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Soheila Shokrollahzade ,MSc Department Of Medical Biotechnology , Iums Activity-Based Protein Profiling: From Enzyme Chemistry to Proteomic Chemistry Soheila Shokrollahzade ,MSc Department Of Medical Biotechnology , Iums

Why profiling ? Is there any need ?

Activity-Based Protein Profiling/ Enzyme Substrate Proteomics Definition from “Proteomic biology using LC/MS Book ,2007, Wiley pub “ Activity-based profiling (ABP) provides a strategy for identifying proteins associated with a particular biological activity—typically enzymatic activity and utilizes chemistry to create tools and assays for the characterization of protein samples of high complexity

Facts To date, genome sequences of 639 organisms have been completed, three primates— rhesus, chimpanzee, and man. Over 2000 other genome projects are underway These statistics made us to develop large-scale methods for protein characterization.

Why study at proteomic level ?

Gene expression profiling deducing protein function

Liquid chromatography-mass spectrometry (LC-MS) platforms for shotgun analysis (discussed later ) yeast two-hybrid methods protein microarrays patterns, interaction maps, and in vitro functional properties of proteins

Shotgun….. The term “shotgun” analysis was initially proposed as a general term for a process in which a protein complex was digested into peptides and analyzed via a single dimension of reversed phase column in an ESI-MS/MS system (Fig. 2-2). Accordingly, the strategy for a large scale analysis, using either multi-LC-MS/MS or single LC- MS/MS technology after protease digestion of protein mixtures, is generally called shotgun proteomics and represents a gel-free approach (without using 1D- or 2D- PAGE) based on peptide separation and identifi cation

ABPP , THE BASIC CONCEPT ABPP relies on the design of active-site directed covalent probes to interrogate specific subsets (families) of enzymes in complex proteomes and to provide the basis for a quantitative readout of the functional state of individual enzymes in the family functional state of proteins in cells and tissues

ABPP probe Reactive groups: Electerical group Photoreactive group Reporter tags fluorophores biotin latent analytical handles such as alkynes or azides Spacer

Gel electrophoresis platforms for ABPP 1D or 2D PAGE for probe-treated proteomes in-gel fluorescence scanning (for fluorescent probes) avidin blotting (for biotinylated probes)

Liquid chromatography-mass spectrometry (LC-MS) platforms for ABPP 1-ABPP-MudPIT (ABPP multidimensional protein identification ) Analysis of protein targets of probes by biotinylated probes

Liquid chromatography-mass spectrometry (LC-MS) platforms for ABPP 2- Active-site peptide profiling specifically analysis of probe-labeled peptides derived from these targets

High-throughput ,high-resolution ABPP Minimal sample preparation Higher resoulution ( compared to LC/MS) + CE LIF

CE-LIF superiority in resolution provide superior resolution compared to 1D-SDS-PAGE Enzyme targets that share similar molecular mass Consuming minimal amounts of sample CE run times are very short (15–20 min) many samples can be analyzed in parallel on 96-channel instruments the identity of enzyme targets initially remains unclear most applicable for the repetitive analysis of well-characterized proteomes

Another way to solve minimal sample preparation and low resolution ABPP microarray Binding to antibodies glass slides and as capture tools direct detection of enzymes by fluorescence scanning

ABPP microarray Improved sensitivity compared to gel-based methods for the detection of protease activities in proteomes No need to secondary antibodies Minimal amounts of proteome (<0.01 mg) required for ABPP microarray experiments Many antibodies can be arrayed in parallel on a single slide

Types of Biological Experiments that Can Be Performed with ABPP 1- Comparative ABPP for target discovery 2- Types of Biological Experiments that Can Be Performed with ABPP

Multiple Advantages Of Comparative ABPP For Target Discovery ABPP accounts for myriad posttranslational mechanisms that regulate enzyme activity (but not necessarily expression) in living systems ABPP probes label enzymes using conserved active- site features rather than mere expression level low-abundance proteins in samples of high complexity

Competitive ABPP for inhibitor discovery

Competitive ABPP for inhibitor discovery Multiple advantages of Competitive ABPP for inhibitor discovery 1-enzymes are tested in native proteomes 2-enzymes that lack known substrates are amenable to analysis 3-because ABPP tests inhibitors against many enzymes in parallel, potency and selectivity factors can be simultaneously assigned to these compounds

Characterization of enzyme active sites by ABPP

ENZYME CLASSES ADDRESSABLE BY ABPP Serine hydrolases Serine protease Cysteine protease Metallohydrolases HDACs Kinase Glycosidase Phosphatase . etc.

Serine hydrolases functional roles in biological systems Cancer Atherosclerosis immune cell activation nervous system signaling

serine hydrolases in cancer research controlling extracellular matrix structure, growth factor activation, metabolism of small-molecule signals, malignant behavior of aggressive cancer cells