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In the present study, click chemistry and Schiff base reactions were simultaneously applied to prepare polymer brush (PEG)–functionalized MOF materials (UiO-66-NH2) and immobilized with Ti4+ (MOF-Brush-THBA-Ti4+) for phosphopeptide analysis. The material has a detection limit of 0.5 fmol, a selectivity of 2000:1, and a loading capacity of 133 mg/g for phosphopeptides. It also demonstrated great repeatability (10 cycles) and recovery rate (96.7 ± 1.4%). During the analysis of bio-samples, 4 specific phosphopeptides were identified in endogenous breast cancer serum, while 11 phosphopeptides were identified in skimmed milk. Moreover, 47 phosphopeptides correlated with 29 phosphorylated proteins were selectively identified from normal control serum, and 66 phosphopeptides correlated with 26 phosphorylated proteins were identified from breast cancer serum. Further analysis of gene ontology (GO) revealed that the detected phosphorylated proteins associated with breast cancer included positive regulation of receptor-mediated endocytosis, proteolysis, extracellular exosome, heparin binding, and chaperone binding. These findings suggest that these associated pathways might contribute to the etiology of breast cancer. Overall, this application exhibits enormous potential in the identification of phosphorylated peptides within bio-samples. [ABSTRACT FROM AUTHOR]

A porphyrin-based titanium-rich porous organic polymer (Th-PPOPs@Ti4+) was designed based on immobilized metal ion affinity chromatography technique and successfully applied to phosphopeptide enrichment with 5,10,15,20-tetrakis(4-carboxyphenyl) porphine tetramethyl ester (TCPTE), 2,3-dihydroxyterephthalaldehyde (DHTA), and 2,3,4-trihydroxybenzaldehyde (THBA) as raw materials. Th-PPOPs@Ti4+ exhibited remarkable sensitivity (0.5 fmol), high selectivity (β-casein: BSA = 1:2000, molar ratio), outstanding recovery (95.0 ± 1.9%), reusability (10 times), and superior loading capacity (143 mg·g−1). In addition, Th-PPOPs@Ti4+ exhibited excellent ability to specifically capture phosphopeptides from the serum of colorectal cancer (CRC) individuals and normal subjects. Sixty phosphopeptides assigned to 35 phosphoproteins were obtained from the serum of CRC individuals, and 43 phosphopeptides allocated to 28 phosphoproteins were extracted in the serum of healthy individuals via nano-LC–MS/MS. Gene ontology assays revealed that the detected phosphoproteins may be inextricably tied to CRC-associated events, including response to estrogen, inflammatory response, and heparin binding, suggesting that it is possible that these correlative pathways may be implicated in the pathogenesis of CRC. [ABSTRACT FROM AUTHOR]

The reasonable design of metal–organic framework (MOF)–derived nanomaterial has important meaning in increasing the enrichment efficiency in the study of protein phosphorylation. In this work, a polyoxometalate (POM) functionalized magnetic MOF nanomaterial (Fe3O4@MIL-125-POM) was designed and fabricated. The nanomaterial with multi-affinity sites (unsaturated metal sites and metal oxide clusters) was used for the enrichment of phosphopeptides. Fe3O4@MIL-125-POM had high-efficient enrichment performance towards phosphopeptides (selectivity, a mass ratio of bovine serum albumin/α-casein/β-casein at 5000:1:1; sensitivity, 0.1 fmol; satisfactory repeatability, ten times). Furthermore, Fe3O4@MIL-125-POM was employed to enrich phosphopeptides from non-fat milk digests, saliva, serum, and A549 cell lysate. The enrichment results illustrated the great potential of Fe3O4@MIL-125-POM for efficient identification of low-abundance phosphopeptides. [ABSTRACT FROM AUTHOR]

Protein phosphorylation, a common post-translational modification (PTM), is fundamental in a plethora of biological processes, most importantly in modulating cell signaling pathways. Matrix-assisted laser desorption/ionization (MALDI) coupled to tandem mass spectrometry (MS/MS) is an attractive method for phosphopeptide characterization due to its high speed, low limit of detection, and surface sampling capabilities. However, MALDI analysis of phosphopeptides is constrained by relatively low abundances in biological samples and poor relative ionization efficiencies in positive ion mode. Additionally, MALDI tends to produce singly charged ions, generally limiting the accessible MS/MS techniques that can be used for peptide sequencing. For example, collision induced dissociation (CID) is readily amendable to the analysis of singly charged ions, but results in facile loss of phosphoric acid, precluding the localization of the PTM. Electron-based dissociation methods (e.g., electron capture dissociation, ECD) are well suited for PTM localization, but require multiply charged peptide cations to avoid neutralization during ECD. Conversely, phosphopeptides are readily ionized using MALDI in negative ion mode. If the precursor ions are first formed in negative ion mode, a gas-phase charge inversion ion/ion reaction could then be used to transform the phosphopeptide anions produced via MALDI into multiply charged cations that are well-suited for ECD. Herein we demonstrate a multistep workflow combining a charge inversion ion/ion reaction that first transforms MALDI-generated phosphopeptide monoanions into multiply charged cations, and then subjects these multiply charged phosphopeptide cations to ECD for sequence determination and phosphate bond localization. [ABSTRACT FROM AUTHOR]

Summary Identification and quantification of phosphorylation sites are essential for biological interpretation of a phosphoproteomics experiment. For data independent acquisition mass spectrometry-based (DIA-MS) phosphoproteomics, extracting a site-level report from the output of current processing software is not straightforward as multiple peptides might contribute to a single site, multiple phosphorylation sites can occur on the same peptides, and protein isoforms complicate site specification. Currently only limited support is available from a commercial software package via a platform-specific solution with a rather simple site quantification method. Here, we present sitereport, a software tool implemented in an extendable Python package called msproteomics to report phosphosites and phosphopeptides from a DIA-MS phosphoproteomics experiment with a proven quantification method called MaxLFQ. We demonstrate the use of sitereport for downstream data analysis at site level, allowing benchmarking different DIA-MS processing software tools. Availability and implementation sitereport is available as a command line tool in the Python package msproteomics, released under the Apache License 2.0 and available from the Python Package Index (PyPI) at https://pypi.org/project/msproteomics and GitHub at https://github.com/tvpham/msproteomics. [ABSTRACT FROM AUTHOR]

In this work, a new type of FE-1 COF material is prepared by a reversible imine condensation reaction with diaminoferrocene and diaminodiformaldehyde as materials. The material is connected by imine bonds to form a COF skeleton, and the presence of plenty of nitrogen-containing groups gives the material good hydrophilicity; the presence of metal Fe ions provides the material application potential in the enrichment of phosphopeptides. According to the different binding abilities of N-glycopeptide and phosphopeptide on FE-1 COF, it can simultaneously enrich N-glycopeptide and phosphopeptide through different elution conditions to realize its controllable and selective enrichment. Using the above characteristics, 18 phosphopeptides were detected from α-casein hydrolysate, 8 phosphopeptides were detected from β-casein hydrolysate and 21 glycopeptides were detected from IgG hydrolysate. Finally, the gradual elution strategy was used; 16 phosphopeptides and 19 glycopeptides were detected from the α-casein hydrolysate and IgG hydrolysate. The corresponding glycopeptides and phosphopeptides were identified from the human serum. It proves that the FE-1 COF material has a good enrichment effect on phosphopeptides and glycopeptides. [ABSTRACT FROM AUTHOR]

