Multiplexing the Quantitation of MAP Kinase Activities Using Differential Sensing

Abstract

Protein kinases are therapeutic targets for many human diseases, but the lack of user-friendly quantitative assays limits the ability to follow the activities of numerous kinases at once (multiplexing). To develop such an assay, we report an array of sulfonamido-oxine (SOX)-labeled peptides showing cross-reactivity to different mitogen-activated protein kinases (MAPKs) for use in a differential sensing scheme. We first verified using linear discriminant analysis that the array could differentiate MAPK isoforms. Then, using principal component analysis, the array was optimized based on the discrimination imparted by each SOX-peptide. Next, the activity of individual MAPK families in ternary mixtures was quantified by support vector machine regression. Finally, we multiplexed the quantification of three MAPK families using partial least squares regression in A549 cell lysates, which has possible interference from other kinase classes. Thus, our method simultaneously quantifies the activity of multiple kinases. The technique could be applied to other protein kinase families and the monitoring of diseases.

One-Component Multichannel Sensor Array for Rapid Identification of Bacteria

“…17−22 Array-based sensing systems that mimic mammalian olfactory and taste systems, which allow for the parallel detection of multiple analytes based on cross-reactive artificial receptors, have attracted extensive attention. 23 Sensor arrays can perform fingerprint recognition of individual analytes according to the differences in physicochemical properties, which is particularly advantageous in distinguishing analytes with similar structures and properties in complex environments. 24−27 At present, several research efforts have been reported regarding array-based detection of multiple pathogenic microorganisms.…”

“…Fluorescent analysis techniques with high sensitivity has been widely applied in the field of sensing. , Conventional sensors based on the “lock and key” mechanism have difficulty in achieving simultaneous detection of multiple analytes, especially for the detection of analytes with highly similar structures in complex media, such as distinguishing multiple bacteria in biological samples. − Array-based sensing systems that mimic mammalian olfactory and taste systems, which allow for the parallel detection of multiple analytes based on cross-reactive artificial receptors, have attracted extensive attention . Sensor arrays can perform fingerprint recognition of individual analytes according to the differences in physicochemical properties, which is particularly advantageous in distinguishing analytes with similar structures and properties in complex environments. − At present, several research efforts have been reported regarding array-based detection of multiple pathogenic microorganisms. − Sensor array-based detection of bacteria covers a variety of recognition mechanisms, such as differences in the composition of volatile gases of bacteria or differences in physicochemical properties of bacteria and their metabolites. − …”

Linear motif specificity in signaling through p38α and ERK2 mitogen-activated protein kinases

“…The copyright holder for this preprint this version posted August 24, 2022. ; https://doi.org/10.1101/2022.08. 23.505039 doi: bioRxiv preprint respectively, the extent to which forward D-sites distinguish between members of these two MAPK subfamilies is less clear 13,30 . In particular, specific features within D-sites conferring selective binding to their cognate MAPKs, and how their respective docking grooves encode specificity, are not known.…”

“…In particular, unique features of the JNK DRS facilitate substantial discrimination between cognate and non-cognate motifs 13,19,21 . While reverse D-sites peptides from the homologous kinases RSK1 and MK2 bind with a high degree of selectivity (>10-fold) to ERK and p38 MAPKs respectively, the extent to which forward D-sites distinguish between members of these two MAPK subfamilies is less clear 13,30 . In particular, specific features within D-sites conferring selective binding to their cognate MAPKs, and how their respective docking grooves encode specificity, are not known.…”

Using a Single Peptide to Electrochemically Sense Multiple Kinases

“…Several platforms for detecting multiple disease biomarkers were developed . These include an electrical device based on antibodies for detecting Alzheimer’s disease-associated proteins, a plasmonic biosensor based on antibodies for detecting bacteria and a spectroscopic method based on fluorophore-labeled peptides for detecting different kinases . The common feature of all these detection methods is that they utilize multiple sensing elements for detecting multiple analytes.…”