Improved Cross-Linking Coverage for Protein Complexes Containing Low Levels of Lysine by Using an Enrichable Photo-Cross-Linker.

Chemical cross-linking coupled with mass spectrometry (XL-MS) is an important technique for the structural analysis of protein complexes where the coverage of amino acids and the identification of cross-linked sites are crucial. Photo-cross-linking has multisite reactivity and is valuable for the structural analysis of chemical cross-linking. However, a high degree of heterogeneity results from this multisite reactivity, which results in samples with higher complexity and lower abundance. Additionally, the applicability of photo-cross-linking is limited to purified protein complexes. In this work, we demonstrate a photo-cross-linker, alkynyl-succinimidyl-diazirine (ASD) with the reactive groups of N-hydroxysuccinimide ester and diazirine, as well as the click-enrichable alkyne group. Photo-cross-linkers can provide higher site reactivity for proteins that contain only a small number of lysine residues, thereby complementing the more commonly used lysine-targeting cross-linkers. By systematically analyzing proteins with differing lysine contents and differing flexibilities, we demonstrated clear enhancement in structure elucidation for proteins containing less lysine and with high flexibility. In addition, enrichment approaches of alkynyl-azide click chemistry conjugated with biotin-streptavidin purification (coinciding with parallel orthogonal digestion) improved the identification coverage of cross-links. We show that this photo-cross-linking approach can be used for membrane proteome-wide complex analysis. This method led to the identification of a total of 14066 lysine-X cross-linked site pairs from a total of 2784 proteins. Thus, this cross-linker is a valuable addition to a photo-cross-linking toolkit and improves the identification coverage of XL-MS in functional structure analysis.

Developing an Affinity-Based Chemical Proteomics Method to In Situ Capture Amorphous Aggregated Proteome and Profile Its Heterogeneity in Stressed Cells.

Stress induced amorphous proteome aggregation is a hallmark for diseased cells, with the proteomic composition intimately associated with disease pathogenicity. Due to its particularly dynamic, reversible, and dissociable nature, as well as lack of specific recognition anchor, it is difficult to capture aggregated proteins in situ. In this work, we develop a chemical proteomics method (AggLink) to capture amorphous aggregated proteins in live stressed cells and identify the proteomic contents using LC-MS/MS. Our method relies on an affinity-based chemical probe (AggLink 1.0) that is optimized to selectively bind to and covalently label amorphous aggregated proteins in live stressed cells. Especially, chaotrope-compatible ligation enables effective enrichment of labeled aggregated proteins under urea denaturation and dissociation conditions. Compared to conventional fractionation-based method to profile aggregated proteome, our method showed improved enrichment selectivity, detection sensitivity, and identification accuracy. In HeLa cells, the AggLink method reveals the constituent heterogeneity of aggregated proteome induced by inhibition of pro-folding (HSP90) or pro-degradation (proteasome) pathway, which uncovers a synergistic strategy to reduce cancer cell viability. In addition, the unique fluorogenicity of our probe upon labeling aggregated proteome detects its cellular location and morphology. Together, the AggLink method may help to expand our knowledge of the previously nontargetable amorphous aggregated proteome.

In-Depth In Vivo Crosslinking in Minutes by a Compact, Membrane-Permeable, and Alkynyl-Enrichable Crosslinker.

Chemical crosslinking coupled with mass spectrometry (CXMS) has emerged as a powerful technique to obtain the dynamic conformations and interaction interfaces of protein complexes. Limited by the poor cell membrane permeability, chemical reactivity, and biocompatibility of crosslinkers, in vivo crosslinking to capture the dynamics of protein complexes with finer temporal resolution and higher coverage is attractive but challenging. In this work, a trifunctional crosslinker bis(succinimidyl) with propargyl tag (BSP), involving compact size, proper amphipathy, and enrichment capacity, was developed to enable better cell membrane permeability and efficient crosslinking in 5 min without obvious cellular interference. Followed by a two-step enrichment method based on click chemistry at the peptide level, 13,098 crosslinked peptides (5068 inter-crosslinked peptides and 8030 intra-crosslinked peptides) were identified under the data threshold of peptide-spectrum matches (PSMs) ≥2 on the basic of the FDR control of 1%, which was the most comprehensive dataset for homo species cells by a non-cleavable crosslinker. Besides, the interactome network comprising 1519 proteins connected by 2913 interaction edges in various intracellular compartments, as well as 80S ribosome structural dynamics, were characterized, showing the great potential of our in vivo crosslinking approach in minutes. All these results demonstrated that our developed BSP could provide a valuable toolkit for the in-depth in vivo analysis of protein-protein interactions (PPIs) and protein architectures with finer temporal resolution.