The elucidation of disease pathogenesis can be achieved by analyzing the low-abundance phosphopeptides in organisms. Herein, we developed a novel and easy-to-prepare polymer-coated nanomaterial. By improving the hydrophilicity and spatial conformation of the material, we effectively enhanced the adsorption of phosphopeptides and demonstrated excellent enrichment properties. The material was able to successfully enrich the phosphopeptides in only 1 min. Meanwhile, the material has high selectivity (1:2000), good loading capacity (100 μg/mg), excellent sensitivity (0.5 fmol), and great acid and alkali resistance. In addition, the material was applied to real samples, and 70 phosphopeptides were enriched from the serum of Parkinson's disease (PD) patients and 67 phosphopeptides were enriched from the serum of normal controls. Sequences Logo showed that PD is probably associated with threonine, glutamate, serine, and glutamine. Finally, gene ontology (GO) analysis was performed on phosphopeptides enriched in PD patients' serum. The results showed that PD patients expressed abnormal expression of the cholesterol metabolic process and cell–matrix adhesion in the biological process (BP), endoplasmic reticulum and lipoprotein in the cellular component (CC), and heparin-binding, lipid-binding, and receptor-binding in the molecular function (MF) as compared with normal individuals. All the experiments indicate that the nanomaterials have great potential in proteomics studies. [ABSTRACT FROM AUTHOR]

Magnetic nanoparticles attached to hydrophilic polymer brushes containing immobilized Fe3+ ions were synthesized and used as a new strategy for phosphopeptide enrichment from digested tryptic proteins. To test the performance of nanoparticles in phosphopeptide enrichment, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used. Magnetic nanoparticles with the following features such as high adsorption capacity (about 360 mg/g), easy control and transfer by external magnetic field (high magnetic saturation, about 50 emu/g), excellent phosphopeptide recovery (93.4%) and no shadow effect (due to their non-porous structure), were synthesized. It has been shown that the presence of other proteins in high concentrations does not affect on the performance of nanoparticles. Furthermore, the lowest concentration of digested β-casein phosphopeptides detectable by nanoparticles was determined to be approximately 0.5 fmol µL− 1. The function of synthesized nanoparticles to enrich phosphopeptides in complex biological samples was also demonstrated by isolating four endogenous phosphopeptides from human serum. [ABSTRACT FROM AUTHOR]

Endogenous peptides, particularly those with post-translational modifications, are increasingly being studied as biomarkers for diagnosing various diseases. However, they are weakly ionizable, have a low abundance in biological samples, and may be interfered with by high levels of proteins, peptides, and other macromolecular impurities, resulting in a high limit of detection and insufficient amounts of post-translationally modified peptides in real biological samples to be examined. Therefore, separation and enrichment are necessary before analyzing these biomarkers using mass spectrometry. Mesoporous materials have regular adjustable pores that can eliminate large proteins and impurities, and their large specific surface area can bind more target peptides, but this may result in the partial loss or destruction of target peptides during centrifugal separation. On the other hand, magnetic mesoporous materials can be used to separate the target using an external magnetic field, which improves the separation efficiency and yield. Core–shell magnetic mesoporous materials are widely utilized for peptide separation and enrichment due to their biocompatibility, efficient enrichment capability, and excellent recoverability. This paper provides a review of the latest progress in core–shell magnetic mesoporous materials for enriching glycopeptides and phosphopeptides and compares their enrichment performance with different types of functionalization methods. [ABSTRACT FROM AUTHOR]

Members of the calcium-dependent protein kinase (CDPK/CPK) and SNF-related protein kinase (SnRK) superfamilies are commonly found in plants and some protists. Our knowledge of client specificity of the members of this superfamily is fragmentary. As this family is represented by over 30 members in Arabidopsis thaliana, the identification of kinase-specific and overlapping client relationships is crucial to our understanding the nuances of this large family of kinases as directed towards signal transduction pathways. Herein, we used the kinase client (KiC) assay—a relative, quantitative, high-throughput mass spectrometry-based in vitro phosphorylation assay—to identify and characterize potential CPK/SnRK targets of Arabidopsis. Eight CPKs (1, 3, 6, 8, 17, 24, 28, and 32), four SnRKs (subclass 1 and 2), and PPCK1 and PPCK2 were screened against a synthetic peptide library that contains 2095 peptides and 2661 known phosphorylation sites. A total of 625 in vitro phosphorylation sites corresponding to 203 non-redundant proteins were identified. The most promiscuous kinase, CPK17, had 105 candidate target proteins, many of which had already been discovered. Sequence analysis of the identified phosphopeptides revealed four motifs: LxRxxS, RxxSxxR, RxxS, and LxxxxS, that were significantly enriched among CPK/SnRK clients. The results provide insight into both CPK- and SnRK-specific and overlapping signaling network architectures and recapitulate many known in vivo relationships validating this large-scale approach towards discovering kinase targets. [ABSTRACT FROM AUTHOR]

Overexpression of the 14-3-3ε protein is associated with suppression of apoptosis in cutaneous squamous cell carcinoma (cSCC). This antiapoptotic activity of 14-3-3ε is dependent on its binding to CDC25A; thus, inhibiting 14-3-3ε – CDC25A interaction is an attractive therapeutic approach to promote apoptosis in cSCC. In this regard, designing peptide inhibitors of 14-3-3ε – CDC25A interactions is of great interest. This work reports the rational design of peptide analogs of pS, a CDC25A-derived peptide that has been shown to inhibit 14-3-3ε–CDC25A interaction and promote apoptosis in cSCC with micromolar IC50. We designed new peptide analogs in silico by shortening the parent pS peptide from 14 to 9 amino acid residues; then, based on binding motifs of 14-3-3 proteins, we introduced modifications in the pS(174–182) peptide. We studied the binding of the peptides using conventional molecular dynamics (MD) and steered MD simulations, as well as biophysical methods. Our results showed that shortening the pS peptide from 14 to 9 amino acids reduced the affinity of the peptide. However, substituting Gln176 with either Phe or Tyr amino acids rescued the binding of the peptide. The optimized peptides obtained in this work can be candidates for inhibition of 14-3-3ε – CDC25A interactions in cSCC. [ABSTRACT FROM AUTHOR]

Developing free-standing and mechanical robust membrane materials capable of superior enrichment of phosphopeptides for analyzing and identifying the specific phosphoproteome of cancer cells is significant in understanding the molecular mechanisms of cancer development and exploring new therapeutic approaches, but still a significant challenge in materials design. To this end, we firstly constructed highly flexible ZrTiO4 nanofibrous membranes (NFMs) with excellent mechanical stability through a cost-effective and scalable electrospinning and subsequent calcination technique. Then, to further increase the enrichment capacity of the phosphopeptide, the biomimetic TiO2@ZrTiO4 NFMs with root hair or leaf like branch microstructure are developed by the hydrothermal post-synthetic modification of ZrTiO4 NFMs through growing unfurling TiO2 nanosheets onto the ZrTiO4 nanofibers. Importantly, remarkable flexibility and mechanical stability enable the resulting TiO2@ZrTiO4 NFMs excellent practicability, while the biomimetic microstructure allows it outstanding enrichment ability of the phosphopeptide and identification ability of the specific phosphoproteins in the digest of cervical cancer cells. Specifically, 6770 phosphopeptides can be enriched by TiO2@ZrTiO4 NFMs (2205 corresponding phosphoproteins can be identified), and the value is much higher than that of ZrTiO4 NFMs (6399 phosphopeptides and 2132 identified phosphoproteins) and commercial high-performance TiO2 particles (4525 phosphopeptides and 1811 identified phosphoproteins). These results demonstrate the super ability of TiO2@ZrTiO4 NFMs in phosphopeptide enrichment and great potential for exploring the pathogenesis of cancer. [ABSTRACT FROM AUTHOR]

