Peptide Code-on-a-Microplate for Protease Activity Analysis via MALDI-TOF Mass Spectrometric Quantitation

Abstract

A peptide-encoded microplate was proposed for MALDI-TOF mass spectrometric (MS) analysis of protease activity. The peptide codes were designed to contain a coding region and the substrate of protease for enzymatic cleavage, respectively, and an internal standard method was proposed for the MS quantitation of the cleavage products of these peptide codes. Upon the cleavage reaction in the presence of target proteases, the coding regions were released from the microplate, which were directly quantitated by using corresponding peptides with one-amino acid difference as the internal standards. The coding region could be used as the unique "Protease ID" for the identification of corresponding protease, and the amount of the cleavage product was used for protease activity analysis. Using trypsin and chymotrypsin as the model proteases to verify the multiplex protease assay, the designed "Trypsin ID" and "Chymotrypsin ID" occurred at m/z 761.6 and 711.6. The logarithm value of the intensity ratio of "Protease ID" to internal standard was proportional to trypsin and chymotrypsin concentration in a range from 5.0 to 500 and 10 to 500 nM, respectively. The detection limits for trypsin and chymotrypsin were 2.3 and 5.2 nM, respectively. The peptide-encoded microplate showed good selectivity. This proposed method provided a powerful tool for convenient identification and activity analysis of multiplex proteases.

Peptide codes for multiple protease activity assay via high‐resolution mass spectrometric quantitation

“…). Their detection limits (3σ) were 3.8 pM and 2.2 nM, respectively, which were lower than those of 2.3 and 5.2 nM for the previous MALDI‐TOFMS analysis and comparable with some fluoresence, phosphorescence, or label‐free colormetric assays . All these results implied the effectiveness of the proposed method for multiple protease assay.…”

“…However, preparation and purification of the probes is still time‐consuming. Our group proposed a peptide‐encoded microplate for matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOFMS) analysis of trypsin and chymotrypsin, but internal standards were required for quantitation . Considering the fact that high‐resolution mass spectrometry (HRMS) can operate in the full‐scan operation mode with the special advantages of high throughput and can be used for accurate peptide quantitation without an internal standard, this work applied the peptide‐encoding technique for HRMS analysis of protease activities.…”

“…The peptide codes were designed to contain a coding region as the “Protease ID” and the substrate for enzymatic cleavage respectively as previously reported (Fig. ) . Upon cleavage by different target proteases, the formed “Protease ID” was identified and quantified using HRMS.…”

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 .…”

MALDI‐MS Patterning of Caspase Activities and Its Application in the Assessment of Drug Resistance

“…Inrecent years,mass spectrometry (MS) has been proven to be apowerful method for enzyme activity assays owing to the advantages of high-throughput and label-free analyses. [1][2][3] However,i ts application is often limited by complicated sample pretreatments,including separation, purification, and desalting procedures. [4][5][6] To solve these problems,several gold chips with covalently assembled oligoethylene disulfide chains have been proposed for profiling the activities of kinases, [7] proteases, [8] and glycotransferases [9] and for identifying enzyme inhibitors, [10] and some aluminum oxide,g old, and ITOslides with immobilized glycan substrates have been developed for studying enzymatic reactions and protein binding.…”