Surface-Charged Hybrid Monolithic Column for MS-Compatible Peptide Separation with High Peak Capacity and Its Application in Proteomic Analysis.

For bottom-up proteomics, peptide separation with high peak capacity under MS-compatible conditions is of vital significance to increase proteome coverage. Herein, a surface-charged ethane-bridged hybrid monolithic column was prepared based on the efficient ring-opening reaction of N-methyl-aza-2,2,4-trimethyl-silacyclopentane after C18-functionalization. The existence of secondary amino groups on the surface was beneficial to reduce the secondary interactions of silanol groups and increase peak capacity for peptide separation with MS-compatible mobile phases (e.g., using 0.1% FA as the mobile phase modifier). Such columns offered a 4-fold increase in peak capacity compared with ethane-bridged hybrid monolithic columns without surface charge modification. By a 100 cm length surface-charged ethane-bridged hybrid capillary column, high peak capacity of 700 was achieved within a 240 min gradient for the separation of Hela tryptic peptides with 0.1% FA-containing mobile phases, under the low backpressure of ∼200 bar. On average, 44493 ± 459 peptides corresponding to 5148 ± 47 proteins were identified from 750 ng Hela tryptic digests. Finally, the surface-charged ethane-bridged hybrid monolithic column was successfully applied in the quantitative proteomic analysis of dopaminergic neuron death model of N-methyl-4-phenylpyridinium iodide induced SH-SY5Y cells. These results demonstrated great promise of such surface-charged ethane-bridged hybrid monolithic columns for bottom-up proteomic analysis in complex samples.

Alkynyl-Enrichable Carboxyl-Selective Crosslinkers to Increase the Crosslinking Coverage for Deciphering Protein Structures.

The coverage of chemical crosslinking coupled with mass spectrometry (CXMS) is of great importance to determine its ability for deciphering protein structures. At present, N-hydroxysuccinimidyl (NHS) ester-based crosslinkers targeting lysines have been predominantly used in CXMS. However, they are not always effective for some proteins with few lysines. Other amino acid residues such as carboxyl could be crosslinked to complement lysines and improve the crosslinking coverage of CXMS, but the low intrinsic chemical reactivity of carboxyl compromises the application of carboxyl-selective crosslinkers for complex samples. To enhance the crosslinking efficiency targeting acidic residues and realize in-depth crosslinking analysis of complex samples, we developed three new alkynyl-enrichable carboxyl-selective crosslinkers with different reactive groups such as hydrazide, amino, and aminooxy. The crosslinking efficiencies of the three crosslinkers were systematically evaluated, giving the best reactivity of the amino-functionalized crosslinker BAP. Furthermore, BAP was extended to the crosslinking analysis of Escherichia coli lysate in combination with efficient crosslink enrichment. A total of 1291 D/E-D/E crosslinks involved in 392 proteins were identified under a false discovery rate (FDR) of ≤1%. Obvious structural complementarity of BAP was exhibited to the lysine-targeting crosslinker, facilitating the capability of CXMS for protein structure elucidation. To the best of our knowledge, this was the first time for the carboxyl-selective crosslinker to achieve proteome-wide crosslinking analysis of the whole cell lysate. Collectively, we believe that this work not only expands on a promising toolkit of CXMS targeting acidic residues but also provides a valuable guideline to advance the performance of carboxyl-selective crosslinkers.

Moisture-resistant MXene-sodium alginate sponges with sustained superhydrophobicity for monitoring human activities.

Wearable mechanical sensors are easily influenced by moisture resulting in inaccuracy for monitoring human health and body motions. Though the superhydrophobic barrier has been extensively explored as passive water repel strategy on the sensor surface, the dense superhydrophobic surface not only limits the sensor working under large deformations but also inevitable degradation in high humidity or saturation water vapor environments. This work reports a superhydrophobic MXene-sodium alginate sponge (SMSS) pressure sensor with a low voltage Joule heating effect to provide sustain moisture-insensitive property for both sensing performance and superhydrophobicity by heating-driven water molecules away. Because of the positive temperature coefficient under pressure applied, the Joule heating can provides a stable temperature to the moisture-insensitivity property during the whole dynamic pressure cycled. Therefore, the pressure sensor with a simple spray-coating superhydrophobic coating on the outer layer demonstrates key capabilities even in extreme use scenarios with high humidity or water vapor and also provides stable and reliable bio-signal monitoring.

