5 research outputs found
Implementing the MSFragger Search Engine as a Node in Proteome Discoverer
Here, we describe the implementation
of the fast proteomics search
engine MSFragger as a processing node in the widely used Proteome
Discoverer (PD) software platform. PeptideProphet (via the Philosopher
tool kit) is also implemented as an additional PD node to allow validation
of MSFragger open (mass-tolerant) search results. These two nodes,
along with the existing Percolator validation module, allow users
to employ different search strategies and conveniently inspect search
results through PD. Our results have demonstrated the improved numbers
of PSMs, peptides, and proteins identified by MSFragger coupled with
Percolator and significantly faster search speed compared to the conventional
SEQUEST/Percolator PD workflows. The MSFragger-PD node is available
at https://github.com/nesvilab/PD-Nodes/releases/
Implementing the MSFragger Search Engine as a Node in Proteome Discoverer
Here, we describe the implementation
of the fast proteomics search
engine MSFragger as a processing node in the widely used Proteome
Discoverer (PD) software platform. PeptideProphet (via the Philosopher
tool kit) is also implemented as an additional PD node to allow validation
of MSFragger open (mass-tolerant) search results. These two nodes,
along with the existing Percolator validation module, allow users
to employ different search strategies and conveniently inspect search
results through PD. Our results have demonstrated the improved numbers
of PSMs, peptides, and proteins identified by MSFragger coupled with
Percolator and significantly faster search speed compared to the conventional
SEQUEST/Percolator PD workflows. The MSFragger-PD node is available
at https://github.com/nesvilab/PD-Nodes/releases/
HDAC8 Substrates Identified by Genetically Encoded Active Site Photocrosslinking
The histone deacetylase family comprises
18 enzymes that catalyze
deacetylation of acetylated lysine residues; however, the specificity
and substrate profile of each isozyme remains largely unknown. Due
to transient enzyme–substrate interactions, conventional co-immunoprecipitation
methods frequently fail to identify enzyme-specific substrates. Additionally,
compensatory mechanisms often limit the ability of knockdown or chemical
inhibition studies to achieve significant fold changes observed by
acetylation proteomics methods. Furthermore, measured alterations
do not guarantee a direct link between enzyme and substrate. Here
we present a chemical crosslinking strategy that incorporates a photoreactive,
non-natural amino acid, <i>p</i>-benzoyl-l-phenylalanine,
into various positions of the structurally characterized isozyme histone
deacetylase 8 (HDAC8). After covalent capture, co-immunoprecipitation,
and mass spectrometric analysis, we identified a subset of HDAC8 substrates
from human cell lysates, which were further validated for catalytic
turnover. Overall, this chemical crosslinking approach identified
novel HDAC8-specific substrates with high catalytic efficiency, thus
presenting a general strategy for unbiased deacetylase substrate discovery
HDAC8 Substrates Identified by Genetically Encoded Active Site Photocrosslinking
The histone deacetylase family comprises
18 enzymes that catalyze
deacetylation of acetylated lysine residues; however, the specificity
and substrate profile of each isozyme remains largely unknown. Due
to transient enzyme–substrate interactions, conventional co-immunoprecipitation
methods frequently fail to identify enzyme-specific substrates. Additionally,
compensatory mechanisms often limit the ability of knockdown or chemical
inhibition studies to achieve significant fold changes observed by
acetylation proteomics methods. Furthermore, measured alterations
do not guarantee a direct link between enzyme and substrate. Here
we present a chemical crosslinking strategy that incorporates a photoreactive,
non-natural amino acid, <i>p</i>-benzoyl-l-phenylalanine,
into various positions of the structurally characterized isozyme histone
deacetylase 8 (HDAC8). After covalent capture, co-immunoprecipitation,
and mass spectrometric analysis, we identified a subset of HDAC8 substrates
from human cell lysates, which were further validated for catalytic
turnover. Overall, this chemical crosslinking approach identified
novel HDAC8-specific substrates with high catalytic efficiency, thus
presenting a general strategy for unbiased deacetylase substrate discovery
Oxidase Activity of the Barnacle Adhesive Interface Involves Peroxide-Dependent Catechol Oxidase and Lysyl Oxidase Enzymes
Oxidases
are found to play a growing role in providing functional chemistry
to marine adhesives for the permanent attachment of macrofouling organisms.
Here, we demonstrate active peroxidase and lysyl oxidase enzymes in
the adhesive layer of adult Amphibalanus amphitrite barnacles through live staining, proteomic analysis, and competitive
enzyme assays on isolated cement. A novel full-length peroxinectin
(AaPxt-1) secreted by barnacles is largely responsible for oxidizing
phenolic chemistries; AaPxt-1 is driven by native hydrogen peroxide
in the adhesive and oxidizes phenolic substrates typically preferred
by phenoloxidases (POX) such as laccase and tyrosinase. A major cement
protein component AaCP43 is found to contain ketone/aldehyde modifications
via 2,4-dinitrophenylhydrazine (DNPH) derivatization, also called
Brady’s reagent, of cement proteins and immunoblotting with
an anti-DNPH antibody. Our work outlines the landscape of molt-related
oxidative pathways exposed to barnacle cement proteins, where ketone-
and aldehyde-forming oxidases use peroxide intermediates to modify
major cement components such as AaCP43