Covalent post-translational modification of proteins is the primary modulator of protein function in the cell. It greatly expands the functional potential of the proteome compared to the genome. In the past few years shotgun proteomics-based research, where the proteome is digested into peptides prior to mass spectrometric analysis has been prolific in this area. It has determined the kinetics of tens of thousands of sites of covalent modification on an equally large number of proteins under various biological conditions and uncovered a transiently active regulatory network that extends into diverse branches of cellular physiology. In this review, we discuss this work in light of the concept of protein speciation, which emphasizes the entire post-translationally modified molecule and its interactions and not just the modification site as the functional entity. Sometimes, particularly when considering complex multisite modification, all of the modified molecular species involved in the investigated condition, the protein species must be completely resolved for full understanding. We present a mathematical technique that delivers a good approximation for shotgun proteomics data.