Proteolysis represents one of the most tightly controlled physiological 
processes, as proteases create events that will typically commit pathways in an 
irreversible manner. Despite their implication in nearly all biological systems, 
our understanding of the role of proteases in disease pathology is often 
limited. Several approaches to studying proteolytic activity as it relates to 
biology, pathophysiology, and drug therapy have been published, including the 
recently described terminal amine isotopic labeling of substrates (TAILS) 
strategy by Kleifeld and colleagues. Here, we investigate TAILS as a methodology 
based on targeted enrichment and mass spectrometric detection of endogenous 
N-terminal peptides from clinically relevant biological samples and its 
potential to provide quantitative information on proteolysis and elucidation of 
the protease cleavage sites. While optimizing the most current protocol, by 
switching to a streamlined one-tube format and simplifying the reagents' removal 
steps, we demonstrate the advantages over previously published methods and 
provide solutions to some of the technical challenges presented in the Kleifeld 
publication. We also identify some of the current and unresolved limitations. We 
use human plasma as a model system to provide data, which illustrates some of 
the key analytical parameters of the modified TAILS procedure, including 
specificity, sensitivity, quantitative precision, and accuracy.