Work flow for the LAXIC strategy to quantify the phosphorylation change in response to osmotic stress. Work flow for the LAXIC strategy to quantify the.

Slides:

Advertisements

Similar presentations

binding sites 58 of the 473 unambiguously assigned phosphorylation sites are predicted by Scansite to be sites for binding. 50 of these correspond.

Advertisements

Quantification of C-terminal phosphorylation of AtPIP2;1 upon NaCl (A) and H2O2 (B) treatments.A, plants were either untreated (white bars) or treated.

Sequence alignment of C-terminal phosphorylated plant aquaporins

Principle for quantification of phosphorylation of the C-terminal tail of AtPIP2;1. Principle for quantification of phosphorylation of the C-terminal tail.

MALDI-TOF MS spectrum of phosphopeptides from plant PM aquaporins.

Phosphopeptide sequencing by MALDI-TOF/TOF of the C-terminal tail of AtPIP2;1.A, MS/MS spectrum of singly phosphorylated 277SLGSFRSAANV287 (m/z ).

Distribution of disorder in the cytosolic phosphoproteome

Experimental overview A sequential protein and peptide IMAC enrichment was analyzed by four different LC-MS/MS strategies. Experimental overview A sequential.

Phosphorylation and sequence disorder in microtubule-associated protein Tau.A, schematic illustration of the domain profile of Tau with all known phosphorylation.

Distribution of phosphorylation sites identified in the cytosolic phosphoproteome.A, numbers of approved phosphopeptides, previously phosphorylated peptides,

Phosphopeptide sequencing by MALDI-TOF/TOF of the C-terminal tail of AtPIP2;4.A, MS/MS spectrum of singly phosphorylated 277ALGSFGSFGSFRSFA291 (m/z ).

Phosphopeptide sequencing by MALDI-TOF/TOF of the C-terminal tail of AtPIP2;7.A, MS/MS spectrum of singly phosphorylated 270ALGSFRSNATN280 (m/z ).

A quantitative proteomics strategy to identify SUMO-conjugated proteins. A quantitative proteomics strategy to identify SUMO-conjugated proteins. HeLa.

Percentage of proteins identified in envelope membrane extracts according to the purification method and the number of transmembrane domains. Percentage.

Novel phosphorylation sites on H+-ATPase proteins

Phosphorylation reduces peptide solution charge state and alters SCX elution at low pH.A, at pH 2.7, a theoretical tryptic peptide without histidine residues.

Frequency distribution of the GRAVY of the theoretical proteins (open bars) and of 110 genes encoding proteins identified on a 2-D electrophoresis gel,

Separation of embryonic brain proteins and peptides by sequential preparative SDS-PAGE and SCX chromatography.A, 6 mg of embryonic day 16.5 murine brain.

Relative quantitation of phosphopeptides from conditioned media from subtype specific breast cancer cell lines. Relative quantitation of phosphopeptides.

Time course of phosphorylation changes at Ser-293, Ser-300, and Ser-232 in PDHE1α following kinase inhibition with DCA. A, relative quantitation over three.

Schematic of MS1 filtering.

Significantly enriched phosphorylation motifs from up-regulated phosphopeptides by Motif-X analysis. Significantly enriched phosphorylation motifs from.

Visual validation of the computational outputs.

Epitope Mapping Performance using a single peptide microarray.

Schematic representation of proteogenomic annotation strategy.

Novel p53 target genes identified by RNA-Seq, pSILAC and ChIP-Seq.

Skyline MS1 filtering graphical user interface.

Representative example of LAXIC performance for complex plant phosphoproteome. Representative example of LAXIC performance for complex plant phosphoproteome.A.

Results from the Morris water task.

Technical reproducibility and biological variability.

Degree of glycosylation of human milk LF from individual donors across lactation. Degree of glycosylation of human milk LF from individual donors across.

A, Base peak chromatogram of apomyoglobin digest generated by 0

The evolutionary conservation of the phosphoproteomes.a, E. coli. b, B. subtilis. The evolutionary conservation of the phosphoproteomes.a, E. coli. b,

Colonopshere-enriched proteins display functional interactions.

Identification of SUMO3peptides from 2D-LC-MS/MS analyses of a tryptic digest of HEK293-SUMO3 cells using DDA and DIA methods. Identification of SUMO3peptides.

A, schematic presentation of fetuin-A domains.

Proteins previously reported in published MALDI IMS studies and their frequency of observation in the present study. Proteins previously reported in published.

N-terminal extension of a gene using peptides mapping upstream to an annotated start site. N-terminal extension of a gene using peptides mapping upstream.

Analysis of newly synthesized proteins by combined pulsed SILAC and click chemistry enrichment. Analysis of newly synthesized proteins by combined pulsed.

Schematic summarizing the various functions and features of MASH Suite Pro. Schematic summarizing the various functions and features of MASH Suite Pro.