Comparative Proteomic Analysis of Histone Modifications upon Acridone Derivative 8a-Induced CCRF-CEM Cells by Data Independent Acquisition.

The lead compound acridone derivative 8a showed potent antiproliferative activity by inducing DNA damage through direct stacking with DNA bases and triggering ROS in CCRF-CEM cells. To define the chromatin alterations during DNA damage sensing and repair, a detailed quantitative map of single and coexisting histone post-translational modifications (PTMs) in CCRF-CEM cells affected by 8a was performed by the Data Independent Acquisition (DIA) method on QE-plus. A total of 79 distinct and 164 coexisting histone PTMs were quantified, of which 16 distinct histone PTMs were significantly altered when comparing 8a-treated cells with vehicle control cells. The changes in histone PTMs were confirmed by Western blotting analysis for three H3 and one H4 histone markers. The up-regulated dimethylation on H3K9, H3K36, and H4K20 implied that CCRF-CEM cells might accelerate DNA damage repair to counteract the DNA lesion induced by 8a, which was verified by an increment in the 53BP1 foci localization at the damaged DNA. Most of the significantly altered PTMs were involved in transcriptional regulation, including down-regulated acetylation on H3K18, H3K27, and H3K122, and up-regulated di- and trimethylation on H3K9 and H3K27. This transcription-silencing phenomenon was associated with G2/M cell cycle arrest after 8a treatment by flow cytometry. This study shows that the DIA proteomics strategy provides a sensitive and accurate way to characterize the coexisting histone PTMs changes and their cross-talk in CCRF-CEM cells after 8a treatment. Specifically, histone PTMs rearrange transcription-silencing, and cell cycle arrest DNA damage repair may contribute to the mechanism of epigenetic response affected by 8a.

Open-pFind Verified Four Missing Proteins from Multi-Tissues.

The Chromosome-Centric Human Proteome Project (C-HPP) was launched in 2012 to perfect the annotation of human protein existence by identifying stronger evidence of the expression of missing proteins (MPs) at the protein level. After an 8 year effort all over the world, the number of MPs in the neXtProt database significantly decreased from 5511 (2012-02-24) to 1899 (2020-01-17). It is now more difficult to provide confident evidence of the remaining MPs because of their specific characteristics, including low abundance, low molecular weight, unexpected modifications, transmembrane structure, tissue-expression specificity, and so on. A higher resolution mass spectrometry (MS) interpretation engine might provide an opportunity to identify these buried MPs in complex samples by the combination with multi-tissue large-scale proteomics. In this study, open-pFind was used to dig MPs from 20 pairs of healthy human tissues by Wang et al. ( Mol. Syst. Biol. 2019, 15 (2), e8503) combined with our large-scale testis data set digested by three enzymes (Glu-C, Lys-C, and trypsin) with specificity for different amino acid residues ( J. Proteme Res. 2019, 18 (12), 4189-4196). A total of 1 535 536 peptides with 17 283 477 peptide-spectrum matches (PSMs) were mapped to 14 279 protein entries at a false discovery rate of <1% at the PSM, peptide, and protein levels. A total of 103 MP candidates were identified, among which 86 candidates had more unique peptide numbers compared with our single testis tissue. After rigorous screening, manual checks, peptide synthesis, and matching with documented peptides from PeptideAtlas, we validated four MPs, P0C7T8 (duodenum and small intestine), Q8WWZ4 (stomach and rectum), Q8IV35 (fallopian tube), and O14921 (tonsil), at the protein level. All MS raw files have been deposited to the ProteomeXchange with identifier PXD021391.

Adaptive predictive scanning method based on a high-precision automatic microscopy system.

