Profiling Deacetylase Activities in Cell Lysates with Peptide Arrays and SAMDI Mass Spectrometry

Abstract

The development of arrays that can profile molecular activities in cells is important to understanding signaling pathways in normal and pathological settings. While oligonucleotide arrays are now routinely used to profile global gene expression, there is still a lack of tools for profiling enzyme activities in cell lysates. This paper describes the combination of peptide arrays formed on self-assembled monolayers and mass spectrometry to provide a label-free approach for identifying patterns of enzyme activities in cell lysates. The approach is demonstrated by profiling lysine deacetylase (KDAC) activities in cell lysates of the CHRF megakaryocytic (Mk) cell line. Class-specific deacetylase inhibitors were used to show that terminal Mk differentiation of CHRF cells is marked by a pronounced decrease in sirtuin activity and by little change in activity of KDACs 1-11. This work establishes a platform that can be used to identify changes in global activity profiles of cell lysates for a wide variety of enzymatic activities.

SIRT1 is a critical regulator of K562 cell growth, survival, and differentiation

“…1B). The decrease in SIRT activity with PMA differentiation is consistent with our earlier report examining a different leukemic cell line, CHRF, that is already committed to the MK lineage [12]. This suggests that there may be a significant relationship between E/MK specification and SIRT activity, and especially SIRT1/2, as these were the most highly expressed isoforms at the time of inducing differentiation.…”

“…This assay consists of incubating a panel of short, acetylated peptides with cell lysate, followed by covalent attachment to a self-assembled monolayer of alkane-thiolates on a gold surface, and then the proportion of peptide that has been deacetylated is quantified by mass spectrometry. The 8 peptide substrates chosen were shown to be substrates for human deacetylases in earlier work [12, 29, 30]. The total cell lysate reveals deacetylase activity contributed from both SIRTs and the class I/II/IV HDACs.…”

“…Cell lysates were prepared as previously described [12]. Lysates were diluted in 3X protein loading buffer (187.5 mM Tris (pH 6.8), 30% glycerol, 6% SDS, 0.005% pyronin Y, 5% beta-mercaptoethanol) and boiled for 5 minutes at 95°C.…”

“…The peak areas for the acetylated and deacetylated forms of the peptide were integrated and used to calculate percent deacetylation (Deacetylase activity = I D /(I D + I A ) where subscript D refers to the deacetylated peptide product peak and A refers to the acetylated peptide substrate peak). Details of the mass spectrometry and peptide synthesis have been previously described [12]. …”

Genetically Encoded Fluorescent Probe for Detecting Sirtuins in Living Cells

“…[3] As a consequence, there has been interest in the development of chemical probes of sirtuin activity. In vitro methods including the use of radiolabeled histones [4] and mass spectrometry, [5] have been used to measure the deacetylation activity of sirtuins. More recently, fluorescent probes composed of an acetylated peptide and a fluorophore, have been developed to evaluate sirtuin activities in vitro and in living cells.…”