Multiplexed protease assays using element-tagged substrates

Abstract

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...

Inductively coupled plasma mass spectrometry‐based immunoassay: A review

“…After in situ hybridization, commercial antibodies conjugated to Eu and gold NPs together with biotinylated oligonucleotide probes reacted with terbium labeled streptavidin, were used to demonstrate simultaneous mRNA and protein detection by ICPMS in leukemia cells (Ornatsky et al, ). The single and multiplexed protease assays were realized by Nitz's group (Lathia et al, ; Lathia et al, ) and Wang's group (Yan et al, ), separately. A lanthanide‐coded protease‐specific peptide‐nanoparticle probe was well designed as shown in Figure (Yan et al, ).…”

In vitro methods for testing antiviral drugs

“…Some recently described arrangements employ nanoparticles (Feltrup and Singh, 2012;Khalilzadeh et al, 2016;Udukala et al, 2016;Wang et al, 2014;Zeng et al, 2015) or quantum dot bioconjugates (Lee and Kim, 2015;Li et al, 2014;Medintz et al, 2006) with immobilized fluorescently or luminescently labeled peptide substrates. Alternatively, cleavage products may be monitored by analysis of proteolytic products by mass spectrometric methods (Hu et al, 2015;Joshi et al, 2017;Lathia et al, 2011;Rumlová et al, 2003), analytical HPLC (Teruya et al, 2016), or electrochemical methods based on the difference in penetration of substrate and cleavage products through the membrane of a polyionselective sensor (Gemene and Meyerhoff, 2011;Han et al, 1996). To study the specificity of inhibitor binding and to extend the research to rational design of inhibitors, X-ray or NMR structures of proteases in complex with the inhibitor may be determined, as reported in numerous cases for the proteases of HIV-1 [reviewed in (Ghosh et al, 2016)], HCV (Yilmaz et al, 2016), and MERS .…”

Recent developments in protease activity assays and sensors

“…Conversely, heterogeneous assays consist of protease substrates that are immobilised on a solid platform, while samples are in the aqueous phase. On the basis of detection methods, homogeneous assays can be further classified into colorimetric assays, 20,21 mass spectrometry-based assays, [22][23][24] and fluorescence resonance energy transfer (FRET) assays. [25][26][27][28][29][30][31][32][33][34] More recently, nanomaterials such as noble metal nanoparticles, [35][36][37][38][39][40][41][42][43][44][45][46] quantum dots (QDs), [47][48][49][50][51][52][53][54][55][56][57][58][59] and graphene oxide (GO) [60][61][62][63][64] are also used in the homogeneous protease assays with impressive detection limits.…”