Skyline MS1 Filtering for label-free quantification targeting posttranslational modifications OHOPO 3 H - NH 3 + HNCOCH 3 kinases phosphatase acetyl transferases.

Slides:

Advertisements

Similar presentations

Platform Independent and Label-free Quantitation of Protein Acetylation and Phosphorylation using MS1 Extracted Ion Chromatograms in Skyline OHOPO 3 H.

Advertisements

Protein Quantitation II: Multiple Reaction Monitoring

Protein Quantitation II: Multiple Reaction Monitoring

From Genome to Proteome Juang RH (2004) BCbasics Systems Biology, Integrated Biology.

Characterization of Recombinant Glycoprotein by Mass Spectrometry Min Xie Spring, 2001.

Pox Proteomics: Identification of the Proteins Associated with the Membranous Fraction of Vaccinia Virus Cliff Gagnier Dr. Dennis Hruby Department of Microbiology.

Proteomic, Metabolomic and Lipidomic Research Capability at ICH and ION UCL Label Free Quantitative Proteomics “biomarker discovery”

Previous Lecture: Regression and Correlation

Proteomics Informatics (BMSC-GA 4437) Course Director David Fenyö Contact information

Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS Gygi et al (2003) PNAS 100(12), presented by Jessica.

Proteomics Informatics Workshop Part III: Protein Quantitation

© 2010 SRI International - Company Confidential and Proprietary Information Quantitative Proteomics: Approaches and Current Capabilities Pathway Tools.

Proteomics and Biomarker Discovery “Research-grade” Targeted Proteomics Assay Development: PRMs for PTM Studies with Skyline or, “How I learned to ditch.

Bringing Metrology to Clinical Proteomic Research David Bunk Chemical Science and Technology Laboratory National Institute of Standards and Technology.

Additional file 1 1.1Workflow of large-scale proteomic analysis of normal human kidney glomerulus 1.2Detailed procedure of LC-MS/MS analysis Additional.

Quantification of Membrane and Membrane- Bound Proteins in Normal and Malignant Breast Cancer Cells Isolated from the Same Patient with Primary Breast.

Repository for Targeted Proteomics Assays Josh Eckels Skyline Users Group - June 9, 2013.

I.Introduction to PTMs II.Protein Phosphorylation (Ser/Thr) A.Methods―enrichment. B.Applications i.Membrane “shaving” ii.Label-free quantitative analysis.

June 9th, 2013 Matthew J. Rardin June 9th, 2013 Matthew J. Rardin MS1 and MS2 crosstalk in label free quantitation of mass spectrometry data independent.

Overview of Mass Spectrometry

Isotope Labeled Internal Standards in Skyline

Proteomics Informatics (BMSC-GA 4437) Instructor David Fenyö Contact information

Multiplexed Data Independent Acquisition for Comparative Proteomics

Agenda  Welcome from the Skyline team!  PRM Targeted Proteomics Using Full-Scan MS2  Introduction with Brendan MacLean  PRM Introduced by Bruno Domon.

Proteomics Informatics (BMSC-GA 4437) Course Directors David Fenyö Kelly Ruggles Beatrix Ueberheide Contact information

What is proteomics? Richard Mbasu and Ben Richards.

Protein quantitation I: Overview (Week 5). Fractionation Digestion LC-MS Lysis MS Sample i Protein j Peptide k Proteomic Bioinformatics – Quantitation.

Ho-Tak Lau, Hyong Won Suh, Martin Golkowski, and Shao-En Ong

Target Analyses in Parallel Reaction Monitoring Mode (PRM)

Agenda Welcome from the Skyline team!

Agenda  Welcome from the Skyline team!  Targeted Method Design  Introduction with Brendan MacLean  Tutorial with Brendan MacLean  Targeted proteomics.

Shotgun protein identification Creative Proteomics offers iTRAQ protein quantification service suited for unbiased untargeted biomarker discovery. Relative.

Richard Mbasu and Ben Richards

Large Scale DIA With Skyline

Jarrett Egertson, Ph.D. MacCoss Lab

Agenda Welcome from the Skyline team!

Pinpointing phosphorylation sites using Selected Reaction Monitoring and Skyline Christina Ludwig group of Ruedi Aebersold, ETH Zürich.

Targeted Proteomics Environment

Thomas BOTZANOWSKI & Blandine CHAZARIN

Bioinformatics Solutions Inc.

V. Protein Chips 1. What is Protein Chips 2. How to Make Protein Chips

Proteomics Informatics David Fenyő

Proteomic Approaches to Cancer Biomarkers

Interpretation of Mass Spectra I

A perspective on proteomics in cell biology

Proteomics Informatics –

Targeted Method Refinement

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.

