Absolute proteomic quantification of the activity state of proteases and proteolytic cleavages using proteolytic signature peptides and isobaric tags

作者：Fahlman Richard P; Chen Wei; Overall Christopher M^*

来源：Journal of Proteomics, 2014, 100: 79-91.

DOI：10.1016/j.jprot.2013.09.006

• Protease
• Proteolytic cleavages
• MMPs
• Proteomics
• iTRAQ synthesis
• Degradomics

Proteolytic processing alters the structure and function of a wide range of proteins in the proteome. We describe a method for the absolute quantification of proteolysis that is compatible with existing quantitative proteomic applications and could be applied on a protein-family wide scale. A tryptic peptide spanning a cleavage site differentiates this intact form of the protein from the corresponding semi-tryptic peptides of a protease cleaved protein. We term such proteomic signatures of specific proteolytic events "proteolytic signature peptides" (PSPs). By quantifying both the tryptic and semi-tryptic PSPs simultaneously with proteotypic peptides common to all forms of the protein both the relative and the absolute amounts of the intact and cleaved protein can be determined. Using synthetic PSP standards of cleavage sites in intact and cleaved proteins the absolute amounts of each form of the protein can be determined. The technique was demonstrated by the simultaneous identification and quantification of matrix metalloproteinase zymogens and their proteolytically activated forms in parallel with conventional absolute quantification of their TIMP inhibitors. For quantification we synthesized a pair of isobaric mass tags, we term CLIP-TRAQ using C-13 labeled reagents that when fragmented during CID generate signature ions at 113.1 or 114.1 respectively. As an expandable platform this allows for the simultaneous identification of multiple proteins and their proteolytic state in complex proteomes on a family-wide scale in parallel with conventional proteomic analysis. This article is part of a Special Issue entitled: CNPN 2013.
Biological significance
Proteolysis is key to various biological processes and the activity and function of many proteins are dictated by their proteolytic state. The development of methods to quantify protein abundance in conjunction to determining their proteolytic.state and hence activity is essential for the complete understanding of the processes for which proteolysis is involved.

• 出版日期2014-4-4