Plants are remarkable in their ability to adapt to changing environments, with receptor-like kinases (RLKs) playing a pivotal role in perceiving and transmitting environmental cues into cellular responses. Despite extensive research on RLKs from the plant kingdom, the function and activity of many kinases, i.e., their substrates or "clients", remain uncharted. To validate a novel client prediction workflow and learn more about an important RLK, this study focuses on P2K1 (DORN1), which acts as a receptor for extracellular ATP (eATP), playing a crucial role in plant stress resistance and immunity. We designed a Kinase-Client (KiC) assay library of 225 synthetic peptides, incorporating previously identified P2K phosphorylated peptides and novel predictions from a deep-learning phosphorylation site prediction model (MUsite) and a trained hidden Markov model (HMM) based tool, HMMER. Screening the library against purified P2K1 cytosolic domain (CD), we identified 46 putative substrates, including 34 novel clients, 27 of which may be novel peptides, not previously identified experimentally. Gene Ontology (GO) analysis among phosphopeptide candidates revealed proteins associated with important biological processes in metabolism, structure development, and response to stress, as well as molecular functions of kinase activity, catalytic activity, and transferase activity. We offer selection criteria for efficient further in vivo experiments to confirm these discoveries. This approach not only expands our knowledge of P2K1's substrates and functions but also highlights effective prediction algorithms for identifying additional potential substrates. Overall, the results support use of the KiC assay as a valuable tool in unraveling the complexities of plant phosphorylation and provide a foundation for predicting the phosphorylation landscape of plant species based on peptide library results. [ABSTRACT FROM AUTHOR]

In this study, titanium (IV)-immobilized magnetic nanoparticles (Ti4+-PTL-MNPs) were firstly synthesized via a one-step aqueous self-assembly of lysozyme nanofilms for efficient phosphopeptide enrichment. Under physiological conditions, lysozymes readily self-organized into phase-transitioned lysozyme (PTL) nanofilms on Fe3O4@SiO2 and Fe3O4@C MNP surfaces with abundant functional groups, including –NH2, –COOH, –OH, and –SH, which can be used as multiple linkers to efficiently chelate Ti4+. The obtained Ti4+-PTL-MNPs possessed high sensitivity of 0.01 fmol μL−1 and remarkable selectivity even at a mass ratio of β-casein to BSA as low as 1:400 for phosphopeptide enrichment. Furthermore, the synthesized Ti4+-PTL-MNPs can also selectively identify low-abundance phosphopeptides from extremely complicated human serum samples and their rapid separation, good reproducibility, and excellent recovery were also proven. This one-step self-assembly of PTL nanofilms facilitated the facile and efficient surface functionalization of various nanoparticles for proteomes/peptidomes. [ABSTRACT FROM AUTHOR]

Blast-induced neurotrauma (BINT) is a pressing concern for veterans and civilians exposed to explosive devices. Affected personnel may have increased risk for long-term cognitive decline and developing tauopathies including Alzheimer's disease-related disorders (ADRD) or frontal-temporal dementia (FTD). The goal of this study was to identify the effect of BINT on molecular networks and their modulation by mutant tau in transgenic (Tg) mice overexpressing the human tau P301L mutation (rTg4510) linked to FTD or non-carriers. The primary focus was on the phosphoproteome because of the prominent role of hyperphosphorylation in neurological disorders. Discrimination learning was assessed following injury in the subsequent 6 weeks, using the automated home-cage monitoring CognitionWall platform. At 40 days post injury, label-free phosphoproteomics was used to evaluate molecular networks in the frontal cortex of mice. Utilizing a weighted peptide co-expression network analysis (WpCNA) approach, we identified phosphopeptide networks tied to associative learning and mossy-fiber pathways and those which predicted learning outcomes. Phosphorylation levels in these networks were inversely related to learning and linked to synaptic dysfunction, cognitive decline, and dementia including Atp6v1a and Itsn1. Low-intensity blast (LIB) selectively increased pSer262tau in rTg4510, a site implicated in initiating tauopathy. Additionally, individual and group level analyses identified the Arhgap33 phosphopeptide as an indicator of BINT-induced cognitive impairment predominantly in rTg4510 mice. This study unveils novel interactions between ADRD genetic susceptibility, BINT, and cognitive decline, thus identifying dysregulated pathways as targets in potential precision-medicine focused therapeutics to alleviate the disease burden among those affected by BINT. [ABSTRACT FROM AUTHOR]

Calcium is one of the important elements for human health. Calcium deficiencies can lead to numerous diseases. Calcium chelating peptides have shown potential application in the management of calcium deficiencies. Casein phosphopeptides (CPP) are phosphoseryl-containing fragments of casein by enzymatic hydrolysis or fermentation during manufacture of milk products as well as during intestinal digestion. An increasing number of CPP with the ability to facilitate and enhance the bioavailability of calcium are being discovered and identified. In this review, 249 reported CPP derived from four types of bovine casein (αs1, αs2, β and κ) were collected, and the amino acid sequence and phosphoserine group information were sorted out. This review outlines the current enzyme hydrolysis, detection methods, purification, structure-activity relationship and mechanism of intestinal calcium absorption in vitro and in vivo as well as application of CPP. [ABSTRACT FROM AUTHOR]

Reasonable design and construction of functionalized materials are of great importance for the enrichment of global phosphopeptides. In this work, Ti4+ functionalized hydrophilic covalent organic frameworks by introducing glutathione (GSH) and 2,3,4‐trihydroxy benzaldehyde (THBA) via click chemistry and Schiff base reaction (COF‐V@GSH‐THBA‐Ti4+) was constructed and applied for selective enrichment of phosphopeptides in serum. Benefit from the high surface area, excellent hydrophilicity as well as regular mesoporous structure, COF‐V@GSH‐THBA‐Ti4+ displayed high selectivity (molar ratio of 2000:1), low limit of detection (0.5 fmol), high load capacity (100.0 mg/g) and excellent size‐exclusion effect (1:10000) for enrichment of phosphopeptides. For actual bio‐sample analysis, 15 phosphopeptides assigned to 10 phosphoproteins with 16 phosphorylated sites and 33 phosphopeptides assigned to 25 phosphoproteins with 34 phosphorylated sites were detected from the serum of patients with chronic obstructive pulmonary disease (COPD), and normal controls. Biological processes and molecular functions analysis further disclosed the difference of serums with phosphoproteomics between COPD and normal controls. [ABSTRACT FROM AUTHOR]

