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Hou, Z.; Liu, H. Mapping the Protein Kinome. Encyclopedia. Available online: https://encyclopedia.pub/entry/43469 (accessed on 13 July 2025).
Hou Z, Liu H. Mapping the Protein Kinome. Encyclopedia. Available at: https://encyclopedia.pub/entry/43469. Accessed July 13, 2025.
Hou, Zhanwu, Huadong Liu. "Mapping the Protein Kinome" Encyclopedia, https://encyclopedia.pub/entry/43469 (accessed July 13, 2025).
Hou, Z., & Liu, H. (2023, April 25). Mapping the Protein Kinome. In Encyclopedia. https://encyclopedia.pub/entry/43469
Hou, Zhanwu and Huadong Liu. "Mapping the Protein Kinome." Encyclopedia. Web. 25 April, 2023.
Mapping the Protein Kinome
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This entry is adapted from the peer-reviewed paper 10.3390/cells12060925

The kinome includes over 500 different protein kinases, which form an integrated kinase network that regulates cellular phosphorylation signals. The kinome plays a central role in almost every cellular process and has strong linkages with many diseases. Thus, the evaluation of the cellular kinome in the physiological environment is essential to understand biological processes, disease development, and to target therapy. A number of strategies for kinome analysis have been developed, which are based on monitoring the phosphorylation of kinases or substrates. They have enabled researchers to tackle increasingly complex biological problems and pathological processes, and have promoted the development of kinase inhibitors. Additionally, with the increasing interest in how kinases participate in biological processes at spatial scales, it has become urgent to develop tools to estimate spatial kinome activity. With multidisciplinary efforts, a growing number of novel approaches have the potential to be applied to spatial kinome analysis. 

kinome phosphorylation activity assay spatial

1. Introduction

Protein kinases (PKs) are key regulators of cell function, and they are also one of the largest and most functionally diverse gene families [1]. Humans contain around 560 PKs, of which about 500 eukaryotic PKs are divided into eight major groups and about 60 atypical PKs [2]. PKs catalyze the transfer of the γ phosphate group from ATP to a protein acceptor, mainly to serine, threonine, and tyrosine residues. Then, the negatively or positively charged amino acids, which are close to the phosphorylated residues, will be repulsed or attracted, respectively. The phosphate group will introduce a very hydrophilic and polar region in the affected protein. It may lead to conformational changes in the proteins, altering their functions or interactions with other biomolecules [3]. The phosphorylation network mediated by PKs is involved in nearly all eukaryotic cellular pathways. Thus, PKs are crucial to cell growth and development [4]. Dysregulation of kinase signaling, therefore, leads to cancers and a variety of other human disorders, including neurological, metabolic, cardiovascular, infectious, and immunological diseases [5][6]. As a result, PKs have been recognized as the most attractive pharmaceutical targets. Indeed, an increasing number of kinase inhibitors have been developed and used for cancer therapy [7].
There are currently 72 small molecule kinase inhibitors [8] approved by the FDA for the treatment of disease (Figure 1). Unfortunately, fewer than 10% of all PKs are targeted by Food and Drug Administration (FDA)-approved inhibitors. Some challenges limit the full potential of kinases as drug targets, including validating novel kinase targets [9], obtaining target selectivity to reduce off-target-mediated toxicity [10], and developing efficient compound screening technologies [11]. Overcoming these limitations relies on sensitive kinase activity detection to discover new targets, to profile inhibitor selectivity, and to perform inhibitor screening [12]. With the efforts of academia and industry, sensitivity and high-throughput detection of most kinase activities have been achieved by monitoring the phosphorylation of substrate peptides with a variety of sensitive detection methods, such as fluorescence, electrochemistry, and absorbance [13][14].
/media/item_content/202304/6448e1bd74d13cells-12-00925-g001.png
Figure 1. US Food and Drug Administration (FDA)-approved small molecular inhibitors that target kinases.

2. Technologies for Kinome Analysis

The kinome profile correlates with the inhibitor design and therapy strategy. Therefore, numerous techniques have been developed to compare kinome activities in normal and aberrant tissues. Such techniques include a kinome peptide library based on kinase substrates, kinome enrichment that relies on kinase inhibitor conjugated beads, a reactive chemical probe for kinome competition labelling, and kinome activity-representing phosphorylation sites. Each method has its own applications and limitations, but it is certain that a combination of these techniques can cover a larger portion of the kinome than any technique alone.