Skyline MS1 filtering graphical user interface.

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

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.

Relative quantification of cis and trans PSP gp10040–42/47–52 variants

Volume 8, Issue 5, Pages (September 2014)

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.

Dorota F. Zielinska, Florian Gnad, Jacek R. Wiśniewski, Matthias Mann

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

Protein identification using MS/MS.

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

Quantitative Proteomics II: Targeted Quantitation

Proteomics Informatics David Fenyő

Identification of Post Translational Modifications

Interpretation of Mass Spectra

Kuen-Pin Wu Institute of Information Science Academia Sinica

Sample preparation Protein and peptide separation techniques Karel Bezstarosti (Proteomics Center, Erasmus MC)

ASPP2 hydroxylation is detectable in vivo and the amount of hydroxylation depends on FIH-1 abundance. ASPP2 hydroxylation is detectable in vivo and the.

Presentation transcript:

Skyline MS1 Filtering for label-free quantification targeting posttranslational modifications OHOPO 3 H - NH 3 + HNCOCH 3 kinases phosphatase acetyl transferases deacetylases Birgit Schilling Buck Institute for Research on Aging Birgit Schilling Buck Institute for Research on Aging Webinar #1: Getting the Most Out of DDA Data with Skyline

Stem Cells (AMD) Prokaryotic Metabolic Regulation Tissue Homeostasis (‘Secretomics’) Tissue Homeostasis (‘Secretomics’) Biology of Aging (NIA) Biology of Aging (NIA) Alzheimer’s Cancer Biology (R01) Cancer Biology (R01) Gibson lab Chemistry Core Ellerby Hughes Brand Mooney Clinical Proteomics, Biomarkers Clinical Proteomics, Biomarkers Fisher (UCSF) Benz Mooney E. Verdin (Gladstone) R. Kahn (Joslin) C. Newgard (Duke) E. Goetzman (Pittsburg) Benz Fisher Infection Disease & Innate Immunity (RO1, PPG) Infection Disease & Innate Immunity (RO1, PPG) Methods Development Apicella, Nauseef (Iowa),.. MacCoss (UW) Vitek (Northeastern U) Tabb (Vanderbilt) Held (WU) BioMarin Hughes, Lithgow Kapahi, Campisi Melov, Jasper, S. Ghaemmaghami (SUNY), others… Campisi, Kapahi C. Adams (Iowa) Bredesen Melov Brand Ellerby/Gan GIBSON LAB 2014 Huntington (CHDI ) Huntington (CHDI ) Metabolic Disorders (R24) Metabolic Disorders (R24) October, 2014 Lamda Wolfe (Loyola) Rao (Illinois)

Schilling, Rardin, MacLean et al., MCP, 2012 Rardin et al., PNAS, 2013 Data-dependent acquisitions (discovery workflows) depend on dynamic sampling of MS/MS spectra. MS1 scans are truly data-independent and can be used for relative quantification. MS1 Filtering uses ion extracted chromatograms of peptide precursor ions for relative quantification Easy interfacing in Skyline with other data independent, targeted workflows, i.e. MRM, MRM-HR (PRM), SWATH-MS2. Interface with Panorama DATA examples Development and Examples for MS1 Filtering Workflows

E. coli acetylation regulation - Background and Workflow Protein lysates Proteolytic Peptides Acetyl-enriched Peptides Anti-acetyl-K- IP Trypsin Digestion 3x technical LC MS/MS replicates Triple TOF 5600 WTackAcobBothers … ackAWTcobB 4x biological replicates of: acCoAacPace + ATP PtaAckA Database Search Skyline MS1 Filtering MS Analysis anti-acetyl blots (Kuhn, Schilling, Gibson, Wolfe et al., PlosOne 2014) acP: acetyl phosphate (chemical acetylation)

PHASE 1: Biomarker Discovery Using Conditioned Media from Cancer Cell Lines to predict cancer-specific plasma glycosites candidates Luminal Triple negative 4 claudin-low (basal B) 1 basal A Centrifugation of CMs 3K MWCO; dialysis Amino acid analysis (normalized) Trypsin Lectin Chromatography (2x) SNA AAL (2x) Bound fractions (glycopeptides) PNGaseF 10 SNA + 10 AAL deglycopeptide fractions 2x 80 HPLC MS/MS Drake, Schilling et al., JPR 2012 (+ phosphatase inhibitors) TiO 2 Phosphopeptide affinity chromatography Bound fractions (P-peptides) 2x HPLC MS/MS HILIC fractionation Zawadzka, Schilling et al., MCP 2014