Protein phosphorylation regulates a variety of important cellular and physiological processes in plants. In-depth profiling of plant phosphoproteomes has been more technically challenging than that of animal phosphoproteomes. This is largely due to the need to improve protein extraction efficiency from plant cells, which have a dense cell wall, and to minimize sample loss resulting from the stringent sample clean-up steps required for the removal of a large amount of biomolecules interfering with phosphopeptide purification and mass spectrometry analysis. To this end, we developed a method with a streamlined workflow for highly efficient purification of phosphopeptides from tissues of various green organisms including Arabidopsis , rice, tomato, and Chlamydomonas reinhardtii , enabling in-depth identification with high quantitative reproducibility of about 11 000 phosphosites, the greatest depth achieved so far with single liquid chromatography-mass spectrometry (LC–MS) runs operated in a data-dependent acquisition (DDA) mode. The mainstay features of the method are the minimal sample loss achieved through elimination of sample clean-up before protease digestion and of desalting before phosphopeptide enrichment and hence the dramatic increases of time- and cost-effectiveness. The method, named GreenPhos, combined with single-shot LC–MS, enabled in-depth quantitative identification of Arabidopsis phosphoproteins, including differentially phosphorylated spliceosomal proteins, at multiple time points during salt stress and a number of kinase substrate motifs. GreenPhos is expected to serve as a universal method for purification of plant phosphopeptides, which, if samples are further fractionated and analyzed by multiple LC–MS runs, could enable measurement of plant phosphoproteomes with an unprecedented depth using a given mass spectrometry technology. GreenPhos, with a streamlined and robust workflow, was developed for rapid enrichment of plant phosphopeptides with high sensitivity and selectivity. GreenPhos enables quantitative measurement of phosphoproteomes of different plants with unprecedented coverage achieved through single LC–MS runs and high reproducibility. Thus, it is expected that GreenPhos will serve as a universal method for plant phosphoproteomics. [ABSTRACT FROM AUTHOR]

Subfornical organ (SFO) is vital in chronic kidney disease (CKD) progression caused by high salt levels. The current study investigated the effects of high salt on phosphoproteomic changes in SFO in CKD rats. 5/6 nephrectomized rats were fed a normal-salt diet (0.4%) (NC group) or a high-salt diet (4%) (HC group) for three weeks, while sham-operated rats were fed a normal-salt diet (0.4%) (NS group). For phosphoproteomic analysis of SFO in different groups, TiO2 enrichment, isobaric tags for relative and absolute quantification (iTRAQ) labeling, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used. There were 6808 distinct phosphopeptides found, which corresponded to 2661 phosphoproteins. NC group had 168 upregulated and 250 downregulated phosphopeptides compared to NS group. Comparison to NC group, HC group had 154 upregulated and 124 downregulated phosphopeptides. Growth associated protein 43 (GAP43) and heat shock protein 27 (Hsp27) were significantly upregulated phosphoproteins and may protect against high-salt damage. Differential phosphoproteins with tight functional connection were synapse proteins and microtubule-associated proteins, implying that high-salt diet disrupted brain's structure and function. Furthermore, differential phosphoproteins in HC/NC comparison group were annotated to participate in GABAergic synapse signaling pathway and aldosterone synthesis and secretion, which attenuated inhibitory neurotransmitter effects and increased sympathetic nerve activity (SNA). This large scale phosphoproteomic profiling of SFO sheds light on how salt aggravates CKD via the central nervous system. [ABSTRACT FROM AUTHOR]

A new general approach called in situ digestion-assisted multi-template imprinting is proposed for preparation of phospho-specific molecularly imprinted nanoparticles. Through the novel templating strategy and controllable imprinting process, imprinted nanoparticles specific to the intact phosphoprotein and its phosphopeptides were synthesized. The prepared imprinted nanoparticles exhibited excellent specificity (cross reactivity < 10%), high affinity (10−6 M), high efficiency (47.5%), and good generality (both intact phosphoprotein and phosphopeptides). We also realized the fine tuning of the recognition at peptide level of the imprinted nanoparticles by adjusting the imprinting time. Based on the selective enrichment of the imprinted nanoparticles, the MS identification of both the intact phosphoprotein (Tau) and phosphopeptides (angiotensin II and peptides of Tau) in real complex samples could be achieved. Therefore, we believe that the in situ digestion-assisted multi-template imprinting strategy holds promising future in both phosphorylation analysis and proteomics applications. [ABSTRACT FROM AUTHOR]

β-Arrestins (βarrs) are functionally versatile proteins that play critical roles in the G-protein-coupled receptor (GPCR) signaling pathways. While it is well established that the phosphorylated receptor tail plays a central role in βarr activation, emerging evidence highlights the contribution from membrane lipids. However, detailed molecular mechanisms of βarr activation by different binding partners remain elusive. In this work, we present a comprehensive study of the structural changes in critical regions of βarr1 during activation using 19F NMR spectroscopy. We show that phosphopeptides derived from different classes of GPCRs display different βarr1 activation abilities, whereas binding of the membrane phosphoinositide PIP2 stabilizes a distinct partially activated conformational state. Our results further unveil a sparsely-populated activation intermediate as well as complex cross-talks between different binding partners, implying a highly multifaceted conformational energy landscape of βarr1 that can be intricately modulated during signaling. The molecular basis for the functional versatility of β-arrestins in the G-protein coupled receptor signaling pathway is not well understood. Here, the authors use19F NMR spectroscopy to show that different binding partners activate β-arrestin-1 through distinct mechanisms. [ABSTRACT FROM AUTHOR]

Recent studies challenge the dogma that a 21-mer phosphopeptide P140 protects against direct cell damage in the phase-III clinical trial (NCT02504645) for lupus, involving reactive oxygen species (ROS)-dependent release of citrullinated histone H3 (H3cit)-linked neutrophil extracellular traps. An open question is the cellular location of ROS production and H3cit formation in lupus. In this study, we examined the effects of P140 peptides on ROS production and H3cit location in lupus with in vivo and situ fluorescence imaging with subcellular resolution. We developed a mouse model of the B6 strain harbouring a bioluminescent reporter under the control of the Lysozyme M promoter. Based on the imiquimod-induced disease model of B6 mice, we used bioluminescent imaging, flow cytometry analysis, and immunohistology staining to study the effects of P140 peptides in lupus. We found a profound accumulation of CX3CR1-positive macrophages in the lungs of lupus mice after the application of P140, accompanied by lung fibrosis formation. The defined P140-mediated macrophage responses were associated with an increase of H3cit in the cytosol, interleukin-1 receptor type 1 on the extracellular membrane, and intracellular production of ROS. Of interest, the disease of imiquimod-induced lupus was prevented with an antioxidant drug apocynin. This study shows that P140 peptides play a role in aggravated murine lupus in a manner dependent on ROS production and H3cit upregulation through pulmonary macrophages. [ABSTRACT FROM AUTHOR]

Cerium ions immobilized magnetic graphite nitride material have been prepared using L-Alanyl-L-Glutamine as the new chelator. The resulting Fe3O4/g-C3N4-L-Ala-L-Gln-Ce4+, as an immobilized metal ion affinity chromatography (IMAC) sorbent, was reusable. This is due to the strong coordination interaction between L-Alanyl-L-Glutamine and cerium ions. After a series of characterizations, the magnetic nanocomposite showed high surface area, good hydrophilicity, positive electricity, and magnetic response. Fe3O4/g-C3N4-L-Ala-L-Gln-Ce4+ had high sensitivity (0.1 fmol), selectivity (α-/β-casein/bovine serum albumin, 1:1:5000), and good recyclability (10 cycles). A total of 647 unique phosphopeptides mapped to 491 phosphoproteins were identified from A549 cell lysate by nano LC–MS analysis. [ABSTRACT FROM AUTHOR]