2.1. Kinome Substrate Peptide Library

Monitoring kinome substrate library phosphorylation sensitively is quite an effective way to represent kinome activity [15]. As the physiological substrates of most kinases are proteins with considerable diversity, this poses a serious challenge in constructing a stable kinase substrate library. Most kinases recognize and modify their targets based on residues near the phosphorylation site through highly conserved catalytic mechanisms, which is largely in the disordered region. In particular, the maximum reaction rate (Vmax) and Michaelis–Menten constant (Km) of the catalytic reaction with peptide mimic kinase substrates were close to those of native protein substrates [16]. This makes it possible to monitor kinase activity using short peptides of specific sequence as alternative substrates. The use of peptides for kinome analysis has great advantages such as simple synthesis, low cost, and high chemical stability. Based on these advantages, the use of a kinome substrate peptide library (KsPL) to quantify kinome activity has been promoted [17][18].
There are a variety of kinome detection methods based on KsPL. They can be classified into three categories: planar peptide array, 3D peptide array, and an in-solution peptide library (Figure 2) [15]. The planar peptide array refers to kinome detection, which is formed by attaching peptides to a planar physical support. To evaluate the reproducibility of the technique, each peptide is printed as multiple spots. The 3D peptide array is the peptide library immobilized on a 3D support [19]. Compared to the conventional planar array, the 3D support provides a larger surface area for peptide presentation and a higher reaction rate, improving the sensitivity and accuracy of the kinome activity assessment [20][21]. The activated alumina surface used as a support for the 3D array is called PamChip [22]. This technique has been applied for a number of important studies including kinome profiling of endothelial inflammation [23], mechanisms of drug resistance [24] and druggable targets identification for cholangiocarcinoma [25]. The disadvantage is that it displays fewer spots compared to a planar array, which has more than 1000 spots. An in-solution peptide library is another option for assessing kinome activity.
/media/item_content/202304/6448e1ebdaec4cells-12-00925-g002.png
Figure 2. Overview of kinome profiling approaches based on the substrate peptide library.
Designing a proper peptide library for kinome analysis is one of the most crucial aspects in ensuring its accuracy. The proper peptide can be screened via one-bead-one-compound [26], phage display [27], or PeSA [28]. The PeSA is a software tool, which uses the peptide array data for sequence analysis. It has the potential for substrate peptide generation. These design methods can generate suitable peptides without the limitation of known sequences. However, they are time-consuming and labor-intensive and this limits their widespread application. The peptide library information can also be obtained from previous phosphoproteome investigations, or from publicly available databases such as PhosphoSitePlus [29] or PhosphoELM [30]. However, online databases contain many phosphorylation sites for the human and mouse, but have only limited information available for other species. Besides, phosphorylation sites are not fully conserved between human and other species. Thus, many computational tools have been developed to enable the prediction of phosphorylation events based on phosphorylation events described in other species with a similar sequence [31].

