Organelle Proteomics by Label-Free and SILAC-Based Protein Correlation Profiling - LC-MS/MS in Proteomics: Methods and Applications - page 252

Chapter 15

Organelle Proteomics by Label-Free and SILAC-Based
Protein Correlation Profiling

Joern Dengjel, Lis Jakobsen, and Jens S. Andersen

Abstract

The ability to purify cell organelles and protein complexes on a large scale, combined with advances in
protein identification using mass spectrometry, has provided a wealth of information regarding protein
localization and function. A major challenge in these studies has been the ability to identify bona fide
organelle components from a background of co-purifying contaminants because none of the available
biochemical purification protocols afford pure preparations. Since this situation is unlikely to change
alternative strategies have been devised to meet this challenge by making use of the information inherent
in the fractionation profile of organelles isolated by density gradient centrifugation. In this chapter we
describe strategies based on protein correlation profiling and quantitative mass spectrometry to sort
out likely candidates. The organelle inventories defined by these methods are suitable to guide future
functional experiments.

Key words: Organelle, protein complex, mass spectrometry, proteomics, SILAC, protein

correlation profiling.

1. Introduction

Mass spectrometry-based proteomics has become a powerful
method to determine the composition of organelles and pro-
tein complexes ( 1 , 2 ) . The most widely used approaches involve
biochemical or affinity purification of the structure of interest,
protein separation by 1D gel electrophoresis, enzymatic diges-
tion of the proteins, followed by separation and analysis of the
resulting peptides by liquid chromatography-mass spectrometry
(LC-MS). Nevertheless, the analysis of cellular structures remains
challenging because such structures are difficult to purify. At best,