Plants are remarkable in their ability to adapt to changing environments, with receptor-like kinases (RLKs) playing a pivotal role in perceiving and transmitting environmental cues into cellular responses. Despite extensive research on RLKs from the plant kingdom, the function and activity of many kinases, i.e., their substrates or “clients”, remain uncharted. To validate a novel client prediction workflow and learn more about an important RLK, this study focuses on P2K1 (DORN1), which acts as a receptor for extracellular ATP (eATP), playing a crucial role in plant stress resistance and immunity. We designed a Kinase-Client (KiC) assay library of 225 synthetic peptides, incorporating previously identified P2K phosphorylated peptides and novel predictions from a deep-learning phosphorylation site prediction model (MUsite) and a trained hidden Markov model (HMM) based tool, HMMER. Screening the library against purified P2K1 cytosolic domain (CD), we identified 46 putative substrates, including 34 novel clients, 27 of which may be novel peptides, not previously identified experimentally. Gene Ontology (GO) analysis among phosphopeptide candidates revealed proteins associated with important biological processes in metabolism, structure development, and response to stress, as well as molecular functions of kinase activity, catalytic activity, and transferase activity. We offer selection criteria for efficient further in vivo experiments to confirm these discoveries. This approach not only expands our knowledge of P2K1’s substrates and functions but also highlights effective prediction algorithms for identifying additional potential substrates. Overall, the results support use of the KiC assay as a valuable tool in unraveling the complexities of plant phosphorylation and provide a foundation for predicting the phosphorylation landscape of plant species based on peptide library results.

In this work, a new type of FE-1 COF material is prepared by a reversible imine condensation reaction with diaminoferrocene and diaminodiformaldehyde as materials. The material is connected by imine bonds to form a COF skeleton, and the presence of plenty of nitrogen-containing groups gives the material good hydrophilicity; the presence of metal Fe ions provides the material application potential in the enrichment of phosphopeptides. According to the different binding abilities of N-glycopeptide and phosphopeptide on FE-1 COF, it can simultaneously enrich N-glycopeptide and phosphopeptide through different elution conditions to realize its controllable and selective enrichment. Using the above characteristics, 18 phosphopeptides were detected from α-casein hydrolysate, 8 phosphopeptides were detected from β-casein hydrolysate and 21 glycopeptides were detected from IgG hydrolysate. Finally, the gradual elution strategy was used; 16 phosphopeptides and 19 glycopeptides were detected from the α-casein hydrolysate and IgG hydrolysate. The corresponding glycopeptides and phosphopeptides were identified from the human serum. It proves that the FE-1 COF material has a good enrichment effect on phosphopeptides and glycopeptides.

AbstractObjectives Subfornical organ (SFO) is vital in chronic kidney disease (CKD) progression caused by high salt levels. The current study investigated the effects of high salt on phosphoproteomic changes in SFO in CKD rats.Methods 5/6 nephrectomized rats were fed a normal-salt diet (0.4%) (NC group) or a high-salt diet (4%) (HC group) for three weeks, while sham-operated rats were fed a normal-salt diet (0.4%) (NS group). For phosphoproteomic analysis of SFO in different groups, TiO2 enrichment, isobaric tags for relative and absolute quantification (iTRAQ) labeling, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used.Results There were 6808 distinct phosphopeptides found, which corresponded to 2661 phosphoproteins. NC group had 168 upregulated and 250 downregulated phosphopeptides compared to NS group. Comparison to NC group, HC group had 154 upregulated and 124 downregulated phosphopeptides. Growth associated protein 43 (GAP43) and heat shock protein 27 (Hsp27) were significantly upregulated phosphoproteins and may protect against high-salt damage. Differential phosphoproteins with tight functional connection were synapse proteins and microtubule-associated proteins, implying that high-salt diet disrupted brain’s structure and function. Furthermore, differential phosphoproteins in HC/NC comparison group were annotated to participate in GABAergic synapse signaling pathway and aldosterone synthesis and secretion, which attenuated inhibitory neurotransmitter effects and increased sympathetic nerve activity (SNA).Discussion This large scale phosphoproteomic profiling of SFO sheds light on how salt aggravates CKD via the central nervous system.

A Ti4+-functionalized magnetic covalent organic framework material with flexible branched polymers (mCOF@ε-PL@THBA-Ti4+) built via an immobilized metal ion affinity chromatography (IMAC) enrichment strategy was proposed through post-synthesis modification. Hydrophilic ε-poly- L -lysine (ε-PL) rich in amino active groups was first introduced in the fabrication of the phosphopeptide enrichment material to increase the hydrophilicity while providing more functional modification pathways of the material. 2,3,4-Trihydroxy-benzaldehyde (THBA) provides abundant binding sites for the immobilization of numerous Ti4+, which is advantageous for the subsequent efficient phosphopeptide enrichment. The magnetic nanocomposite exhibited outstanding performance of phosphopeptide enrichment with good selectivity (1 : 5000), a low detection limit (2 fmol), and relatively high loading capacity (66.7 mg g−1). What's more, after treatment with mCOF@ε-PL@THBA-Ti4+, 16 endogenous phosphopeptides from fresh saliva of healthy people were recognized by MALDI-TOF MS, and 50 phosphopeptides belonging to 35 phosphoproteins from the serum of uremia patients were detected by nano-LC-MS/MS. Proteomics data analysis for the differential protein selection between uremia and normal controls was conducted using R software, and four down-regulated and three up-regulated proteins were obtained. The results suggested that the prepared material has potential applications in biomarker discovery. [ABSTRACT FROM AUTHOR]

A Ti4+ functionalized β-cyclodextrin covalent organic framework nanoparticle (named as β-CD-COF@Ti4+) was synthesized using a one-pot method successfully realizing the enrichment of phosphorylated peptides and exosomes based on the immobilized metal ion affinity chromatography strategy. The functionalized β-CD-COF@Ti4+ exhibited superior performance on the enrichment of phosphopeptides, including high selectivity (1:1000), low detection limit (0.5 fmol), and loading capacity for phosphopeptides (100 mg·g−1). After treatment with β-CD-COF@Ti4+, 9 phosphopeptides from defatted milk, 29 phosphopeptides related to 23 phosphoproteins from normal group serum, and 24 phosphopeptides related to 22 phosphoproteins from the serum of uremia patients were captured. Through the analysis of Gene Ontology, the captured phosphoprotein is closely related to kidney disease, including lipoprotein metabolism, very-low-density lipoprotein particle, high-density lipoprotein particle, and lipid binding activity process. Furthermore, western blot verification showed that this nanoparticle could successfully capture exosomes from human serum. This study demonstrates great prospects for the enrichment of phosphopeptides and exosomes from actual bio-samples. [ABSTRACT FROM AUTHOR]

