High Resolution Isolelectric Focussing - HiRIEF Group 4.

Slides:

Advertisements

Similar presentations

- DATA DEPENDENT ANALYSIS - Proteomics Unit. Centro Nacional de Biotecnología sHPP Miguel Marcilla Goldaracena.

Advertisements

Control St Control St Control St 3 dai 7 dai 11 dai Protein extraction Tryptic digestion and TMT six-plex labeling Phosphopeptides enrichment by TiO 2.

PROTEORED Multicentric Study QUANTITATIVE PROTEOMICS METHOD SPECTRAL COUNT 2010 UNIVERSITY OF BARCELONA Salamanca 16 th March.

Protein Quantitation II: Multiple Reaction Monitoring

Post-Translational Modifications: CrossTalk Robert Chalkley Chem 204.

Proteomics Informatics – Protein characterization I: post-translational modifications (Week 10)

An enzyme is a biological catalyst that speeds up the rate of reactions in biological processes All enzymes are proteins, coded for by genes Enzymes are.

MN-B-C 2 Analysis of High Dimensional (-omics) Data Kay Hofmann – Protein Evolution Group Week 5: Proteomics.

Proteored Multicentric Experiment 8 (PME8) Quantitative Targeted Analysis in Proteomics. An Assesment Study (QTAPAS) ProteoRed WG1-WG2 Meeting Pamplona,

LCM and Proteomics Tissue heterogeneity: Farm  Haystack LCM: pure(r) cell populations Avoid potential expression artifacts a/w sorting Proteins: closer.

Proteomics Informatics Workshop Part III: Protein Quantitation

Fa 05CSE182 CSE182-L9 Mass Spectrometry Quantitation and other applications.

Magic Lysis Buffer Improves the Efficiency of Immunoprecipitation-LC/MS/MS (IP-MS) with Less Non-Specific Interactions and Stronger Retention of Binding.

Introduction of SILAC and its applications Stable Isotope Labeling of Amino acids in Culture Develop and promoted by Matthias Mann Two papers: –Lipid Rafts.

Yin-Yang Column for Global Protein Identification Column Technology Inc.

Overview of 2DE Complex mixture of proteins

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

Phosphopeptides identification Column technology Inc.,

ProteoRed WG1-WG2 Meeting Salamanca, March,16th 2010 PME5: Quantitative LC-MS differential analysis F. Canals.

Multiple flavors of mass analyzers Single MS (peptide fingerprinting): Identifies m/z of peptide only Peptide id’d by comparison to database, of predicted.

A b c d 30% AcN 30% Organic (143 Features) FT #Features CV R % Organic (237 Features) FT.

FIRST APPROACH TO THE SHOTGUN UNKNOWN PROTEINS sHPP CHROMOSOME 16 MEETING August, 28, 2012 La Cristalera, Miraflores de la Sierra.

Treatment of A375 cells with PA (15 μM) for different time points iTRAQ Labeling Pool, SCX, Desalting and Offline 2DLC MS/MS analysis Cell Lysis, Reduction,

ProteoRed WG1-WG2 Meeting Salamanca, March,16th 2010 PME5: Quantitative LC-MS differential analysis F. Canals.

Salamanca, March 16th 2010 Participants: Laboratori de Proteomica-HUVH Servicio de Proteómica-CNB-CSIC Participants: Laboratori de Proteomica-HUVH Servicio.

Lecture 6 Comparative analysis Oct 2011 SDMBT.

Oct 2011 SDMBT1 Lecture 11 Some quantitation methods with LC-MS a.ICAT b.iTRAQ c.Proteolytic 18 O labelling d.SILAC e.AQUA f.Label Free quantitation.

Click to add Text Sample Preparation for Mass Spectrometry Sermin Tetik, PhD Marmara University July 2015, New Orleans.

Quantitative Proteomic Profiling by Mass Spectrometry Paolo Lecchi, Ph.D. Dept. of Pharmacology George Washington University Emerging Technologies in Protein.

Deducing protein composition from complex protein preparations by MALDI without peptide separation.. TP #419 Kenneth C. Parker SimulTof Corporation, Sudbury,

Quantitation using Pseudo-Isobaric Tags (QuPIT) and Quantitation using Pseudo-isobaric Amino acids in Cell culture (QuPAC) Parimal Samir Andrew J. Link.

Protein Identification using 2D LC/MS/MS based on pH Gradient and Reverse Phase Separation Column Technology Inc.,

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

Goals in Proteomics Identify and quantify proteins in complex mixtures/complexes Identify global protein-protein interactions Define protein localizations.