Predicting the focal plane is an effective method to increase the scanning speed of an automatic microscopy system. However, the image easily defocuses when using traditional predictive scanning methods. In this paper, we introduce an adaptive predictive scanning method (APSM) that greatly improves the accuracy of predictive scanning. Instead of using a fixed planar model to predict the focal plane position, APSM updates the predicted focal plane in real time based on the focal position of the reference point during the scanning process, thus predicting the focal position of each local view more accurately. Using the APSM, the average image defocus value is 0.39 µm, while conventional predictive scanning methods reach 1.05 µm. APSM greatly improves focal accuracy and can be applied to a high-precision automatic microscopy system.

Improved autofocus method for human red blood cell images.

This paper presents an improved autofocus method for human red blood cell images in a microscope. The products of the sum modulus difference and the real-valued fast Fourier transform function are multiplied to obtain an improved sharpness evaluation using the properties of a Gaussian function. It is superior to traditional evaluations in terms of unimodality, steepness, and sensitivity. A new quantitative criterion is proposed to represent the ability of sharpness evaluation against noise. An adaptive focus window with great robustness is proposed that can reduce the computation cost and adverse effects of the background. The better performances of the proposed algorithms are all proved by experiment results, and they can help to find the quasi-focus position more quickly and accurately.

Predicting the Prognosis of Bladder Cancer Patients Through Integrated Multi-omics Exploration of Chemotherapy-Related Hypoxia Genes.

Bladder cancer is a prevalent malignancy with high mortality rates worldwide. Hypoxia is a critical factor in the development and progression of cancers. However, whether and how hypoxia-related genes (HRGs) could affect the development and the chemotherapy response of bladder cancer is still largely unexplored. This study comprehensively explored the complex molecular landscape associated with hypoxia in bladder cancer by analyzing 260 hypoxia genes based on transcriptomic and genomic data in 411 samples. Employing the 109 dysregulated hypoxia genes for consensus clustering, we delineated two distinct bladder cancer clusters characterized by disparate survival outcomes and distinct oncogenic roles. We defined a HPscore that was correlated with a variety of clinical features, including TNM stages and pathologic grades. Tumor immune landscape analysis identified three immune clusters and close interactions between hypoxia genes and the various immune cells. Utilizing a network-based method, we defined 129 HRGs exerting influence on apoptotic processes and critical signaling pathways in cancer. Further analysis of chemotherapy drug sensitivity identified potential drug-target HRGs. We developed a Risk Score model that was related to the overall survival of bladder cancer patients based on doxorubicin-target HRGs: ACTG2, MYC, PDGFRB, DHRS2, and KLRD1. This study not only enhanced our understanding of bladder cancer at the molecular level but also provided promising avenues for the development of targeted therapies, representing a significant step toward the identification of effective treatments and addressing the urgent need for advancements in bladder cancer management.

Interindividual- and blood-correlated sweat phenylalanine multimodal analytical biochips for tracking exercise metabolism.

In situ monitoring of endogenous amino acid loss through sweat can provide physiological insights into health and metabolism. However, existing amino acid biosensors are unable to quantitatively assess metabolic status during exercise and are rarely used to establish blood-sweat correlations because they only detect a single concentration indicator and disregard sweat rate. Here, we present a wearable multimodal biochip integrated with advanced electrochemical electrodes and multipurpose microfluidic channels that enables simultaneous quantification of multiple sweat indicators, including phenylalanine and chloride, as well as sweat rate. This combined measurement approach reveals a negative correlation between sweat phenylalanine levels and sweat rates among individuals, which further enables identification of individuals at high metabolic risk. By tracking phenylalanine fluctuations induced by protein intake during exercise and normalizing the concentration indicator by sweat rates to reduce interindividual variability, we demonstrate a reliable method to correlate and analyze sweat-blood phenylalanine levels for personal health monitoring.

A Zero-Voltage-Writing Artificial Nervous System Based on Biosensor Integrated on Ferroelectric Tunnel Junction.

The artificial nervous system proves the great potential for the emulation of complex neural signal transduction. However, a more bionic system design for bio-signal transduction still lags behind that of physical signals, and relies on additional external sources. Here, this work presents a zero-voltage-writing artificial nervous system (ZANS) that integrates a bio-source-sensing device (BSSD) for ion-based sensing and power generation with a hafnium-zirconium oxide-ferroelectric tunnel junction (HZO-FTJ) for the continuously adjustable resistance state. The BSSD can use ion bio-source as both perception and energy source, and then output voltage signals varied with the change of ion concentrations to the HZO-FTJ, which completes the zero-voltage-writing neuromorphic bio-signal modulation. In view of in/ex vivo biocompatibility, this work shows the precise muscle control of a rabbit leg by integrating the ZANS with a flexible nerve stimulation electrode. The independence on external source enhances the application potential of ZANS in robotics and prosthetics.