2.2. Kinase Inhibitor Conjugated Beads

Using kinase inhibitor conjugated beads to enrich PKs from total cell extracts is an important way to improve the sensitivity of the kinome analysis. It has been used for the simultaneous enrichment of the kinome (Figure 3) [32]. By immobilizing multiple pan-kinase inhibitors with different specificities and affinities on agarose beads, kinase inhibitor conjugated beads enabled the capture of a large proportion of the kinome in a single experiment. Then, the captured kinome was subsequently eluted and identified by MS or Western blotting [33]. Many kinase inhibitors have been derivatized to facilitate rapid coupling to modified agarose or other supports without losing binding groups. In addition, inhibitor bead compositions have been customized to meet the needs of specific experiments [34].
/media/item_content/202304/6448e216ee544cells-12-00925-g003.png
Figure 3. Workflow of kinome enrichment. Protein lysate is passed through an inhibitor conjugated beads column. The activated kinases are enriched and eluted in the column.
The selection of inhibitors or inhibitor derivatives is essential to improve the depth of the kinome assay. Kinase capture with CDK or p38 inhibitors has been used to identify CDK or p38-associated kinases. Some novel intracellular targets can also been identified by that method [35][36]. Unfortunately, only 30 kinases have been identified. An improvement in the original kinase capture has resulted in a multichannel inhibitor beads (MIBs) approach, which uses individual, layered, immobilized kinase inhibitors in a column [37]. The use of a layer-wise approach allows more abundant kinases to be bound by highly selective inhibitors, providing additional binding “space” for lower abundance kinases. Thus, the kinome detection depth can be further improved. The original components of MIBs from top to bottom are Bisindoylmaleimide-X, SB203580, Lapatinib, Dasatinib, Purvalanol B, VI16832, and PP58 [38]. This approach has revealed 50–60% of expressed kinome activity, and identified a reprogramming of the kinome in response to MEK inhibitors.
Several quantification methods for an enriched kinome have been explored and which contributed to a wide application of kinase inhibitor conjugated beads. Data dependent acquisition (DDA) is a typical mode used for kinome quantification, and around 110–125 kinases can be assayed in any single LC-MS/MS run [39]. In addition, isobaric tags, such as TMT and iTRAQ, depending on DDA mode, have been used for the relative quantitation of downstream MIBs enrichment [40]. SILAC labeling based on DDA is also an alternative means of quantification and has been used in the study of kinases associated with the cell cycle [41]. However, despite kinome enrichment being performed, quantification based on DDA mode still suffers from highly complex peptide mixtures. In addition, it is not conducive to the quantitative detection of low abundance kinases. Data independent acquisition (DIA) via Sequential Window Acquisition of all Theoretical fragment ions (SWATH) can improve the detection of low-abundance proteins. SWATH increases the number of kinase identifications relative to DDA [42]. DIA via parallel reaction monitoring (PRM), which is a targeted proteomic methodology, allows for the quantitation of a selected set of target peptides with an improved sensitivity in detection. A PRM strategy, which exploits meter-scale monolithic silicone-C18 column chromatography, has demonstrated the reliable quantification of more than 150 kinases in a single run [43]. The kinome detection coverage of each method is often not identical, so the combination of multiple methods can often further increase kinome detection [44].

2.3. Chemical Reactive Probe

The chemical probe labeling strategy is a widely used method in kinome analysis [45]. Such chemical probes used in the kinome assay generally have three main functions, which are the selectivity function—to recognize the functional domain of the target protein; reactivity function—forms irreversible covalent binding with the target protein after activation; sorting function—enables the capture of the kinome from complex protein mixtures (Figure 4a) [46]. This labeling method has a wide application in the purification, enrichment and identification of the kinome [47]. The most commonly used probe for kinome analysis is the desthiobiotin-ATP affinity probe (Figure 4b) [48]. The design of this probe is based on two important characteristics of kinase. One is that most kinases contain an ATP-binding domain that recognizes ATP specifically and broadly. The other is that most kinases have at least one conserved lysine residue within the active site indicated by sequence comparisons [49]. Therefore, the desthiobiotin-ATP recognizes the ATP-binding domain of the kinase and binds to the pocket. The acyl phosphate reactive group of the probe will be close and react with the lysine residue to yield a stable amide bond, which results in the covalent attachment of desthiobiotin together with a linker to the kinase. Finally, the bound peptides or proteins in the composite samples are enriched by streptavidin resin and identified by MS [50]. The probe was first reported in 2006 and the ATP analogue FSBA was used for target recognition. A total of 132 proteins were identified by the probe, of which 6 were protein kinases [51].
/media/item_content/202304/6448e236a7688cells-12-00925-g004.png
Figure 4. The principle of a chemical reactive probe. (a) Depiction of schematic kinome capture compounds; (b) A schematic diagram showing the conjugation between reactive ATP affinity probes with an ATP-binding protein including kinase.

2.4. Kinome Activity-Representing Phosphorylation Sites

The conformational changes in the functional domain of kinases can be caused by the phosphorylation of the key amino acid residues, which results in activity alteration. Thus, kinase activity can be reflected by quantifying kinome activity-representing phosphorylation sites to some extent (Figure 5) [52]. Phospho-specific antibody-based kinase assays can quantify kinase activity. Unfortunately, the throughput is too low. An antibody-based human phospho-kinase array enables a kinome high-throughput assay through a membrane-based sandwich immunoassay [53]. However, the antibody-dependent detection effect is closely related to the quality of the antibody, and the availability is limited to well-studied kinases. An alternative approach to phosphorylation sites detection is the MS-based large-scale phosphoproteomics study. Due to the low abundance of the corresponding peptides, few sites can be accurately quantified. Thus, PRM is used for quantifying the kinome activity-representing phosphorylation sites. It is sensitive and accurate in low abundance peptide analysis, and has achieved the activation profiles of 178 kinases in various biology samples [54]. Tyrosine kinases (TKs) are the main targets of kinase inhibitors; however, the coverage of the above methods on TK is limited.
/media/item_content/202304/6448e256b9d40cells-12-00925-g005.png
Figure 5. Quantification of kinase activity-representing phosphorylation sites to determine kinome activity. Antibody-based kinase array and target MS-based methods are used for the quantification of phosphorylated kinase.