Manual assessment of the quality of peptide spectra with scores ranging from 5 to 10 of OFFGEL electrophoresis fractions 3 and 4 that were rejected by.

Testing the effectiveness of the three-step peptide fractionation method.A, μLC mass chromatograms of SCX fractions for an acidic FFE fraction. Testing.

Altered pathways in prostate cancer.

Resolution and mass accuracy of A, a peptide isotope cluster (m/z 558

Putative targets of miRNAs directly induced by p53 with down-regulated mRNA- and de novo protein synthesis or reduced de novo protein synthesis only. Putative.

Interaction networks of the regulated phosphoproteins.

Standard concentration curves for stable isotope-labeled and acetyllysine containing peptides. Standard concentration curves for stable isotope-labeled.

2D-LC-MS/MS analysis of tryptic digest of HEK293-SUMO3 cells (2 μg inj

Overview of the analytical workflow used in this study and a representative MS/MS spectrum.a, Overview of the analytical workflow used in this study. Overview.

Identification of chaperonin GroEL (Rv0440) with representative MS/MS spectrum. Identification of chaperonin GroEL (Rv0440) with representative MS/MS spectrum.A,

Plot of the deviation of the predicted pI value of every peptide spectrum from the average pI calculated for each fraction for validated (a) and non-validated.

Distribution of the phosphoproteins based on GO analysis, including biological process (Left) and cellular component (Right). Distribution of the phosphoproteins.

Comparison of mapped epitopes and peptides identified in immuno-SILAC screening of polyclonal antibodies against trypsin-digested PrESTs. Comparison of.

Serum LAMC2 levels in pancreatic adenocarcinoma (PDAC) and other samples from Japan. Serum LAMC2 levels in pancreatic adenocarcinoma (PDAC) and other samples.

The principle of the immuno-SILAC method.

Chromatograms, MS and MS/MS spectra obtained by LC-MS/MS (Q-TOF) of peptides from UPIII identified after Western blotting followed by on-membrane digestion.

Analytical metrics of yeast proteome analysis using the Q-OT-qIT (Fusion) as compared with qIT-OT (Orbitrap Elite) and Q-OT (Q-Exactive) hybrids. Analytical.

Schematic of the Q-OT-qIT hybrid mass spectrometer (Fusion).

Biochemical characterization of the protein phosphatases Saci-PTP.

Identification and quantification of cathepsin D in chronic pancreatitis.A, identification of two peptides, AIGAVPLIQGEYMIPCEK (A) and LLDIACWIHH (MS/MS.

Illustration of chromatography metric C-2A applied to LC-MS/MS data from three Thermo LTQ systems in analyses of yeast proteome samples in CPTAC Study.

Quantitation of phosphopeptides binding to and recovered from different types of IMAC material.32P-labeled phosphopeptides were produced by in vitro autophosphorylation.

Classification of the 1458 identified proteins into molecular functions. Classification of the 1458 identified proteins into molecular functions. The pie.

2-D gel images visualized by Coomassie Brilliant Blue staining representing total proteins extracted from HCT-8 under apoptotic conditions in 2 mm Gln.

GeneGoTM-based signaling pathway annotations of proteins identified in CD56+ NK cell subsets. GeneGoTM-based signaling pathway annotations of proteins.

The average median S.D. and PEV reduction after applying different normalization methods compared with raw data. The average median S.D. and PEV reduction.

Monitoring the acetylation profile of mitochondrial proteins in SIRT3 KO mice. Monitoring the acetylation profile of mitochondrial proteins in SIRT3 KO.

Occurrence of fur−-only genes and expected sensitivity to Fur.

MS3 for peptide identification and mapping phosphorylation sites

SCX chromatography at low pH permits strong enrichment of phosphopeptides of distinct solution charge states.A, the number of phosphopeptides (blue triangles)

Presentation transcript:

Work flow for the LAXIC strategy to quantify the phosphorylation change in response to osmotic stress. Work flow for the LAXIC strategy to quantify the phosphorylation change in response to osmotic stress.A, Schematic representation of the LAXIC algorithm. First, all the chromatographic peaks were aligned and the ratios were calculated. Second, the normalization factors which equal to ratios of library peptide peaks between MS runs were chosen to construct normalization curve. Third, sample peptide peak ratios were normalized against predicted normalization factor corresponding to certain retention time. B, Schematic representation of quantitative phosphoproteomics. Plants either treated with mannitol or PBS were lysed and mixed proportionally at first. Following peptide digestion and enrichment, phosphopeptides were identified and further quantified through LAXIC incorporated with self-validating process using thelinear regression model to analyze the fold change (fold), linearity (R2) and accuracy (%Acc). Liang Xue et al. Mol Cell Proteomics 2013;12:2354-2369 © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.