Development of inductively coupled plasma–mass spectrometry-based protease assays

Lathia, Urja S.; Ornatsky, Olga; Baranov, Vladimir; Nitz, Mark

doi:10.1016/j.ab.2009.11.010

Cited by 34 publications

(15 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This assay also demonstrates that background protease activity in the lysate can be detected, as a small amount of cleavage of the calpain-1 substrate was observed in the control assay (top four bars Figure 1B). This is consistent with our previous study showing that HeLa cell lysates have significant chymotrypsin-like activity 8. The calpain-1 substrate contains a Tyr∼Gly linkage which is susceptible to cleavage by chymotrypsin-like enzymes.…”

supporting

confidence: 93%

“…Recently, we demonstrated the use of a lanthanide-tagged protease substrate in a novel ICP-MS based protease assay 8. Here we demonstrate that this ICP-MS assay can be readily adapted to a multiplexed protease assay.…”

mentioning

confidence: 88%

“…Four β-alanine residues were introduced between the N-terminal DTPA chelator and the peptide substrate sequence. In our previous studies we observed that it was necessary to place a short repeat of β-alanine residues between the DTPA-Ln complex and the peptide substrate to ensure the DTPA ligand does not interfere with protease substrate recognition 8. Fmoc-Lys(biotin)-OH was used during the solid phase synthesis to introduce a biotin label at the C-terminus of the peptide substrates.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Multiplexed protease assays using element-tagged substrates

Lathia

Ornatsky

Baranov

et al. 2011

Analytical Biochemistry

Self Cite

View full text Add to dashboard Cite

Inductively coupled plasma-mass spectrometry (ICP-MS) based assays lend themselves to multiplexing due to the high resolution between mass channels, the sensitivity, and the reliability of the technique. Here the potential of ICP-MS based protease assays is demonstrated with a quadruplex assay of cysteine proteases and metalloproteases. Four orthogonal peptide substrates were synthesized for the proteases calpain-1, caspase-3, MMP-9, and ADAM10. Each substrate carries a biotin tag at the C-terminus and a DTPA-based lanthanide complex at the N-terminus. The results demonstrate that this is simple and reproducible analysis technique with excellent correlation between the single and the multiplex assay formats. Keywords ICP-MS; Multiplex; Peptidase; Element-Tagging; LanthanidesProteases play crucial roles in all biological systems ranging from non-specific catabolism to specific signalling events. Disruption or acceleration of proteolysis of various protease substrates is observed in many diseases, making protease activity an important biomarker and a potential target for therapeutic intervention. 1, 2 Proteases rarely act alone but function in a 'protease web' and thus, the multiplex analysis of many proteases in a single assay would aid in the diagnosis of diseases through the identification of characteristic patterns of proteolytic activity. 3 Here we demonstrate a multiplex protease assay based upon lanthanide tagged protease substrates and ICP-MS analysis. There have been successful examples reporting multivariate bioassays for the detection of surface cell antigens 4 , glycoproteins 5 , and proteins 6, 7 using ICP-MS. The potential of ICP-MS based assays for multiplexed enzyme assays has not yet been realized.Recently, we demonstrated the use of a lanthanide-tagged protease substrate in a novel ICP-MS based protease assay. 8 Here we demonstrate that this ICP-MS assay can be readily adapted to a multiplexed protease assay. A quadruplex assay of two cysteine proteases, calpain-1 and caspase-3, and two metalloproteinases, MMP-9 and ADAM10, was carried out to demonstrate the potential of the multiplexed assay for the simultaneous detection of four different families of protease activity each with different requirements for activity.The design of the multiplex protease assay using ICP-MS is shown in Scheme 1. The assay is based on dual labeled peptide substrates that contain an N-terminal lanthanide chelate and a C-terminal biotin tag. All of the protease substrates were readily synthesized using standard Corresponding author: Mark Nitz, mnitz@chem.utoronto.ca, 416-946-0640. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and...

