GeLCMS for in-depth protein characterization and advanced analysis of proteomes

TY - CHAP

T1 - GeLCMS for in-depth protein characterization and advanced analysis of proteomes

AU - Lundby, Alicia

AU - Olsen, Jesper V

PY - 2011/1/1

Y1 - 2011/1/1

N2 - In recent years the array of mass spectrometry (MS) applications to address questions in molecular and cellular biology has greatly expanded and continues to grow. Modern mass spectrometers allow for identification, characterization, as well as quantification of protein compositions and their modifications in complex biological samples. Prior to MS analysis any biological sample needs to be properly prepared for the experiment. Here we present a protocol that combines pre-separation of proteins by 1D gel electrophoresis followed by analysis of in situ digested protein products by tandem mass spectrometry (MS/MS). All steps of the sample preparation are explained in detail, and the procedure is compatible with downstream analysis on any mass spectrometer available. With minor adjustments the protocol can be used with 2D gels as well. The protocol provided can be applied to analyze specific proteins of particular interest as well as entire proteomes. If SILAC-labeled protein samples are mixed prior to gel separation, the protein content of the sample can furthermore be accurately quantified.

AB - In recent years the array of mass spectrometry (MS) applications to address questions in molecular and cellular biology has greatly expanded and continues to grow. Modern mass spectrometers allow for identification, characterization, as well as quantification of protein compositions and their modifications in complex biological samples. Prior to MS analysis any biological sample needs to be properly prepared for the experiment. Here we present a protocol that combines pre-separation of proteins by 1D gel electrophoresis followed by analysis of in situ digested protein products by tandem mass spectrometry (MS/MS). All steps of the sample preparation are explained in detail, and the procedure is compatible with downstream analysis on any mass spectrometer available. With minor adjustments the protocol can be used with 2D gels as well. The protocol provided can be applied to analyze specific proteins of particular interest as well as entire proteomes. If SILAC-labeled protein samples are mixed prior to gel separation, the protein content of the sample can furthermore be accurately quantified.

U2 - 10.1007/978-1-61779-148-2_10

DO - 10.1007/978-1-61779-148-2_10

M3 - Book chapter

C2 - 21604121

VL - 753

SP - 143

EP - 155

BT - Gel-Free Proteomics Methods and Protocols

ER -