Spectral Counting Label-Free Proteomics

Abstract

Label-free proteome quantification methods used in bottom-up mass-spectrometry based proteomics are gaining more popularity as they are easy to apply and can be integrated into different workflows without any extra effort or cost. In the label-free proteome quantification approach, samples of interest are prepared and analyzed separately. Mass-spectrometry is generally not recognized as a quantitative method as the ionization efficiency of peptides is dependent on composition of peptides. Label-free quantification methods have to overcome this limitation by additional computational calculations. There are several algorithms available that take into account the sequence and length of the peptides and compute the predicted abundance of proteins in the sample. Label-free methods can be divided into two categories: peptide peak intensity based quantification and spectral counting quantification that relies on the number of peptides identified from a given protein. This protocol will concentrate on spectral counting quantification—exponentially modified protein abundance index (emPAI). Normalized emPAI, most commonly derived from Mascot search results, can be used for broad comparison of entire proteomes. Absolute quantification of proteins based on emPAI values with or without added standards will be demonstrated. Guidelines will be given on how to easily integrate emPAI into existing data; for example, calculating emPAI based absolute protein abundances from iTRAQ data without added standards.