Microproteomics: Quantitative Proteomic Profiling of Small Numbers of Laser-Captured Cells

INTRODUCTION

During the last decade, significant progress in the analysis of whole genomes and transcriptomes has triggered efforts to analyze the proteome. Advancements in protein extraction, purification, and identification have been driven by the development of mass spectrometers with greater sensitivity and resolution. Nevertheless, comparative and quantitative proteomic technologies have not progressed to the extent of genomic and transcriptomic technologies for accessing gene expression differences. Unlike the genome, which is similar throughout all cells in a given organism, the proteome varies in different cells. Also, there is no self-replicating amplification mechanism for proteins such as the polymerase chain reaction (PCR) for DNA. Therefore, developing methods that extract, separate, detect, and identify proteins from extremely small samples are needed. The advent of laser capture microdissection (LCM) has expanded the analytical capabilities of proteomics. LCM has proven an effective technique to harvest pure cell populations from tissue sections. This protocol describes a microproteomic platform that uses nanoscale liquid chromatography/tandem mass spectrometry (nano-LC-MS/MS) to simultaneously identify and quantify hundreds of proteins from LCMs of tissue sections from small tissue samples containing as few as 1000 cells. The LCM-dissected tissues are subjected to protein extraction, reduction, alkylation, and digestion, followed by injection into a nano-LC-MS/MS system for chromatographic separation and protein identification. The approach can be validated by secondary screening using immunological techniques such as immunohistochemistry or immunoblots.