SPS TMT Method Development- What is New on Lumos

Multiplexing On the Benchtop Orbitrap System (Q Exactive Series)

•Take advantage of segmented quadrupole (QE Plus and HF), for more efficient isolation in narrow windows

•Suited for the analysis of low and medium complexity samples

•Pre-fractionation recommended for high complexity samples to improve quantification accuracy and precision (Pierce Spin Column)

Data Analysis

Proteome Discoverer 2.1

•New method TMT quantification

  •TMT correction factors (for all TMT reagents) with new user interface

  •Use of “Razor” peptides for protein quantification

  •Modified form of Gygi group’s S/N-based approach to TMT quantification

  •Custom ratio generation

  •New heat map-like coloring of ratios and scaled abundances

Note that almost of all these changes are also applied to isotope-labeled quantification (e.g. SILAC)

TMT Correction Factor Setup

TMT correction factor certificate for each manufacturing lot

Edit Quantification Method

Certificate of Analysis (CoA) can be found at http://www.thermofisher.com/order/catalog/product/90110?ICID=search-product using the lot number displayed on the reagents packages.

Study Management Setup

•Select quan method and assign study factors

•Input data

•Specify how to group quantification results

•Specify Quan method, match data files and Quan Channels with study factors

•Select processing and consensus workflows and make modifications

•Run

Accommodates the most complex study designs

Select Workflows and Modify Parameters

Select Workflows and Modify Parameters (Con’d)

New Custom Ratio Calculation in PD 2.1

Grouping and Quantification

Results From Biological Replicate Search

•Replicates grouped into ratios + standard errors

Zoomed Ratios and Scaled Abundances

Summary

Comprehend

the fundamentals of isobaric labelling and the dramatically increased throughput enabled by multiplexed quantitation as well as the ease of sample preparation

Configure

an LCMS method for the high accuracy quantitation of TMT labeled samples using the Orbitrap Fusion Lumos with SPS MS3 with the ideal settings

Quantify

peptides labeled with TMT using Proteome Discoverer 2.1 using SequestHT and MS3 quantitation.

Advocate

the complete workflow from sample preparation to data analysis for the multiplexed quantitation of complex samples using TMT and the highly differentiated SPS MS3 on the Orbitrap Fusion Series Instruments

Additional Resources

•Online Resources

    •http://portal.thermo-brims.com/ (Software, Manuals, Tutorial Help Videos, Discussion Forum.)

    •http://planetorbitrap.com/ (Published Articles, Posters, Brochures, Product Support Bulletins, Technical Guides, Webinars, Protocols, Application Workflows.)

•Some More Publications

    Relative Quantitation of TMT-Labeled Proteomes - Focus on Sensitivity and Precision

    Viner R, Scigelova M, Zeller M, Oppermann M, Moehring T, Zabrouskov V.

    Application Note 566

    Increasing the multiplexing capacity of TMTs using reporter ion isotopologues with isobaric masses

    McAlister GC, Huttlin EL, Haas W, Ting L, Jedrychowski MP, Rogers JC, Kuhn K, Pike I, Grothe R, Blethrow JD, Gygi SP.

    Anal Chem. 2012 Sep 4;84(17):7469-78.

    MS3 eliminates ratio distortion in isobaric multiplexed quantitative proteomics

    Ting L, Rad R, Gygi SP, Haas W.

    Nat Methods. 2011 Oct 2;8(11):937-40.

    Evaluating multiplexed quantitative phosphopeptide analysis on a hybrid quadrupole mass filter/linear ion trap/orbitrap mass spectrometer

    Erickson BK , Jedrychowski MP, McAlister GC, Everley RA, KUNZ R, Gygi SP

    Anal Chem.2015 Jan 20;87(2):1241-9.

Contact: Dr. Xiaoyue Jiang(xiaoyue.jiang@thermofisher.com)

Or Dr. Rosa Viner (rosa.viner@thermofisher.com)

Acknowledgements

•Thermo-Life Sciences
•Ryan Bomgarden
•Sergei Snovida
•Paul Haney
•John Rogers

•Thermo-LSMS
•Rosa Viner
•Xiaoyue Jiang
•Michael Blank
•Andreas Huhmer
•Graeme McAllister
•Tabiwang Arrey
•Vlad Zabrouskov
•Michaela Scigelova
•David Horn
•Torsten Ueckert

•TMT Collaborators
•Steve Gygi, Harvard Medical School
•Josh Coon, University of Wisconsin, Madison
•Jennifer Van Eyk, Johns Hopkins School of Medicine
•Zezong Gu, University of Missouri-Columbia
•Kay-Hooi Khoo, Academia Sinica
•Bernhard Kuster, Technische Universität München
•Somi Afiuni & Tim Griffin, University of Minnesota