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Multiple studies with a single experiment: The Power of Quantitative Multiplexing
Multiple studies with a single experiment: The Power of Quantitative Multiplexing
Overview
Part1：Introduction
An overview of multiplexed isobaric labeling, the basics of Tandem Mass Tags and the SPS MS3 technique
Part2：Sample Prep
A summary of the steps involved for a complete TMT workflow including labeling and fractionation
Part3：Instrument Configuration
Getting started with building an instrument method on the Orbitrap Tribrids and Benchtops using nanoHPLC
Part4：Data Analysis
Data analysis of SPS MS3 data using Proteome Discoverer 2.1 workflow
Introduction
Moving Beyond Qualitative Proteomics
Problem: Quantitative information about expression level of a protein is essential to understanding its biological role in response to change or disease.
Add another dimension to any experiment by determining the relative abundance of each identified protein
Alterations in expression can reveal a meaningful biological pattern not apparent in a pure identification experiment, which provides only a list of detected proteins
Label Free Quantitation
Several well established pipelines for the quantitation of label-free data from a data dependent (or DDA informed DIA experiment) exist. Among these:
Label Free
*Multiple LC/MS Runs
*Compare a few conditions
*Requires replicate sample material
Problem: Requires multiple LC/MS analyses and is thus sample intensive
A differential analysis of 2 biological conditions with 3 technical replicates each would require six LC/MS injections and analyses:
Problem: Substantial instrument time to compare only a few conditions simultaneously
Comparing just two conditions with a two hour gradient would take more than 14 hours of instrument time
Problem: Irreproducibility due to less than 100% sample overlap
Improving Quantitation Throughput: SILAC
SILACWorkflow
SILAC MS1 Quantitation
Geiger T., et al, Nature protocols(2011):147-157
SILAC Quantitation
Problem: Increases MS1 Spectral Complexity
High resolution and intelligent precursor selection (i.e. selection of only one SILAC labeled peptide per pair or triad) is required for best quantitative results
Problem: requires cell labeling in culture
Proteins must be able to be metabolically labelled and thus is not suitable for all organisms/conditions
With SILAC began a trend towards increased multiplexing…
A Better Multiplexing Method– Isobaric Mass Tagging
•Less MS1 Complexity
•Increased Throughput Concurrent MS analysis of multiple samples
•Less consumed samples and less instrument time
•Fewer Missing Values
• Identification and quantification achieved in a single run
•No worries about irreproducibility
•Sample Origin Flexibility
•Samples can be derived from cells, tissues or biological fluids
•Increased Multiplexing
•Compare more than 3 conditions
•Multiple Comparisons and Improved Statistics
•Incorporate replicates with multiple conditions: dose-response, time-course, multiple tissues, subcellular fractions, etc
Thermo Scientific Tandem Mass Tag (TMT) Isobaric Tag Family
*13C and 15N labeled reporter
*Fragments by ETD or HCD
*Isotopes balanced between linker region and reporter region keeping all tags exactly isobaric
*Up to 10 different tags
*Other reactive tags :Iodo TMT and Aminoxy TMT
The Multiplexing Revolution –Not Only Consumables…
High Performance Depends Upon High Resolution Instruments
HIGH RESOLVING POWER IS ESSENTIAL FOR ACCURATE QUANTITATION OF THE TMT10PLEX REAGENTS
Result: Get accurate quantitation using the high resolution of Orbitrap Mass Analyzer
A Real Example
Sample: Mouse mitochondrial extract untreated or treated with phosphatase inhibitor
Orbitrap Elite
*75 um x 50 cm PepMap C18
*210 min gradient: 250 min run
*1 ug of sample on column
Thermo Poster Note : Liver Mitochondria Proteomics Employing High –Resolution MS Technology; J.Ho. et al
Ratio Distortion with Isobaric Multiplexing
Problem: Quantitation of low-abundance proteins in a complex background is distorted by co-isolated interfering precursor ions
Synchronous Precursor Selection (SPS) for Accurate Quantification
TMT3 Experiment, Powered by SPS
Co-isolation of Interfering Ions Affects Accuracy
Results: Best possible accuracy and precision by reducing co-isolated interfering ions.
Enhanced Differences Using SPS MS3 Quantitation
Thermo Poster Note : Towards Mechanism of EGFR Inhibitor Resistance in Non-Small Lung Cancer Cell;M.Blank. et al
…While Still Getting Proteome Coverage
The speed and parallelizable work flow of the Orbitrap Fusion means not choosing between accuracy and coverage…
The Orbitrap Fusion using multinotch can Quantify more proteins than were Identified on the previous generation top tier hybrid
…Don’t Settle
Orbitrap Fusion Lumos Tribrid Mass Spectrometer
2015
Unmatched Analytical Performance
Revolutionary performance
Exceptional versatility
Unprecedented usability
Highest sensitivity
Better Ion Transmission With Segmented Quadrupole
Segmented Quadrupole
•Improved transmission across m/z range and for narrow windows
•Brighter Source and Segmented Quad allows the use of a 0.4 amu isolation without loss of IDs (here for 1 ug HeLa, DD OT IT CID, 2 h runs, n=2)
•Improved performance for TMT quantitation
•Improved performance for PRM and DIA
•Improved performance for top down
Improved TMT SPS MS3 Performance
TMT Dilution
•Standard HeLa digest, labeled with TMT0 analyzed with an 85 min gradient using SPS-MS3
•Sample diluted 1:1, 1:10, 1:30, 1:50, 1:100
•The number of MS3 acquisitions was similar in both analyses
•The number of unique peptides quantified was systematically higher with the Orbitrap Fusion Lumos MS
Improved Low Level Quan: Ubiquitinated Peptides
TMT10 Quantitation of Ubiquitinated Peptides
•Human HTC116 cells were treated with a proteasome inhibitor (Bortezomib) for 16 h and analyzed with TMT 10-plex (5 treated vs. 5 untreated)
•Two fractions were prepared •With higher amount
•With lower amount
•25-73% more quantifiable peptides
ASMS Lecture: Rose et al. Isobaric labeling enables 10-Plex quantitative analysis of ubiquitylated peptides: A diagnostic ion to improve identification and quantification
SPS MS3 Quantification on Orbitrap Fusion Lumos MS
Results: Best possible accuracy and precision by reducing co-isolated interferences.