Fast autofocus method for piezoelectric microscopy system for high interaction scenes.

Piezoelectric objective driver positioners are increasingly used in the field of microscopy. They have the advantages of high dynamic and fast response. This paper presents a fast autofocus algorithm for highly interactive microscope system. First, the Tenengrad gradient of the down-sampled image is used to calculate the image sharpness, and Brent search method is adopted to quickly converge to the correct focal length. At the same time, the input shaping method is used to eliminate the displacement vibration of the piezoelectric objective lens driver and further accelerate the image acquisition speed. Experimental results show that the proposed scheme can improve the speed of the automatic focusing task of the piezoelectric objective driver and improve the real-time focus of the automatic microscopic system. HIGHLIGHTS: A high real-time autofocus strategy. A vibration control method suitable for a piezoelectric objective driver.

Urinary protein biomarkers based on LC-MS/MS analysis to discriminate vascular dementia from Alzheimer's disease in Han Chinese population.

Objective: This study aimed to identify the potential urine biomarkers of vascular dementia (VD) and unravel the disease-associated mechanisms by applying Liquid chromatography tandem-mass spectrometry (LC-MS/MS). Methods: LC-MS/MS proteomic analysis was applied to urine samples from 3 groups, including 14 patients with VD, 9 patients with AD, and 21 normal controls (NC). By searching the MS data by Proteome Discoverer software, analyzing the protein abundances qualitatively and quantitatively, comparing between groups, combining bioinformatics analysis using Gene Ontology (GO) and pathway crosstalk analysis using Kyoto Encyclopedia of Genes and Genomes (KEGG), and literature searching, the differentially expressed proteins (DEPs) of VD can be comprehensively determined at last and were further quantified by receiver operating characteristic (ROC) curve methods. Results: The proteomic findings showed quantitative changes in patients with VD compared to patients with NC and AD groups; among 4,699 identified urine proteins, 939 and 1,147 proteins displayed quantitative changes unique to VD vs. NC and AD, respectively, including 484 overlapped common DEPs. Then, 10 unique proteins named in KEGG database (including PLOD3, SDCBP, SRC, GPRC5B, TSG101/STP22/VPS23, THY1/CD90, PLCD, CDH16, NARS/asnS, AGRN) were confirmed by a ROC curve method. Conclusion: Our results suggested that urine proteins enable detection of VD from AD and VC, which may provide an opportunity for intervention.

Super enhancer-driven core transcriptional regulatory circuitry crosstalk with cancer plasticity and patient mortality in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a clinically aggressive subtype of breast cancer. Core transcriptional regulatory circuitry (CRC) consists of autoregulated transcription factors (TFs) and their enhancers, which dominate gene expression programs and control cell fate. However, there is limited knowledge of CRC in TNBC. Herein, we systemically characterized the activated super-enhancers (SEs) and interrogated 14 CRCs in breast cancer. We found that CRCs could be broadly involved in DNA conformation change, metabolism process, and signaling response affecting the gene expression reprogramming. Furthermore, these CRC TFs are capable of coordinating with partner TFs bridging the enhancer-promoter loops. Notably, the CRC TF and partner pairs show remarkable specificity for molecular subtypes of breast cancer, especially in TNBC. USF1, SOX4, and MYBL2 were identified as the TNBC-specific CRC TFs. We further demonstrated that USF1 was a TNBC immunophenotype-related TF. Our findings that the rewiring of enhancer-driven CRCs was related to cancer immune and mortality, will facilitate the development of epigenetic anti-cancer treatment strategies.

A dynamic parallel image acquisition method for slide scanning process.

Automated microscope systems have played an important role in the screening of numerous diseases. However, it is a very time-consuming process to continuously acquire images under the high magnification objective lens. This paper proposes a dynamic parallel image acquisition method, which can greatly improve image acquisition speed. Due to the relative motion between the x-y stage and the camera, some of the captured images have motion blur To this end, we also designed a motor variable speed motion curve to ensure the quality of the collected images. The experimental results show that the traditional image scanning mode needs 47.3 ms to obtain continuous microscopic images, while the dynamic parallel image acquisition method only needs 25.4 ms, which improves the acquisition speed without affecting the clarity of the acquired images.

