Methods in Enzymology. Volume 642. Proteomics of arthropod soluble olfactory proteins

Shotgun proteomics is a very sensitive bottom-up approach used to study complex mixtures of proteins through a combination of high-performance liquid chromatography (HPLC) combined with High-Resolution Mass Spectrometry (MS). This technique has been successfully applied to the study of olfactory appendixes of insects and of other arthropods. Since extracting soluble proteins from tissues and processing them for shotgun proteomics is quite straightforward, the technique has proved to be very suitable for studying soluble olfactory proteins, such as Odorant Binding Protein, Chemosensory Proteins and Niemann-Pick type 2 proteins. Here we describe the main principles and methods at the basis of shotgun proteomics, including its use to quantify proteins through Label-Free Quantification, and we provide operational protocols for the experimental workflow steps, i.e. insect dissection, proteins extraction, protein enzymatic digestion, sample purification, HPLC-MS analysis, search of mass spectral data against polypeptide databases and first steps in the analysis of the search results.