3. Potential Strategies in Kinase Spatial Assay

Kinase function is closely related to its spatial localization [55]. Alterations in the spatial localization are involved in most cellular biological processes, such as nucleocytoplasmic shuttling of p38 [56] and endocytic uptake of receptor tyrosine kinase [57]. Mislocalization of kinases is frequently associated with cellular dysfunction and diseases, including neurodegeneration, cancer and metabolic disorders [58]. Therefore, a tight control of kinase localization is an important regulatory component of cell physiology. Understanding the spatial distribution of kinases is essential for fully revealing the cell’s biology. However, traditional kinome analysis omits the spatial information in the process of tissue homogenization. The final obtained kinome information is the average level of kinase activity in the tissues or cell lysates [59]. However, it cannot reveal the differences in the spatial distribution of the kinome. In situ protein images based on FRET, confocal, or staining are effective methods for determining kinase localization, but only for individual kinases [60].

3.1. Proximity Labeling

Proximity labeling (PL), an advanced method for protein–protein interaction analysis, has a potential application in spatial kinome analysis. Conceptually, since the interacting proteins must be located in the same spatial location, the interaction of the kinase with the substrate can be considered as a “local” spatial proteome. They participate in various cellular processes at different spatiotemporal levels, such as in cell cycle regulation, protein synthesis and secretion, signal transduction and metabolism, and stress response [61]. Thus, a more detailed and extensive coverage of the protein kinase interactome would bring a better understanding of the biological processes and spatial localization involved in PKs. Some traditional methods for PKs’ interactome analysis, including co-immunoprecipitation (Co-IP) [62] and kinase–substrates crosslinking [63], are limited to stable complexes, or a relatively high affinity, rather than a transient or weak protein–protein interaction. PL, a novel technology for protein interactome analysis, has been developed to be directly performed in living cells under natural conditions [64]. It is conducive to the identification of hydrophobic and low-abundance protein interactions and the analysis of weak or transient protein interactions. It helps researchers better understand the complex kinase interactome and provides insights into kinase spatial information. The principle is to fuse the kinase with a tool enzyme that has a specific catalytic ligation activity. The PL enzyme is able to activate the small molecule substrate, which labels proteins with a sorting moiety (Figure 6) [65]. The labeled proteins are candidate kinase substrates, which are often difficult to be captured by conventional methods. It should be noted that complementary methods must be used to determine whether the interactors are true substrates when using PL.
/media/item_content/202304/6448e2714050ecells-12-00925-g006.png
Figure 6. Kinase–substrate interaction analysis based on proximity labeling. PL enzymes are fused to the kinase of interest, which is expressed in the cells. The PL enzymes catalyze biotin-phenol or biotin into reactive biotin, which diffuses and labels proximal proteins.

3.2. Low Cell Numbers Proteomics

The method of low cell number proteomics contributes to spatial-resolved kinases [66]. Multicellular organisms contain specialized cell types with unique functions and morphological features. The specialization is caused by differences in protein expression [67]. Different cell states may respond differently to the same exogenous signal. Even within the same cell type, the response may differ. Therefore, the spatial and cell type information can be linked with the kinome by analyzing a small number of cells. The spatial kinome can provide insight into the tissue microenvironment and uncover more precise biomarkers and new functional mechanisms [68]. Unlike single-cell analysis at the genomic and transcriptomic level, low cell number proteomic analysis cannot rely on techniques that allow amplifying trace amounts of protein [69]. Therefore, massive innovations and technologies [70] have been developed and applied in sample preparation and mass spectrometry detection, which make kinome analysis on paucicellular samples possible (Figure 7).
/media/item_content/202304/6448e27e616becells-12-00925-g007.png
Figure 7. Schematic workflow of the low cell number proteome method. Samples are collected from tissue or cells via LCAM or FACS, then treated in a single pot with three main steps—cell lysis, reduction and alkylation, and digestion. Peptides are detected by the LC-MS/MS with a sensitivity data acquisition mode, such as BOOST and SWATH.

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Subjects: Biochemistry & Molecular Biology
Contributors MDPI registered users' name will be linked to their SciProfiles pages. To register with us, please refer to https://encyclopedia.pub/register :
Zhanwu Hou
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Huadong Liu
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Update Date: 26 Apr 2023
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