61Peter V. Bozhkov and Guy Salvesen (eds.), Caspases, Paracaspases, and Metacaspases: Methods and Protocols,
Methods in Molecular Biology, vol. 1133, DOI 10.1007/978-1-4939-0357-3_3, © Springer Science+Business Media New York 2014
Chapter 3
Global Identifi cation of Caspase Substrates Using
PROTOMAP (Protein Topography and Migration Analysis
Platform)
Melissa M. Dix , Gabriel M. Simon , and Benjamin F. Cravatt
Abstract
Delineation of the natural substrate scope of proteases is important for understanding the functions of
proteolytic pathways in physiology and disease. Herein we describe the protocol for PROTOMAP, a tech-
nique that combines SDS-PAGE with tandem mass spectrometry to globally identify shifts in protein
migration indicative of proteolytic processing. When applied to cells undergoing apoptosis, this unbiased
global method provides a snapshot of the topography and magnitude of proteolytic events associated with
programmed cell death.
Key words SDS-PAGE , In-gel digestion , Mass spectrometry , Spectral counting , PROTOMAP ,
Peptograph , Substrate identifi cation1 Introduction
The key to elucidating the biological role of proteases is identifi cation
of their physiological substrates and the functional consequences
of protein cleavage. For example, does a proteolytic cleavage event
result in loss-of-function and rapid degradation of the substrate?
Or, does cleavage produce a stable protein fragment that performs
a new biochemical function? If so, what is the topography of the
fragment, how large is it, and does it contain any known functional
domains? The challenge of comprehensively defi ning the topogra-
phy of protein cleavage in physiological systems and thereby pre-
dicting functional consequences of proteolysis has historically
required generation of multiple antibodies that recognize epitopes
throughout the length of proteins. In pursuit of a more general
solution, we developed an unbiased proteomic platform that pro-
vides a global snapshot of proteolytic events occurring upon activa-
tion or inhibition of a protease(s). We have named this platform
PROTOMAP for Protein Topography and Migration Analysis