Immune Cell Subsets Fractionated from Whole Blood of Human Subjects

Mass spectrometry data enhancement software

Jarrett Egertson, Ph.D. MacCoss Lab

2 Dimensional Gel Electrophoresis

Kelly Stecker Sussman Lab

M M Additional file 2 - Silver staining of protein extract and tryptic digest run on SDS-PAGE. The figure shows.

AssayMAP Bravo for Automated Sample Preparation

Lecture 2 Techniques in proteomics By Ms. Shumaila Azam

MCB test 2 Review M. Alex Miranda 11/5/16.

Bioinformatics Solutions Inc.

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

Volume 17, Issue 1, Pages (September 2016)

Quantifying Ubiquitin Signaling

Immuno-Assay (Proteomic and Metabonomic)

Houston et al., 2009 Figure S1 A B

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.

Volume 24, Issue 13, Pages (July 2014)

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.

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

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

Absolute proteome quantification of highly purified populations of circulating reticulocytes and mature erythrocytes by Emilie-Fleur Gautier, Marjorie.

CMTR1 interacts with DHX15.

The principle of the immuno-SILAC method.

Grid Sampling: Point vs. Random

Volume 17, Issue 6, Pages (June 2013)

The Coming Age of Complete, Accurate, and Ubiquitous Proteomes

Mass spectrometric validation of DLG5 as a CDKL5 substrate

Volume 61, Issue 2, Pages (January 2016)

Identification of Post Translational Modifications

Kuen-Pin Wu Institute of Information Science Academia Sinica

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

Proteomic profiling of VCP substrates links VCP to K6‐linked ubiquitylation and c‐Myc function VCP inhibition increases the cellular levels of ubiquitylated.

Volume 3, Issue 4, Pages e4 (October 2016)

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

Presentation transcript:

High Resolution Isolelectric Focussing - HiRIEF Group 4

What we did in the lab

Cell lysates Denaturing proteins Reduction Alkylation Tryptic digestion SCX cleaning Sample preparation

Fractionation step Resuspend peptides in rehydration solution Swell gel-strips with peptide solution overnight Run IPG-strips in the machine overnight Extraction of 72 fractions from the IPG-strip

Run LC- MS No frac. 1.Peptide from SCX cleaning directly HiRIEF 2. Extracted peptide after fractionation

Fractions pooled: 18pH pH 7.074

First 3 fractions

Middle 3

Last 3

Identified peptides – HiRief vs. no frac.

Identified proteins – HiRief vs. no frac.

Summary HiRiefNo Frac. Coverage7%13.7% Unique peptides Shared peptides199 (3%)380 (5%) Total peptides

Identified Phosphopeptides

Misscleavages HiRIEF No frac.

TOP 10 hits No frac.: AreaPSM [ACTB_HUMAN][AHNK_HUMAN] [ACTBL_HUMAN][FLNA_HUMAN] [ACTH_HUMAN][ACTB_HUMAN] [POTEE_HUMAN][MYH9_HUMAN][K2C8_HUMAN] [TBA1B_HUMAN][PLEC_HUMAN] [TBA1C_HUMAN][ENOA_HUMAN] [G3P_HUMAN][HS90B_HUMAN] [H2B1K_HUMAN][FLNB_HUMAN] [H2B1J_HUMAN][K1C18_HUMAN] HiRief.: AreaPSM [ACTB_HUMAN][G3P_HUMAN] [ACTA_HUMAN][ACTB_HUMAN] [H32_HUMAN][FLNA_HUMAN] [ANXA2_HUMAN][PGK1_HUMAN] [FLNA_HUMAN][ENOA_HUMAN] [G3P_HUMAN][ANXA2_HUMAN] [PRDX1_HUMAN][K1C17_HUMAN] [HSP7C_HUMAN][TKT_HUMAN] [K1C17_HUMAN][AHNK_HUMAN] [NDKB_HUMAN][HSP7C_HUMAN]

GO term enrichment – DAVID: HiRief Top 100

Pathway enrichment – REACTOME: HiRief Top 100

GO term enrichment – DAVID: No fractionation Top 100

GO term enrichment – DAVID: No fractination Top 100

Shared proteins of GO enrichment – Top 100

Quantification No internal standard/labels Synthetic peptides, iTRAQ, TMT, SILAC Possible quantification: Area PSM

Protein isolation with HiRIEF Pros Reproducible Decrease complexity More sensitive Cons Missing peptides with PI out of the pH range Extra lab step Higher input necessary

Did you find any proteins that you are interested in for your own research? Mff Mfn2 MRP3 MRP1 IL18