Label-Free Quantitative Shotgun Proteomics Using Normalized Spectral Abundance Factors

Abstract

In this chapter we describe the workflow used in our laboratory for label-free quantitative shotgun proteomics based on spectral counting. The main tools used are a series of R modules known collectively as the Scrappy program. We describe how to go from peptide to spectrum matching in a shotgun proteomics experiment using the XTandem algorithm, to simultaneous quantification of up to thousands of proteins, using normalized spectral abundance factors. The outputs of the software are described in detail, with illustrative examples provided for some of the graphical images generated. While it is not strictly within the scope of this chapter, some consideration is given to how best to extract meaningful biological information from quantitative shotgun proteomics data outputs.