A Novel Feedforward Model of Piezoelectric Actuator for Precision Rapid Cutting.

The piezoelectric actuator has been widely used in modern precision cutting technology due to its fast response speed and high positioning accuracy. In recent years, with the development of precision technology, modern cutting requires higher and higher cutting accuracy and efficiency. Therefore, this paper proposes a feedforward control method based on the modified Bouc-Wen (MBW) model. Firstly, a novel asymmetrical modified Bouc-Wen model with an innovative form of shape control function is developed to model the hysteresis nonlinearity property of piezoelectric actuators. Then, a self-adaptive cooperative particle swarm optimization (PSO) algorithm is developed to identify the parameters of MBW model. The comparative evaluation reveals that the MBW model outperforms the classical Bouc-Wen (CBW) model by 66.4% in modeling accuracy. Compared with traditional PSO algorithm, the self-adaptive cooperative PSO algorithm can obtain minimum fitness in parameter identification. Furthermore, the feedforward control strategy is realized to improve the position tracking accuracy. A position tracking experiment verifies that the feedforward control strategy improves the tracking accuracy of piezoelectric actuators significantly compared with the open-loop control strategy.

Modeling of Rapid Response Characteristics of Piezoelectric Actuators for Ultra-Precision Machining.

Piezoelectric actuators are characterized by high positioning accuracy, high stiffness and a fast response and are widely used in ultra-precision machining technologies such as fast tool servo technology and ultrasonic machining. The rapid response characteristics of piezoelectric actuators often determine the overall quality of machining. However, there has been little research on the fast response characteristics of piezoelectric actuators, and this knowledge gap will lead to low precision and poor quality of the final machining. The fast response characteristics of a piezoelectric actuator were studied in this work. Firstly, the piezoelectric actuator was divided into a no-load state and a load state according to the working state. A fast response analysis and output characteristic analysis were carried out, the corresponding dynamic model was established, and then the model was simulated. Finally, an experimental system was established to verify the dynamic model of the piezoelectric actuator's fast response by conducting an experiment in which the piezoelectric actuator bounces a steel ball. The experimental results verify the correctness of the model and show that the greater the cross-sectional area and height of the piezoelectric actuator, the higher the bouncing height of the ball, and the better the dynamic performance of the piezoelectric actuator. It is believed that this study has guiding significance for the application of the dynamic characteristics of piezoelectric actuators in the machining field.

In vivo cross-linking-based affinity purification and mass spectrometry for targeting intracellular protein-protein interactions.

Comprehensive interactome analysis of targeted proteins is important to understand how proteins work together in regulating functions. Commonly, affinity purification followed by mass spectrometry (AP-MS) has been recognized as the most often used technique for studying protein-protein interactions (PPIs). However, some proteins with weak interactions, which are responsible for key roles in regulation, are easily broken during cell lysis and purification through an AP approach. Herein, we have developed an approach termed in vivo cross-linking-based affinity purification and mass spectrometry (ICAP-MS). By this method, in vivo cross-linking was introduced to covalently fix intracellular PPIs in their functional states to assure all PPIs could be integrally maintained during cell disruption. In addition, the chemically cleavable crosslinkers which were employed enabled unbinding of PPIs for in-depth identification of components within the interactome and biological analysis, while allowing binding of PPIs for cross-linking-mass spectrometry (CXMS)-based direct interaction determination. Multi-level information on targeted PPIs network can be obtained by ICAP-MS, including composition of interacting proteins, as well as direct interacting partners and binding sites. As a proof of concept, the interactome of MAPK3 from 293A cells was profiled with 6.15-fold improvement in identification than by conventional AP-MS. Meanwhile, 184 cross-link site pairs of these PPIs were experimentally identified by CXMS. Furthermore, ICAP-MS was applied in the temporal profiling of MAPK3 interactions under activation by cAMP-mediated pathway. The regulatory manner of MAPK pathways was presented through the quantitative changes of MAPK3 and its interacting proteins at different time points after activation. Therefore, all reported results demonstrated that the ICAP-MS approach may provide comprehensive information on interactome of targeted protein for functional exploration.