Background: Lysine carbamylation is a biomarker of rheumatoid arthritis and kidney diseases. However, its cellular function is understudied due to the lack of tools for systematic analysis of this post-translational modification (PTM). Methods: We adapted a method to analyze carbamylated peptides by co-affinity purification with acetylated peptides based on the cross-reactivity of anti-acetyllysine antibodies. We also performed immobilized-metal affinity chromatography to enrich for phosphopeptides, which allowed us to obtain multi-PTM information from the same samples. Results: By testing the pipeline with RAW 264.7 macrophages treated with bacterial lipopolysaccharide, 7,299, 8,923 and 47,637 acetylated, carbamylated, and phosphorylated peptides were identified, respectively. Our analysis showed that carbamylation occurs on proteins from a variety of functions on sites with similar as well as distinct motifs compared to acetylation. To investigate possible PTM crosstalk, we integrated the carbamylation data with acetylation and phosphorylation data, leading to the identification 1,183 proteins that were modified by all 3 PTMs. Among these proteins, 54 had all 3 PTMs regulated by lipopolysaccharide and were enriched in immune signaling pathways, and in particular, the ubiquitin-proteasome pathway. We found that carbamylation of linear diubiquitin blocks the activity of the anti-inflammatory deubiquitinase OTULIN. Conclusions: Overall, our data show that anti-acetyllysine antibodies can be used for effective enrichment of carbamylated peptides. Moreover, carbamylation may play a role in PTM crosstalk with acetylation and phosphorylation, and that it is involved in regulating ubiquitination in vitro. 9DSWmM7hoJm8b3LaxSLrsC Video Abstract [ABSTRACT FROM AUTHOR]

Overexpression of the 14-3-3ε protein is associated with suppression of apoptosis in cutaneous squamous cell carcinoma (cSCC). This antiapoptotic activity of 14-3-3ε is dependent on its binding to CDC25A; thus, inhibiting 14-3-3ε – CDC25A interaction is an attractive therapeutic approach to promote apoptosis in cSCC. In this regard, designing peptide inhibitors of 14-3-3ε – CDC25A interactions is of great interest. This work reports the rational design of peptide analogs of pS, a CDC25A-derived peptide that has been shown to inhibit 14-3-3ε–CDC25A interaction and promote apoptosis in cSCC with micromolar IC50. We designed new peptide analogs in silico by shortening the parent pS peptide from 14 to 9 amino acid residues; then, based on binding motifs of 14-3-3 proteins, we introduced modifications in the pS(174–182) peptide. We studied the binding of the peptides using conventional molecular dynamics (MD) and steered MD simulations, as well as biophysical methods. Our results showed that shortening the pS peptide from 14 to 9 amino acids reduced the affinity of the peptide. However, substituting Gln176 with either Phe or Tyr amino acids rescued the binding of the peptide. The optimized peptides obtained in this work can be candidates for inhibition of 14-3-3ε – CDC25A interactions in cSCC.

Blast-induced neurotrauma (BINT) is a pressing concern for veterans and civilians exposed to explosive devices. Affected personnel may have increased risk for long-term cognitive decline and developing tauopathies including Alzheimer’s disease-related disorders (ADRD) or frontal-temporal dementia (FTD). The goal of this study was to identify the effect of BINT on molecular networks and their modulation by mutant tau in transgenic (Tg) mice overexpressing the human tau P301L mutation (rTg4510) linked to FTD or non-carriers. The primary focus was on the phosphoproteome because of the prominent role of hyperphosphorylation in neurological disorders. Discrimination learning was assessed following injury in the subsequent 6 weeks, using the automated home-cage monitoring CognitionWall platform. At 40 days post injury, label-free phosphoproteomics was used to evaluate molecular networks in the frontal cortex of mice. Utilizing a weighted peptide co-expression network analysis (WpCNA) approach, we identified phosphopeptide networks tied to associative learning and mossy-fiber pathways and those which predicted learning outcomes. Phosphorylation levels in these networks were inversely related to learning and linked to synaptic dysfunction, cognitive decline, and dementia including Atp6v1a and Itsn1. Low-intensity blast (LIB) selectively increased pSer262tau in rTg4510, a site implicated in initiating tauopathy. Additionally, individual and group level analyses identified the Arhgap33 phosphopeptide as an indicator of BINT-induced cognitive impairment predominantly in rTg4510 mice. This study unveils novel interactions between ADRD genetic susceptibility, BINT, and cognitive decline, thus identifying dysregulated pathways as targets in potential precision-medicine focused therapeutics to alleviate the disease burden among those affected by BINT.

Enrichment of phosphopeptides is a crucial step in phosphoproteomics study. At present, combination of multiple affinity chromatography is regard as an efficient phosphopeptide enrichment strategy. Therefore, designing and preparing composites with multiple affinity sites is of great significance. Herein, the Oxid-Ti3C2Tx/UIO-66-NH2 composites with multiple affinity sites are rapidly synthesized by dielectric barrier discharge (DBD) technology at low temperature and atmospheric pressure. The in-situ nucleation of UIO-66-NH2 and TiO2 can be flexibly regulated through the feed ratio of MOF precursors and the DBD duration individually. The UIO-66-NH2 has large specific surface area, high porosity and plentiful Zr-O clusters. Oxid-Ti3C2Tx provides abundant exposed Ti sites and Ti-O clusters. Based on these, Oxid-Ti3C2Tx/UIO-66-NH2 composites exhibited high sensitivity (0.1 fmol μL−1), satisfactory selectivity (BSA: α-casein = 100: 1, molar ratio) and superior repeatability for phosphopeptide enrichment. In addition, the composites performed well in biological samples like nonfat milk (24 phosphopeptides), human saliva (15 endogenous phosphopeptides) and human serum (all 4 endogenous phosphopeptides), demonstrating a huge application potential in phosphoproteomics study. More importantly, this study opens a new path to synthesize MXene/MOF composites with controllable oxidation degree and flexible compositions regulation efficiently, revealing its broad application prospects in various fields.

In this work, titanium-rich hydrazide-linked porous organic polymers (hydrazide-POPs-Ti4+) were synthesized using hydrazine, 2,3-dihydroxyterephthalaldehyde (DHTA) and trimethyl 1,3,5-benzenetricarboxylate (TP) as the ligands. Hydrazide-POPs-Ti4+ combined with HILIC and IMAC can be used for simultaneous enrichment of glycopeptides and phosphopeptides. The detection limit of this protocol is 0.1 fmol μL−1 for glycopeptides and 0.005 fmol μL−1 for phosphopeptides, and the selectivities are 1 : 1000 and 1 : 2000 for glycopeptides and phosphopeptides, respectively. For practical bio-sample analysis, 201 glycopeptides associated with 129 glycoproteins and 26 phosphopeptides associated with 21 phosphoproteins were selectively captured from healthy human serum, and 186 glycopeptides associated with 117 glycoproteins and 60 phosphopeptides associated with 50 phosphoproteins were enriched in the serum of breast cancer patients. Gene Ontology analysis indicated that the identified glycoproteins and phosphoproteins were linked to breast cancer, including the binding of complement component C1q and low-density lipoprotein particles, protein oxidation and complement activation, suggesting that these connected pathways are probably engaged in the disease pathology of breast cancer. [ABSTRACT FROM AUTHOR]

