Bottom-Up Proteomics

In this chapter we provide an overview of bottom-up proteomic approaches. These allow the identification and characterization of proteins and their amino acid sequences, including post-translational modifications, by proteolytic digestion prior to mass spectrometry (MS) analysis. Intact proteins can be separated by gel electrophoresis followed by in-gel protein digestion to generate peptides which are then analyzed by MS. Alternatively, complex protein mixtures can be digested directly (an approach referred to as “shotgun”) and the resulting peptides can be separated by liquid chromatography prior to MS. Following MS analysis, the comparison of the peptides’ spectra with those predicted from genomics/proteomics sequence databases, or annotated peptide spectral libraries, allows the identification of peptides which are finally assigned to corresponding proteins. After a description of the separation methods and MS acquisition modes, a relevant part of the chapter will be dedicated to data processing pointing to algorithms, computational tools and strategies useful for researchers in the discovery process. In particular, liquid-chromatography (LC) based approaches, including Multidimensinal Protein Identification Technology (MudPIT), will be taken as reference and different aspects, ranging from database search engines to protein-protein interaction (PPI) network analysis, will be addressed. Potential issues will be discussed in the context of cardiovascular research, and specifically the last section will focus on the translational applications (clinical proteomics) of cardiovascular proteomics.