTP are identification and quantification of proteins, global discovery, label free quantification (LFQ), in-gel digest, in-solution digest, enrichment of phosphopeptides and purity of recombinant proteins. (lu.se)
Quantification3
- Comparative quantification and identification of phosphoproteins using stable isotope labeling and liquid chromatography/mass spectrometry. (mpg.de)
- Accurate quantification and enumeration of peptide motifs is hampered by redundancy in peptide identification. (biorxiv.org)
- This next generation system provides you with the highest chroma-tographic resolution resulting in exceptional peptide identification and quantification. (bruker.com)
Bottom-up proteom1
- 2000 phosphopeptides via bottom-up proteomics methods. (polylc.com)
Proteins5
- Here we describe the synthesis of a series of α,β-phosphopeptides, based on the phosphoepitope site on YAP1 (yes-associated protein 1), and the biochemical, biophysical and structural characterization of their binding to 14-3-3 proteins. (rcsb.org)
- 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. (usda.gov)
- The first is a more targeted approach to discover numerous sites of phosphorylation on proteins of interest, whereas the latter is directed towards a more global phosphopeptide discovery based approach. (yale.edu)
- 2013) Identification of missing proteins in the neXtProt database and unregistered phosphopeptides in the PhosphoSitePlus database as part of the Chromosome-centric Human Proteome Project. (phosphosite.org)
- Identification of proteins in complex mixtures using immunoprecipitation, SDS-PAGE and/or 2D/LC. (proteabio.com)
Synthetic1
- In this study Fe(III)-IMAC was qualitatively and quantitatively assessed using a panel of phosphopeptides, both synthetic and derived from proteolysis of known phosphoproteins, to identify the causes of success and failure in the application of this technique. (reading.ac.uk)
Isolation2
- It also permits the selective isolation of phosphopeptides from a tryptic digest. (polylc.com)
- This attraction is not sufficient to overcome the electrostatic repulsion from the two basic groups in a typical tryptic peptide (the N-terminus and the Lys- or Arg- residue at the C-terminus), which is why anion-exchange has not been used for selective isolation of phosphopeptides from tryptic digests. (polylc.com)
IMAC3
- Modulating the loading buffer used for Fe(III)-IMAC significantly affects phosphopeptide binding and suggests that conformational factors that lead to steric hindrance and reduced accessibility to the phosphate are important. (reading.ac.uk)
- The use of 1,1,1,3,3,3-hexa-fluoroisopropanol is shown here to significantly improve Fe(III)-IMAC enrichment and subsequent detection of phosphopeptides by MALDI-MS. (reading.ac.uk)
- We will look at phosphopeptide mapping of your protein, using IMAC and LC/MS/MS. We will identify candidate binding partners for known targets. (proteabio.com)
Mixtures1
- SDS PAGE) or immunoprecipitated and another for more complex phosphopeptide mixtures which are directly extracted from tissue or whole cell extracts. (yale.edu)
Kinase1
- Overall, our results demonstrate an modified phosphopattern in CB2R-IN-1 the glutamatergic synapse, together with the identification of a protein kinase/phosphatase biochemical signature in the Ts65Dn murine model, which may represent novel restorative focuses on for DS synaptopathy. (logic2010.org)
Tryptic5
- These analyses suggest that the tryptic peptide containing S90 and S93 is a major phosphopeptide in human TTP. (usda.gov)
- In addition to isolating the phosphopeptides from the nonphosphopeptides in tryptic digests, ERLIC is also a high-resolution method for separating the phosphopeptides from each other. (polylc.com)
- 20 fmol BSA tryptic digest (Bruker Daltonics 8217498) have been injected onto a PepSep TWENTY-FIVE column (Part No. 1893476, 150 μm ID, 1.5 μm particle size) and separated by a 10 min gradient. (bruker.com)
- 20 fmol BSA tryptic digest (8217498) have been injected onto a PepSep FIFTEEN column (Part No. 1895814, 75 μm ID, 1.9 μm particle size) and separated by a 10 min gradient. (bruker.com)
- More than 6800 protein groups identified using the Bruker ProteoScape realtime search engine with TIMScore™ for dda-PASEF of K562 tryptic digest with a PepSep Twenty-five series column (150 μm ID, 1.5 μm particle size) within a 41 min runtime on the timsTOF HT. (bruker.com)
Assays1
- Collectively, these results indicate the feasibility of using super-SILAC mix-SIM/AIMS assays for targeted verification of phosphopeptides discovered by large-scale phosphoproteome analyses of HCC specimens. (nih.gov)
Mass5
- This is accomplished through phosphopeptide mapping, which involves digestion of a radioactively labeled protein, separation of phosphopeptide products, and finally analysis via high-performance liquid chromatography (HPLC) or mass spectrometry. (wikipedia.org)
- This study generated additional information on TTP phosphorylation using phosphopeptide mapping and mass spectrometry. (usda.gov)
- To expand our verification capability, we evaluated the use of LTQ-Orbitrap run in SIM/Accurate inclusion mass screening (AIMS) mode with a super-SILAC mixture as an internal standard to quantify a subset of phosphopeptide candidates in HCC tissue samples. (nih.gov)
