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J Biomol Tech. 2010 September; 21(3 Suppl): S51.
PMCID: PMC2918217

The Effect of Pressure Cycling on Proteolytic Cleavage Efficiency, Reaction Time and Protein Sequence Coverage

E. Bonneil,2 J. Saba,1 A. Huhmer,1 and P. Thibault2
1Thermo Fisher Scientific, San José, CA, United States;
2Institute for Research in Immunology and Cancer, Université de Montreal, Montreal, QC, Canada

Abstract

RP-96

Over the years many methodologies have been advanced to aid in the enhancement of proteolysis cleavage efficiency, reaction time, and protein sequence coverage for LC-MS/MS based analysis. The series of experiments presented here examine the effect of pressure cycling on each of these factors. Pressure cycling applies alternating hydrostatic pressure between 1 atm and high levels to control molecular interactions. Several individual proteins and a mixture of 9 protein standards were digested with different proteases under high pressure using a Barocycler NEP2320 pressure cycler and compared to overnight at 1 atm and 37°C proteolysis conditions. The resulting peptides were identified and quantified using an LC-MS/MS workflow with a LTQ XL with ETD. For the mixture of 9 protein standards, PCT produces more peptides and more missed cleavages, particularly at lower number of pressure cycles. As a result of digestion under PCT conditions, more distinct peptides with missed cleavages are produced, resulting in additional overlapping sequence coverage and a potential benefit for ETD-based detection. The studies were further extended using an LTQ Orbitrap XL to evaluate in the context of complex cell model system, specifically proteins extracted from human monocyte-like U937 hystiocytic lymphoma cells. The use of a complex protein extract from human monoblastic U937 cells provides an opportunity to evaluate the overall performance of pressure cycling for complex mixtures. In general, lower number of cycles (20 cycle) using PCT produced more peptide identifications (equivalent to 4 hr standard digestion) than the standard overnight digestion and the number of proteins identified were roughly equivalent. We also demonstrate the use of PCT for the analysis of mouse J774 membrane proteins after digestion with chymotrypsin.


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