Many biological processes are regulated through dynamic protein phosphorylation. Monitoring disease‐relevant phosphorylation events in circulating biofluids is highly appealing but also technically challenging. We introduce here a functionally tunable material and a strategy, extracellular vesicles to phosphoproteins (EVTOP), which achieves one‐pot extracellular vesicles (EVs) isolation, extraction, and digestion of EV proteins, and enrichment of phosphopeptides, with only a trace amount of starting biofluids. EVs are efficiently isolated by magnetic beads functionalized with TiIV ions and a membrane‐penetrating peptide, octa‐arginine R8+, which also provides the hydrophilic surface to retain EV proteins during lysis. Subsequent on‐bead digestion concurrently converts EVTOP to TiIV ion‐only surface for efficient enrichment of phosphopeptides for phosphoproteomic analyses. The streamlined, ultra‐sensitive platform enabled us to quantify 500 unique EV phosphopeptides with only a few μL of plasma and over 1200 phosphopeptides with 100 μL of cerebrospinal fluid (CSF). We explored its clinical application of monitoring the outcome of chemotherapy of primary central nervous system lymphoma (PCNSL) patients with a small volume of CSF, presenting a powerful tool for broad clinical applications. [ABSTRACT FROM AUTHOR]

Based on the motifs (RNISY (M) and DEEVELILGDT (D)) in the protein crystal structures of Merlin and CRL4DCAF−1, we phosphorylated the tyrosine residue in M and conjugated M to a self‐assembling motif to produce a phosphopeptide (1P) and examined enzyme‐instructed self‐assembly (EISA) of 1P with and without the presence of D (4). Our results show that EISA of 1P forms a hydrogel at exceedingly low volume fraction (∼0.03 %) even with the presence of the hydrophilic peptide, 4. Unlike 1P, 2P (a diastereomer of 1P) or 3P (the enantiomer of 1P) forms a hydrogel via EISA when their concentration is four or three times that of 1P, respectively. Circular dichroism (CD) spectra show that increasing the concentration of the phosphopeptides lowers the CD signals of the mixtures, and the magnitudes of the CD signals depends on the interaction between M and D. This work provides insight for understanding multi‐component hydrogels formed by self‐assembly, which involves both specific intermolecular interaction and enzymatic reactions. [ABSTRACT FROM AUTHOR]

Cytokinesis partitions the cell contents to complete mitosis. During cytokinesis, polo-like kinase 1 (PLK1) activates the small GTPase RhoA to assemble a contractile actomyosin ring. PLK1 is proposed to pattern RhoA activation by creating a docking site on the central spindle that concentrates the RhoA guanine nucleotide exchange factor ECT2. However, ECT2 targeting to the central spindle is dispensable for cytokinesis, indicating that how PLK1 controls RhoA activation remains unresolved. To address this question, we employed an unbiased approach targeting ∼100 predicted PLK1 sites in two RhoA regulators: ECT2 and the centralspindlin complex, composed of CYK4 and kinesin-6. This comprehensive approach suggested that the only functionally critical PLK1 target sites are in a single cluster in the CYK4 N terminus. Phosphorylation of this cluster promoted direct interaction of CYK4 with the BRCT repeat module of ECT2. However, mutational analysis in vitro and in vivo led to the surprising finding that the interaction was independent of the conserved "canonical" residues in ECT2's BRCT repeat module that, based on structurally characterized BRCT-phosphopeptide interactions, were presumed critical for binding. Instead, we show that the ECT2 BRCT module binds phosphorylated CYK4 via a distinct conserved basic surface. Basic surface mutations mimic the effects on cytokinesis of loss of CYK4 cluster phosphorylation or inhibition of PLK1 activity. Together with evidence for ECT2 autoinhibition limiting interaction with CYK4 in the cytoplasm, these results suggest that a spatial gradient of phosphorylated CYK4 around the central spindle patterns RhoA activation by interacting with ECT2 on the adjacent plasma membrane.

Phosvitin and its phosphopeptides contain a large amount of phosphorylated amino groups and exhibit a series of unique bioactivities including metal-binding, antioxidant, bacteriostatic and biomineralization activities due to strong interactions between phosphate groups and metal ions. In this article, the structures and preparation of phosvitin phosphopeptides, the interactions of phosphopeptides with metal ions, and the biological activities associated with their metal-binding capacity, as well as future potential application prospects are discussed.

A polyhedral MIL-125-NH2@Pd nanoparticle with light-responsive oxidase-like activity was synthesized through a simple solvothermal method and was characterized by field emission scanning electron microscopy, transmission electron microscopy, energy-dispersive X-ray spectroscopy, X-ray powder diffraction, Fourier transform infrared spectroscopy, thermogravimetric analysis, Brunauer–Emmett–Teller analysis, etc. MIL-125-NH2@Pd can catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) under light radiation without H2O2, indicating its intrinsic light-responsive oxidase-like characteristic. MIL-125-NH2@Pd shows a superior catalytic activity and higher affinity to TMB, compared to MIL-125-NH2, owing to the presence of homogenously distributed palladium (Pd) nanoparticles. The results of the mechanism study indicate the existence of ˙O2− and electron holes during the catalytic reaction of MIL-125-NH2@Pd. Furthermore, based on the excellent light-responsive oxidase-like activity and specific recognition capacity for casein phosphopeptides (CPP) of titanium ions (Ti4+) in MIL-125-NH2@Pd, a simple colorimetric sensing platform was developed for the determination of CPP in a linear range of 10–100 μg mL−1 with a limit of detection as low as 2.9 μg mL−1. Finally, the developed method was successfully employed in the sensitive and selective detection of CPP in spiked rabbit and rat plasma samples. This study may provide a reliable and highly efficient strategy to realize the excitation-responsive sensing applications of materials. [ABSTRACT FROM AUTHOR]

Protein glycosylation and phosphorylation play essential roles in biological systems. The crosstalk of both glycosylation and phosphorylation on one protein represents an unveiled biological function. To realize the analyses of both glycopeptides and phosphopeptides, a simultaneous enrichment method of N-glycopeptides, mono-phosphopeptides and multi-phosphopeptides was developed based on a multi-functional dual-metal centered zirconium metal–organic framework that provided multiple interactions for HILIC, IMAC, and MOAC for glycopeptides and phosphopeptides. Based on a careful optimization of sample loading and elution conditions for the simultaneous enrichment of glycopeptides and phosphopeptides with the zirconium metal–organic framework, a total of 1011 N-glycopeptides derived from 410 glycoproteins and 1996 phosphopeptides including 741 multi-phosphopeptides derived from 1189 phosphoproteins could be identified from a HeLa cell digest. The simultaneous enrichment approach for glycopeptides and mono-/multi-phosphopeptides demonstrates the great potential of the combined interactions for HILIC, IMAC, and MOAC in integrated post-translational modification proteomics research. [ABSTRACT FROM AUTHOR]

Inspired by the enrichment preferences of metal ions for phosphopeptides, a layered assembly method was adopted to introduce hydrophilic polyhydroxy ligands tannic acid (TA) to prepare magnetic bimetal ion-functionalized nanomaterials denoted by Fe3O4@TA@Ti4+/Zr4+ to provide the comprehensive enrichment of phosphopeptides. Benefiting from the excellent ion immobilization ability and unbiasedness for phosphopeptide enrichment, Fe3O4@TA@Ti4+/Zr4+ shows excellent properties for the enrichment and identification of global phosphopeptides. The results show that the material possesses high efficiency, outstanding selectivity (the maximum molar ratio β-casein to bovine serum albumin is 1:1000), a low detection limit of 0.4 fmol/μL, and the ability to be recycled at least 10 times. In addition, excellent performance was also demonstrated in the analysis of skimmed milk and human saliva: 13 and 31 phosphopeptides were successfully captured. The results demonstrate that the material has high potential for practical application in phosphoproteomics. [ABSTRACT FROM AUTHOR]