show abstract

supporting

confidence: 93%

mentioning

confidence: 88%

mentioning

confidence: 99%

See 1 more Smart Citation

Multiplexed protease assays using element-tagged substrates

Lathia

Ornatsky

Baranov

et al. 2011

Analytical Biochemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…They play critical roles in a number of biological and physiological processes such as blood clotting, digestion and a variety of cellular activities [1–3]. In addition, they are involved in a number of pathological processes [4–13]. Owing to their specificity, they are excellent biomarkers for disease diagnosis and inhibitors of these proteases are widely employed as therapeutic agents [14–16].…”

Section: Introductionmentioning

confidence: 99%

Detection of protease activities by flash chronopotentiometry using a reversible polycation-sensitive polymeric membrane electrode

Gemene

Meyerhoff

2011

Analytical Biochemistry

View full text Add to dashboard Cite

A novel electrochemical method, termed flash chronopotentiometry (FCP), is used to develop a rapid and sensitive method to detect protease activities. In this method, an appropriate current pulse is applied across a polycation-selective polymer membrane to induce a strong flux of the polycationic peptides from the sample phase into the organic membrane of the electrode. During this current pulse, the cell potential (EMF) is monitored continuously, and is a function of the polypeptide concentration. The imposed current causes a local depletion of the polypeptide at the sample/membrane interface, which yields a drastic potential change in the observed chronopotentiogram at a characteristic time, called the transition time (τ). For a given magnitude of current, the square root of τ is directly proportional to the concentration of the polypeptide. Proteases cleave polypeptides into smaller fragments that are not favorably extracted into the membrane of the sensor. Therefore, a decrease in the transition time is observed during the proteolysis process. The degree of change in the transition time can be correlated to protease activity. To demonstrate this approach, the activities of trypsin and α-chymotrypsin are detected using protamine and synthetic polycationic oligopeptides that possess specific cleavage sites that are recognized by these proteases.

show abstract

“…Proteases play critical roles in cell‐cycle control and programmed cell suicide/death (apoptosis) . They are involved in a wide range of physiological processes such as blood coagulation and protein degradation as well as pathological processes including cancer progression , toxin generation , cardiac diseases , diabetes , genetic diseases of the central nervous system and many other diseases and conditions .…”

Section: Introductionmentioning

confidence: 99%

Pulsed Chronopotentiometric Detection of Thrombin Activity Using Reversible Polyion Selective Electrodes

et al. 2016

View full text Add to dashboard Cite

A simple and rapid electrochemical method for the detection of thrombin activity is presented here for the first time using a synthetic polypeptide substrate and a pulsed chronopotentiometry transduction protocol with polyion selective electrodes. A cathodic current applied across a polyion selective membrane electrode causes the extraction of the polypeptides from the sample into the membrane and the membrane potential, which is a function of the concentration of these polypeptides in the sample, is measured at the same time. Since the polyion extraction is a diffusion‐controlled mass transfer process, depletion of the polypeptides at the membrane‐sample interface at a characteristic transition time occurs. The square root of the transition time is directly proportional to the concentration of the polypeptide substrate according to the Sand equation. Upon addition of thrombin, the polypeptide substrate undergoes proteolysis and yields smaller peptide fragments that exhibit smaller measured electromotive force (emf) responses since they are less preferred by the membrane, and the transition times become shorter. The rate of change of the square root of the transition time is directly proportional to the change in the polypeptide concentration, which in turn relates to the polypeptide hydrolysis time, and can be used for monitoring enzyme activity. Indeed, the square root of transition time was found to be linear with the proteolysis time with R2=0.98. The activity of thrombin was determined to be 3.6 μg substrate per μg thrombin per Min.

show abstract

Development of inductively coupled plasma–mass spectrometry-based protease assays

Cited by 34 publications

References 43 publications

Multiplexed protease assays using element-tagged substrates

Multiplexed protease assays using element-tagged substrates

Detection of protease activities by flash chronopotentiometry using a reversible polycation-sensitive polymeric membrane electrode

Pulsed Chronopotentiometric Detection of Thrombin Activity Using Reversible Polyion Selective Electrodes

Contact Info

Product

Resources

About