Mass spectrometryEnrichmentUnique phosphopeptidesEnrich phosphopeptidesPeptideIsolationBovinePhosphoproteomicsMixturesProteinsIMACAssaysPhosphoproteomeBasalCandidatesDeterminationVerificationMolecularAnalysisQuantitativeDiscoveryPhosphorylation sitesStraightforwardAnalysisExperimentalStepToolsComparisonCellApproximatelyMass
Mass spectrometry2
- This mixture contains 131 mass spectrometry vetted phosphopeptides specifically chosen to cover as many known biologically interesting phosphosites as possible from seven different signaling networks: AMPK signaling, death and apoptosis signaling, ErbB signaling, insulin/IGF-1 signaling, mTOR signaling, PI3K/AKT signaling, and stress (p38/SAPK/JNK) signaling. (stanford.edu)
- To test this hypothesis, we compared the phosphopeptide-enriched fractions obtained from proteins secreted into conditioned media (CM) derived from five luminal and five basal type breast cancer cell lines using label-free quantitative mass spectrometry. (escholarship.org)
Enrichment6
- 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 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)
- 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)
- reported the profiling of endogenous serum phosphorylated peptides by TiO2-enrichment and MALDI-TOF MS detection.21 However, the characterization and description of the content of phosphoprotein analytes in serum were limited. (scienza-under-18.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)
- Assessment of immunoaffinity (anti-phosphotyrosine antibody) versus chemical (TiO2) enrichment methods, as well as solitary versus dual enrichment strategies, yielded an initial assessment of the phosphoprotein/peptide content of human being serum, which can serve as a launch-point for further exploration and analysis. (scienza-under-18.org)
Unique phosphopeptides1
- Using this approach we recognized ~100 unique phosphopeptides with stringent filtering criteria and a lower than 1% false discovery rate. (scienza-under-18.org)
Enrich phosphopeptides1
- 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)
Peptide1
- 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)
Isolation1
- This study utilized IMAC phosphoprotein isolation directly coupled with LC MS/MS analysis. (biomedcentral.com)
Bovine2
- Sequencing of chymotryptic and thermolytic phosphopeptides from bovine DARPP-32 phosphorylated by casein kinase II suggested that the main phosphorylated residues were Ser45 and Ser102. (cornell.edu)
- Proteins from cell lysate, uncooked serum, eluted serum proteins that were depleted of albumin and IgG, and eluted albumin and IgG from resin, were spiked with the phosphoprotein bovine beta-casein as an internal standard, reduced by 10 mM DTT in the presence of 8 M urea for 30 minutes at 37 C, and then alkylated by 50 mM iodoacetamide at space temp. (scienza-under-18.org)
Phosphoproteomics1
- A multipathway phosphopeptide standard for rapid phosphoproteomics assay development. (stanford.edu)
Mixtures1
- SDS PAGE) or immunoprecipitated and another for more complex phosphopeptide mixtures which are directly extracted from tissue or whole cell extracts. (yale.edu)
Proteins1
- 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)
IMAC2
- 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)
- 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)
Assays2
- 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)
- As a research group under the Association for Biomolecular Resource Facilities (ABRF) umbrella, the Proteomics Standards Research Group (sPRG) has worked to develop a multipathway phosphopeptide standard based on a mixture of heavy-labeled phosphopeptides designed to enable researchers to rapidly develop assays. (stanford.edu)
Phosphoproteome1
- 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)
Basal1
- Altogether over 5000 phosphosites derived from 1756 phosphoproteins were identified, several of which have the potential to qualify as phosphopeptide plasma biomarker candidates for the more aggressive basal and also the luminal-type breast cancers. (escholarship.org)
Candidates5
- This analysis yielded 7868 quantifiable phosphopeptides and numerous up- or down-regulated candidates. (nih.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)
- This method could be readily applied to verify dozens of phosphopeptide candidates in a larger HCC sample set. (nih.gov)
