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…