Applications of Nanoscale Liquid Chromatography Coupled to Tandem Mass Spectrometry in Quantitative Studies of Protein Expression, Protein–Protein Interaction, and Protein Phosphorylation

Mass spectrometry can provide a very sensitive and rapid analysis of protein expression and can be used as an alternative to immunochemical methods to study protein–protein interaction and protein posttranslational modifications. In many circumstances, a functional study, such as one that aims to elucidate a specific signaling pathway or disease state, will require the detection and quantification of a specific set of proteins and their modifications. Very often, there will be no available antibody for some of the proteins in the set, and mass spectrometry will be the only option. This chapter describes a robust and efficient protocol for a small-scale sample preparation and a suite of separation and mass spectrometry techniques that allow the quantitative analysis of low femtomolar amounts of proteins that may be obtained from very limited amount of clinical specimens, affinity techniques, and cell sorting. The protocols can be used by researchers in the applied biomedical field and also in basic cell biology.