- Our optimized TiO 2 phosphopeptide enrichment methodologies are combined with a state-of-the-art nanoACQUITY™ Ultra Performance or Ultra-high pressure LC (UPLC) system (Waters Corp.) /LTQ-Orbitrap XL mass spectrometer (Thermo Scientific) platform to provide a very robust phosphoprotein profiling and phosphoproteomics analysis. (yale.edu)
- The second option provides high accuracy mass measurement that is essential for the validation of revised peptide identifications and the reduction of false positive identifications. (scienza-under-18.org)
Analysis4
- Analysis of phosphopeptides can provide information about which amino acids are phosphorylated and how many sites on the primary sequence are phosphorylated. (wikipedia.org)
- This analysis yielded 7868 quantifiable phosphopeptides and numerous up- or down-regulated candidates. (nih.gov)
- phosphopeptides ETTCSKEpSNEELTESCETK and AIPVAQDLNAPSDWDpSR were synthesized from Peptide 2.0 Inc.. The solitary clinical specimen chosen for analysis, a pre-operative, pre-treatment serum sample procured from a male subject with prostate malignancy undergoing radical prostatectomy, was acquired under IRB authorization (University or college of California-Irvine) and individual consent. (scienza-under-18.org)
- Analysis using site-specific anti-phosphopeptide antibodies. (concordia.ca)
Gradient1
- B) Boxplot for protein group and precursor identifications dependent on gradient length (DDA-PASEF - timsTOF Pro 2, and Bruker ProteoScape realtime search engine with TIMScore). (bruker.com)
Zinc finger1
- MS also identified a major phosphopeptide associated with the first zinc-finger region. (usda.gov)
Phosphoproteins1
- This strategy allowed us to generate a quantitative HCC tissue phosphoproteome dataset containing significantly deregulated phosphoproteins that represents a valuable resource for the identification of potential HCC biomarkers and/or therapeutic targets. (nih.gov)
Analyses1
- LC-MS/MS analyses identified 2755 and 3191 phosphopeptides from experiments with activator or inhibitor of PKA. (nih.gov)
Tissue1
- Furthermore, our proof-of-concept experiments demonstrated the feasibility of applying LTQ-Orbitrap, operated in SIM/AIMS mode, to multiplex and targeted verification of phosphopeptides in individual tissue specimens using a super-SILAC mix as an internal phosphopeptide standard. (nih.gov)
Significantly1
- Of these, they identified 121 phosphopeptides that were significantly upregulated and 165 that were significantly downregulated in all replicates, which they used to build a ceritinib effector network consisting of 139 protein nodes and 312 interactions, with RSK1/2, IGF1R, and FAK1 among the most central nodes of this network. (genomeweb.com)
Complex1
- Advances in phosphopeptide enrichment methods enable the identification of thousands of phosphopeptides from complex samples. (hecklab.com)
Protein Identification3
- We offer integrated Protein identification and post-identification services, including Protein characterization, identification of posttranslational modifications, and protein-protein interactions. (proteabio.com)
- Our integrated Protein Identification services are available both for your samples (gel bands or solution), or you may request samples for your use from our own protein sample database. (proteabio.com)
- Protein identification is validated by manual confirmation of the MS/MS spectra. (proteabio.com)
Experiment2
- In sample I used for discovery experiment, we successfully quantified 32 (in SIM mode) and 30 (in AIMS mode) phosphopeptides with median coefficients of variation (CVs) of 7.5% and 8.3%, respectively. (nih.gov)
- When the assay was applied to other three pairs of HCC specimens for verification experiment, 40 target phosphopeptides were quantified reliably (~7.5% CV), and more than half of them were differentially expressed between tumor and adjacent non-tumor tissues. (nih.gov)
Unique1
- Using this approach we recognized ~100 unique phosphopeptides with stringent filtering criteria and a lower than 1% false discovery rate. (scienza-under-18.org)
Cutoff1
- Even more encouraging is the 57% improvement in the number of phosphopeptides identified using the strict 5% FLR cutoff. (przoom.com)
Fraction1
- 1) Low-salt fraction: HyperCarb retains ~ 50% more phosphopeptides than does C-18, and appreciably less nonphosphopeptides. (polylc.com)
Biomarkers2
- More generally, the research demonstrates the potential of such work to enable rational selection of combination therapies as well as identification of candidate biomarkers for predicting patient responses to therapy, said Uwe Rix, an assistant professor at Moffitt and senior author on the paper. (genomeweb.com)
- Biomarkers for identification of these histologically identical WHO II tumors do not exist. (nature.com)
Method1
- This method could be readily applied to verify dozens of phosphopeptide candidates in a larger HCC sample set. (nih.gov)
Cancer1
- Simone Lemeer works on the elucidation of the mechanisms of action of small molecule inhibitors in cancer cells, as well as identification of the intrinsic resistance mechanisms. (hecklab.com)