Multiple studies with a single experiment: The Power of ...（一）
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…
