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

Abstract

Mass spectrometry (MS) has been widely used for enzyme activity assays. Herein, we propose a MALDI-MS patterning strategy for the convenient visual presentation of multiple enzyme activities with an easy-to-prepare chip. The array-based caspase-activity patterned chip (Casp-PC) is fabricated by hydrophobically assembling different phospholipid-tagged peptide substrates on a modified ITO slide. The advantages of amphipathic phospholipids lead to high-quality mass spectra for imaging analysis. Upon the respective cleavage of these substrates by different caspases, such as caspase-1, -2, -3, and -8, to produce a mass shift, the enzyme activities can be directly evaluated by MALDI-MS patterning by m/z-dependent imaging of the cleavage products. The ability to identify drug-sensitive/resistant cancer cells and assess the curative effects of anticancer drugs is demonstrated, indicating the applicability of the method and the designed chip.

Sulfur-Doped Graphene-Based Immunological Biosensing Platform for Multianalysis of Cancer Biomarkers

“…Zhu and co-workers reported a multianalyte electrochemical immunoassay based on metal-ion-functionalized titanium phosphate nanospheres as labels . All of these methods can achieve good performance due to the usage of nanomaterials in the immunoassay, − and biomolecule interactions can exhibit much tightly bonded chemical bonds that is a benefit to the immunoassay fabrication . On the basis of above considerations, the biomolecule-interaction-based immunological biosensing platform for multianalysis can achieve good performance; however, this has not been reported before.…”

In Situ Imaging of Cellular Reactive Oxygen Species and Caspase-3 Activity Using a Multifunctional Theranostic Probe for Cancer Diagnosis and Therapy

“…8−10 Therefore, direct intracellular detection and monitoring of multiple apoptotic target molecules related to apoptotic pathways are quite significant for better understanding the mechanism of cell apoptosis and the further clinical research of apoptosis-targeted cancer therapy. 11,12 So far, various strategies have been reported for detecting apoptotic target molecules, including mass spectrometry, 13 electrochemistry, 14 and chemiluminescence. 15 However, most of these studies are capable of detection of a single apoptotic target molecule, which is usually ruinous and incapable for in situ detection and acquires spatiotemporal variation and distribution information in living cells.…”

“…For example, excessive production of reactive oxygen species (ROS) can activate the apoptotic-signaling pathway and irreversibly destroy macromolecules in cells such as gene expression, lipids, proteins, or DNA, ultimately leading to cell apoptosis. − Caspase-3 is a family of cysteine proteases that catalyze the proteolysis of peptide bonds and initiate specific degradation of many key cellular proteins, which play an essential role in apoptosis pathways, and has been identified as a key mediator of apoptosis. Many therapeutic modalities will result in the activation of caspase-3 from the dormant by proteolytic cleavage in the cytosol. , The apoptotic-signaling pathway is an energy-dependent molecular cascade reaction regulated by the changes and interactions of different apoptotic target molecules (such as ROS, caspases, p53, ATP, and cytochrome C) in cells. − Therefore, direct intracellular detection and monitoring of multiple apoptotic target molecules related to apoptotic pathways are quite significant for better understanding the mechanism of cell apoptosis and the further clinical research of apoptosis-targeted cancer therapy. , So far, various strategies have been reported for detecting apoptotic target molecules, including mass spectrometry, electrochemistry, and chemiluminescence . However, most of these studies are capable of detection of a single apoptotic target molecule, which is usually ruinous and incapable for in situ detection and acquires spatiotemporal variation and distribution information in living cells. , Besides this, both of the fluorescence optical imaging and localized surface plasmon resonance (LSPR) techniques are powerful methods for in situ monitoring of intracellular molecules with high sensitivity and signal-to-noise ratio and suitable for multiple apoptotic target molecules sensing in live cell studies. − …”

Protease-responsive mass barcoded nanotranslators for simultaneously quantifying the intracellular activity of cascaded caspases in apoptosis pathways

“…19 Characterized by high mass resolution and outstanding qualitative and quantitative precision, the mass spectrometry (MS)-based strategy may be able to overcome the above limitation in multiplex protease assays. [20][21][22][23] Since MS is an invasive analytical method for characterizing living cells, thus far most MS-based approaches towards enzyme assay have been restricted to cell extracts, rather than the intracellular physiological environment. 20,[23][24][25] Due to the difference in proteolytic reaction conditions, the activity of proteases measured in cell lysates cannot reveal their actual function and behavior in cells.…”

“…[20][21][22][23] Since MS is an invasive analytical method for characterizing living cells, thus far most MS-based approaches towards enzyme assay have been restricted to cell extracts, rather than the intracellular physiological environment. 20,[23][24][25] Due to the difference in proteolytic reaction conditions, the activity of proteases measured in cell lysates cannot reveal their actual function and behavior in cells. A proteomics experiment could prole cellular proteolytic processing events at the level of neo-N-terminal peptides, but tedious pretreatment procedures and complicated data analysis are always unavoidable.…”