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

Abstract

RATIONALE: Proteases, which are involved in a number of biological processes, play important roles in human health. Alterations in protease activities have been associated with many diseases. Thus, effective methods for multiple protease assay are very essential and may help to discover more about their functions in different physiological processes. Here, we proposed a strategy of peptide codes for label-free analysis of multiple protease activities using high-resolution mass spectrometry (HRMS). METHODS:The peptide codes were designed to contain a coding region and the substrate of the protease, respectively. Upon the cleavage of target proteases, the coding regions could be rapidly identified and directly quantitated by accurate m/z extractions without internal standard. Therefore, the coding region could be used as the unique "Protease ID" for the identification of the corresponding protease, and the amount of the cleavage product was used for protease activity analysis. RESULTS: The method was validated by using trypsin and chymotrypsin as model proteases, with the designed "Trypsin ID" and "Chymotrypsin ID" occurring at m/z 761.270 and 711.243, respectively. The peak area of "Protease ID" was proportional to trypsin and chymotrypsin concentration in the range from 0.01 to 10 nM and 10 to 2000 nM, respectively. The proposed method could also be used for screening of protease inhibitors, which might be of great importance for drug discovery. CONCLUSIONS: This method provided a label-free and accurate quantitative platform for convenient identification and activity analysis of multiple proteases, which could potentially be applied in clinical diagnosis.

Mass Spectrometric Biosensing: A Powerful Approach for Multiplexed Analysis of Clinical Biomolecules

“…In the past decade, tens to hundreds of peptides has been analyzed with mass spectrometry owing to its capability as a promising tool for highly specific, multiplexed, precise, and accurate quantitative analysis . Since the development of proteomics, peptides with specific sequences and molecular weights have been used for protein biomarker identifications, and thus regarded as “bio-barcodes” or “ID numbers” of target molecules. − In addition, as the basic units of peptides, the diversity of amino acids offers more possibilities for the design of peptide sequences and properties. All these characteristics make peptides potential mass tags for biosensing.…”

“…As natural substrates of enzymes, unlabeled peptides have been generally applied for enzyme activity assays. − The specific m / z of substrate and product peptides can act as “codes” for corresponding types of enzymes, while the signal intensity of peptides or the ratio of product to substrate can be used for precise quantitation. A two-step multiplex quantitative Endopep-MS method was developed for the detection of human botulism-causing serotypes of botulinum neurotoxins (BoNTs) with MALDI-MS by measuring the cleavage product at two time points to extend the dynamic range and to reduce the number of experiments and save valuable clinical samples .…”

“…A two-step multiplex quantitative Endopep-MS method was developed for the detection of human botulism-causing serotypes of botulinum neurotoxins (BoNTs) with MALDI-MS by measuring the cleavage product at two time points to extend the dynamic range and to reduce the number of experiments and save valuable clinical samples . Our group designed the peptide codes that contained a coding region and a cleavage site for different proteases for quantitative enzyme activity analysis using high-resolution mass spectrometry (HRMS) . The peptide-encoding technique was further applied by constructing a portable microplate, with the peptide codes immobilized on a microplate .…”

“…82 Our group designed the peptide codes that contained a coding region and a cleavage site for different proteases for quantitative enzyme activity analysis using highresolution mass spectrometry (HRMS). 81 The peptideencoding technique was further applied by constructing a portable microplate, with the peptide codes immobilized on a microplate. 80 Upon cleavage by different target proteases, the coding region of "Protease ID" could be specifically released, identified, and assayed with the MALDI-MS internal standard method (Figure 5A).…”

Novel Method for the Quantitative Analysis of Protease Activity: The Casein Plate Method and Its Applications

“…The analysis of protease activity is an important step in the research and application of protease development and validation of a simple titration method for studying the heat-activated; endogenous protease in arrow tooth flounder fillet mince is described. Protease activity has been detected by the fluorescence method, , but detection based on calorimetry, mass spectrometry (MS), immunoassay, electrophoresis, , amperometry, optics, , and so on.…”