TMT Technology is More Precise than Label Free Quan
“We compared the average and median CVs (calculated for the whole dataset containing ca. 4000 proteins quantified with ≥2 peptides) between the three biological replicates of the same treatment. Ignoring the fact that the cell lines were different, the results are clearly in favor of TMT. In other words, TMT produced two times lower CVs than our label-free quantification, which we thought was pretty good. I am stunned…”
TMT Used for Protein Research in…
Straightforward Workflow
Result: Complete software and method development suite from reagents to data analysis
Competitive Advantages
Trust your quantitation!
Multinotch MS3 quantitation is more accurate than other MS2 Methods
The accuracy of Multinotch MS3 quantitation means not missing important expression level changes due to co-isolated interference
Multinotch MS3 quantitation is only available on the Orbitrap Fusion and Orbitrap Fusion Lumos
Orbitrap Fusion Lumos provides highest sensitivity, highest selectivity and lowest detection limit for best quantitation
Sample Preparation
Sample Preparation: Materials
Sample Preparation: Simple Peptide Labeling
Reduced and alkylated trypsin digested proteins
Use Non-Amine Buffer @ pH ~ 8.0 (e.g. TEAB)
Part No. 88328
Description：HeLa Protein Digest Standard Formulation: Lyophilized peptide mixture from a
tryptic digest of HeLa S3 cell lysate Sufficient For: 20 to 100 analyses
•Add 41μL of anhydrous acetonitrile to each tube. Allow the reagent to dissolve for 5 minutes
with occasional vortexing. Briefly centrifuge the tube to gather the solution.
•Transfer 25-100 uL of the reduced and alkylated protein digest (each condition) to the TMT
Reagent vial (41 uL). Add sufficient 100 mM TEAB buffer to reach a final volume in vial of 141 uL. Vortex briefly
•Incubate the reaction for 1 hour at room temperature.
•Add 8μL of 5% hydroxylamine to the sample and incubate for 15 minutes to quench the reaction.
•Combine samples in a new microcentrifuge tube at equal amounts and speed vacuum to dryness to
remove all TEAB
•Aliquot and Store at -80°C.
A More Complete Workflow For Better Coverage
High pH Fractionation Spin Columns
Thermo Poster Note 64606: High pH Reversed-Phase Peptide Fractionation in a Convenient SpinColumn Format; Snovida S. et al
Thermo Poster Note 64604: Quantitative peptide assay for optimized and reproducible sample preparations for mass spectrometry applications; Jiang X. et al
High pH Reversed Phase vs SCX Fractionation
•SCX and high pH reversed phase fractionation are both orthogonal to low pH C18 LC separation
•Strong cation exchange (SCX) requires sample desalting after fractionation
Reproducibly Identify More TMT-labeled Peptides
•Significantly increase the number of proteins identified and quantified
•The percentage and number of peptides observed in only 1 fraction are within 10% between runs
Two New Peptide Quantitation Assays
Colorimetric peptide assay is more sensitive than BCA
Two New Peptide Quantitation Assays
Thermo Poster Note: Quantitative Peptide Assays for Mass Spectrometry Applications; Haney P. et al
Peptide Quantitation Improves MS Reproducibility
Thermo Poster Note 64604: Quantitative peptide assay for optimized and reproducible sample preparations for mass spectrometry applications; Jiang X. et al
Peptide Assays Provide Consistent Quantification
“We really like those peptide quant kits. We find that peptide mass quantitation is directly relevant for determining the amount of analyte we use for LC-MS/MS analyses, phosphopeptide enrichments, and for TMT labeling.
Instrument Configuration
Introducing A New Powerful Combination
Unlock the performance of your MS with incredibly easy chromatography
[EASY-nLC™ 1200 HPLC System]
•Industry leading 1200 bar system pressure
•Improved system robustness and easier maintenance
•Temperature control of EASY-Spray columns
•Effortless ultra-high performance for every user, every time
[75cm EASY-Spray™ Column]
•Increased peak capacity
•Even more identifications
•Excellent retention time consistency
•Improved quantitative reproducibility
Extend The Performance Of Your MS with 75cm Columns
Improved Protein Identifications
•Peak capacity exceeding 800.
•Deeper proteome coverage with a consistent 5700 protein groups per run.
•9% increase in protein groups over 3 combined runs compared to 50 cm column.
•Identify more proteins per hour – comparable identifications in 120 minutes to a 50 cm column in 240 minutes.
Increased Quantitative Reproducibility
•Less run-to-run variability 91% identification overlap run to run
• More quantifiable proteins 50% increase in the number of quantifiable proteins with CVs <5%
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
•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
•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
•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
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