- The analysis of phosphopeptides from breast cancer patient plasma and controls allowed us to construct a discovery list of phosphosites under rigorous collection conditions, and second to qualify discovery candidates generated from the CM studies. (escholarship.org)
- Together, this approach allowed us to assemble a set of novel cancer subtype specific phosphopeptide candidates for subsequent biomarker verification and clinical validation. (escholarship.org)
Determination1
- Comparison of thermolytic phosphopeptide maps and determination of the phosphorylated residue by manual Edman degradation identified the main phosphorylation site in intact cells as Ser102. (cornell.edu)
Verification3
- 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)
- In this study, we developed a strategy for conducting both discovery and targeted verification of deregulated phosphoproteins in HCC tissue specimens on LTQ-Orbitrap. (nih.gov)
- 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)
Molecular1
- In this study, a phosphoproteomic approach was performed to dissect the molecular mechanism of phosphoprotein regulation, which is involved in the inhibitory effect of parasites on erythroid cell development. (biomedcentral.com)
Analysis1
- Recent advances in methodology have made phosphopeptide analysis a tractable problem for many proteomics researchers. (stanford.edu)
Quantitative1
- Our results suggest that widely available, biologically-relevant phosphopeptide standards can act as a quantitative "yardstick" across laboratories and sample preparations, enabling experimental designs larger than a single laboratory can perform. (stanford.edu)
Discovery1
- 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)
Phosphorylation sites1
- Because phosphorylation is a dynamic process difficult to quantify, we must at first acquire an inventory of phosphoproteins and characterize their phosphorylation sites. (biomedcentral.com)
Straightforward1
- Unfortunately, phosphoproteins analysis is not straightforward for five main reasons. (biomedcentral.com)
Analysis3
- This analysis yielded 375 non-redundant phosphopeptides tied to the PI3K pathway, a number Hendrickson said was somewhat higher than the researchers had expected, particularly given "that the PIK3 pathway has been so well studied to date. (genomeweb.com)
- Analysis of the entire cellular phosphoproteins panel, the so-called phosphoproteome, has been an attractive study subject since the discovery of phosphorylation as a key regulatory mechanism of cell life. (biomedcentral.com)
- Well established methods involving the analysis of 32 P-labeled phosphoproteins by Edman degradation and two-dimensional phosphopeptide mapping have proven to be powerful but not without limitations. (biomedcentral.com)
Experimental1
- One of the main challenges of phosphoproteomic approaches is to deconvolute biological processes that specifically respond to an experimental query from a list of phosphoproteins. (biomedcentral.com)
Step1
- A research team led by Merck scientists has taken what could represent a first step toward using phosphoproteins as biomarkers for cancer drug development. (genomeweb.com)
Tools1
- The results, published in a study in this week's Science Translational Medicine , suggest that biomarker panels comprising some of the drug-responsive phosphoproteins could be used as tools for studying and evaluating the effectiveness of PI3K pathway inhibitors, Ronald Hendrickson, director of proteomics at Merck Research Laboratories and one of the study's authors, told ProteoMonitor . (genomeweb.com)
Comparison1
- Comparison of thermolytic phosphopeptide maps and determination of the phosphorylated residue by manual Edman degradation identified the main phosphorylation site in intact cells as Ser102. (cornell.edu)
Cell1
- Using a label-free approach, we quantified approximately 3400 proteins and 5700 phosphopeptides in cell lysates across four independent donors. (nih.gov)
Approximately1
- the number of peptides and phosphopeptides were approximately 50% and 76% lower in FFPE specimens, respectively, relative to frozen OCT-embedded specimens. (nih.gov)
Mass2
- They then enriched for phosphopeptides using phospho-motif-specific antibodies and sequenced them via electron disassociation mass spectrometry on a Thermo Electron triple quadrupole-ETD instrument. (genomeweb.com)
- Phosphopeptides were further separated by UPLC and analyzed using a Q-Exactive HF mass spectrometer. (nih.gov)