Mass spectrometry for monitoring protease reactions

Schlüter, Hartmut; Hildebrand, Diana; Gallin, C.; Schulz, Anna; Thiemann, Joachim; Trusch, Maria

doi:10.1007/s00216-008-2213-7

Cited by 16 publications

(4 citation statements)

References 80 publications

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“…As reviewed by Caprioli in 1987, these early reports included gas chromatography (GC)/MS, fast‐atom bombardment (FAB)‐MS and thermospray approaches 4. Since that time and with the development of electrospray ionization (ESI) and laser desorption based methods, hundreds of reports have highlighted the advantages and limitations of MS for evaluating enzyme activities and the effects of inhibitors on these reactions, as reviewed recently 5–9. These reviews also cover a number of processes that take enzyme assays a step further by describing how MS approaches can be applied as the primary readout for isolated enzymes in HTS.…”

mentioning

confidence: 99%

Extending matrix‐assisted laser desorption/ionization triple quadrupole mass spectrometry enzyme screening assays to targets with small molecule substrates

Rathore

Corr²,

Lebre³

et al. 2009

Rapid Comm Mass Spectrometry

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Mass spectrometry (MS)-based high-throughput screening (HTS) has tremendous potential as an alternative to current screening methods due to its speed, sensitivity, reproducibility and label-free readout. We recently reported that a new generation matrix-assisted laser desorption/ionization triple quadrupole (MALDI-QqQ) mass spectrometer is ideally suited for a variety of enzyme assays and screening protocols. However, all the targets measured to date had peptide substrates that were easily monitored by selected ion monitoring (SIM) without interference from the MALDI matrix. To further extend the application to enzymes with small molecule, non-peptide substrates, we evaluated this method for measuring enzyme activity and inhibition of acetylcholinesterase (AChE). Due to the potential of MALDI matrix interference, multiple reaction monitoring (MRM) was investigated for selective MS/MS transitions and to accurately measure the conversion of acetylcholine into choline. Importantly, ionization, detection and MRM transition efficiency differences between the substrate and product can be overcome by pre-balancing the MRM transitions during method development, thus allowing for a direct readout of the enzyme activity using the ratio of the substrate and product signals. Further validation of the assay showed accurate concentration-dependent inhibition measurements of AChE with several known inhibitors. Finally, a small library of 1008 drug-like compounds was screened at a single dose (10 microM) and the top 10 inhibitors from this primary screen were validated in a secondary screen to determine the rank order of inhibitory potency for each compound. Collectively, these data demonstrate that a MALDI-QqQMS-based readout platform is amenable to measuring small molecule substrates and products and offers significant advantages over current HTS methods in terms of speed, sensitivity, reproducibility and reagent costs.

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mentioning

confidence: 99%

Extending matrix‐assisted laser desorption/ionization triple quadrupole mass spectrometry enzyme screening assays to targets with small molecule substrates

Rathore

Corr²,

Lebre³

et al. 2009

Rapid Comm Mass Spectrometry

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“…In contrast to the methods described above that primarily aim at the identification of protease network components, few experimental approaches exist that capture the dynamics of proteolytic processing 62 . Classically, chromo- or fluorogenic protease cleavage assays are used to measure the kinetics of individual proteolytic reactions.…”

Section: How To Gain Insights Into the Dynamics Of Proteolytic Procesmentioning

confidence: 99%

Targeting Proteases in Cardiovascular Diseases by Mass Spectrometry-Based Proteomics

Klingler

Hardt

2012

Circ Cardiovasc Genet

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Proteases hydrolyze peptide bonds, thereby controlling the function of proteins and peptides on the posttranslational level. In the cardiovascular system, proteases play pivotal roles in the regulation of blood pressure, coagulation and other essential physiological processes. Accordingly, proteases are prime targets for therapeutic interventions and diagnostics. Proteases are part of complex proteolytic networks comprised of enzymes, inhibitors, activators, substrates and cleavage products. Analyzing these networks on a system-wide level is essential to understanding cardiovascular function and how dysregulation can lead to pathological conditions. Mass spectrometry-based quantitative and dynamic proteomics approaches are leading the way to enhance our knowledge of proteolytic networks such as the renin-angiotensin-system. Here, we critically review proteomics tools utilized in protease biology and provide an overview on how these methods can be used to characterize and validate protease function.

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“…Captured enzymes are subsequently incubated with substrates, whose degradation fates are monitored by MS. By fractionating enzymes from the biological matrices, interfering compounds are removed and defined amounts and combinations of substrates can be used for the temporal analysis step [56]. Other methods for monitoring enzymatic reactions by MS have been reviewed by Liesener and Karst [57] and Schlüter et al [58]. It is noteworthy to point out that label-free protease assays do not necessarily have to rely on MS, as shown by the recent development of a supramolecular tandem protease assay that relies on indicator-replacement technology [59].…”

Section: Introductionmentioning

confidence: 99%

Profiling protease activities by dynamic proteomics workflows

Klingler

Hardt

2012

Proteomics

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Proteases play prominent roles in many physiological processes and the pathogenesis of various diseases, which makes them interesting drug targets. To fully understand the functional role of proteases in these processes, it is necessary to characterize the target specificity of the enzymes, identify endogenous substrates and cleavage products as well as protease activators and inhibitors. The complexity of these proteolytic networks presents a considerable analytic challenge. To comprehensively characterize these systems, quantitative methods that capture the spatial and temporal distributions of the network members are needed. Recently, activity-based workflows have come to the forefront to tackle the dynamic aspects of proteolytic processing networks in vitro, ex vivo and in vivo. In this review, we will discuss how mass spectrometry-based approaches can be used to gain new insights into protease biology by determining substrate specificities, profiling the activity-states of proteases, monitoring proteolysis in vivo, measuring reaction kinetics and defining in vitro and in vivo proteolytic events. In addition, examples of future aspects of protease research that go beyond mass spectrometry-based applications are given.

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Mass spectrometry for monitoring protease reactions

Cited by 16 publications

References 80 publications

Extending matrix‐assisted laser desorption/ionization triple quadrupole mass spectrometry enzyme screening assays to targets with small molecule substrates

Extending matrix‐assisted laser desorption/ionization triple quadrupole mass spectrometry enzyme screening assays to targets with small molecule substrates

Targeting Proteases in Cardiovascular Diseases by Mass Spectrometry-Based Proteomics

Profiling protease activities by dynamic proteomics workflows

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