Effective phosphoproteome of nanoscale sample analysis remains a daunting task, primarily due to significant sample loss associated with non-specific surface adsorption during enrichment of low stoichiometric phosphopeptide. We develop a tandem tip phosphoproteomics sample preparation method that is capable of sample cleanup and enrichment without additional sample transfer, and its integration with our recently developed SOP (Surfactant-assisted One-Pot sample preparation) and iBASIL (improved Boosting to Amplify Signal with Isobaric Labeling) approaches provides a streamlined workflow enabling sensitive, high-throughput nanoscale phosphoproteome measurements. This approach significantly reduces both sample loss and processing time, allowing the identification of >3000 (>9500) phosphopeptides from 1 (10) µg of cell lysate using the label-free method without a spectral library. It also enables precise quantification of ~600 phosphopeptides from 100 sorted cells (single-cell level input for the enriched phosphopeptides) and ~700 phosphopeptides from human spleen tissue voxels with a spatial resolution of 200 µm (equivalent to ~100 cells) in a high-throughput manner. The new workflow opens avenues for phosphoproteome profiling of mass-limited samples at the low nanogram level. A streamlined tandem tip-based workflow for sensitive nanoscale phosphoproteomics is developed, reducing sample loss and processing time, allowing the phosphoproteome profiling of mass-limited samples at the low nanogram level. [ABSTRACT FROM AUTHOR]

The stability of MOFs plays one of the most important roles in material applications, while the delicate structure of MOFs suffers from the limitation of poor alkali tolerance. A new biligand Zr-MOF (biUIO-66–NH2NO2) with alkali-resistance performance and active functional groups has been synthesized in this study. The biUIO-66–NH2NO2 demonstrated a much better stability in 1% NH3·H2O solution than its parent material, UIO-66–NH2. Following further immobilization of Zr4+ ions, the biDZMOF consisting of dual-zirconium centers was prepared and was further applied in global enrichment of phosphopeptides by avoiding the instability of enrichment materials in the essential alkali elution procedure for the phosphopeptide enrichment workflow. The alkali-resistant elution of phosphopeptides from the biDZMOF can be directly coupled to a tandem mass spectrometry system for peptide analysis without desalting treatment. 425 phosphopeptides in total in 3 independent samples were identified from 10 μL human saliva after enrichment with biDZMOF. The improvement in alkali resistance and successful post-modification of biUIO-66–NH2NO2 suggest an efficient strategy to develop new types of MOF materials for application. [ABSTRACT FROM AUTHOR]

Dual‐selective enrichment of phosphopeptides and glycopeptides of post‐translational modifications (PTMs) in the complex biological samples are challenging. In this work, considering the versatile properties including surface abundant metal sites and electrostatic attraction between Ti3C2‐layers and Al‐layers, layered ternary carbides Ti3AlC2 nanomaterials was successfully applied for the first time as an affinity adsorbent for the dual‐selective capture of phosphopeptides and glycopeptides. Especially, the Ti3AlC2 nanomaterials had an excellent detection sensitivity for phosphopeptides (1×10−11 M) and a good selectivity for glycopeptides with a low molar ratio of 1 : 500 of HRP (horseradish peroxidase) to BSA (bovine serum albumin). Furthermore, Ti3AlC2 nanomaterials was also applied for dual‐selective enrichment of phosphopeptides and glycopeptides from mouse brain neocortex lysate and human serum lysate respectively before mass spectrometry (MS) analysis, yielding twenty‐two unique phosphopeptides from thirteen phosphoproteins and fifty‐three unique glycopeptides from thirty‐seven glycoproteins, respectively. This work will open a new avenue and will greatly promote sample preparation for mass spectrometric analysis in phosphoproteomics and glycoproteomics research. [ABSTRACT FROM AUTHOR]

Abnormally hyperphosphorylated tau can be recognized by a variety of phosphoprotein‐binding domains (PBDs) to elicit downstream tau signaling in neuropathology, which has been found to have a potential association with subarachnoid hemorrhage. In this study, the genome‐wide binding behavior of tau phosphorylation sites (p‐sites) to PBDs involved in subarachnoid hyperphosphorylation events was systematically profiled at molecular level by integrating peptide docking, structural minimization, affinity scoring, and binding assay, from which a number of potent PBD–p‐site interaction pairs were identified. It was revealed that the PBD domains exhibit distinct binding preferences for phosphotyrosine, phosphoserine, and phosphothreonine p‐sites; the PBD‐recognition specificity of different tau p‐sites is not overlapped with each other, and their phosphorylations would therefore regulate varying biological functions in tau signaling. A number of PBD–p‐site pairs were identified to have potent binding potency as compared to others. The KCIP–pS[393–399] pair was found as a strong binder, which was further optimized with a rational peptide design protocol to derive a number of affinity‐improved phosphopeptides. Structural analysis revealed diverse noncovalent chemical forces across the complex interface of KCIP domain with a designed high‐affinity pS[393–399]‐d4, which confers both stability and specificity to the domain–peptide complex system, with affinity improved by 10.9‐fold relative to the native pS[393–399]. [ABSTRACT FROM AUTHOR]

In this work, on the basis of an immobilized metal ion affinity chromatography enrichment strategy, a new kind of covalent organic framework (COF) material for enrichment of phosphorylated peptides and exosomes was successfully prepared in a facile method, and Ti4+ and Nb5+ were used as dual-functional ions (denoted as COF-S–S-COOH-Ti4+/Nb5+). With the advantage of unbiased enrichment towards phosphopeptides, COF-S–S-COOH-Ti4+/Nb5+ shows ultra-high selectivity (maximum molar ratio of β-casein: BSA is 1:20,000) and low limit of detection (0.2 fmol). In addition, the material has an excellent phosphopeptide loading capacity (100 μg/mg) and reusability (at least seven times). Furthermore, applying the material to the actual sample, 4 phosphopeptides were selectively extracted from the serum of renal carcinoma patients. At the same time, exosomes with an intact structure in the serum of renal carcinoma patients were successfully isolated rapidly using this strategy. All experiments have shown that COF-S–S-COOH-Ti4+/Nb5+ exhibits exciting potential in practical applications. [ABSTRACT FROM AUTHOR]

Enrichment of phosphopeptides before mass spectrometry (MS) analysis is essential due to the limitations of low abundance and poor ionization efficiency in complex biological samples. Immobilized metal affinity chromatography (IMAC), especially titanium ion (Ti4+)-IMAC, has become a popular strategy for enrichment of phosphopeptides due to high selectivity and sensitivity. Conventional Ti4+-immobilized macroporous adsorption resin (MAR) fabricated by monolayer modification can preferentially capture mono-phosphopeptide over multi-phosphopeptides, which takes on more functions in the regulation of cell behaviors of organism. In this paper, a kind of monodisperse MAR microsphere with functional polymer brush (Ti4+-Brush@MAR) was prepared and modified via surface-initiated atom transfer radical polymerization (SI-ATRP). Compared with common Ti4+-MAR without polymer brush, Ti4+-Brush@MAR exhibited high enrichment specificity not only for mono-phosphopeptides but also for multi-phosphopeptides in β-casein or milk digest samples. As a result, a total of 93 unique phosphopeptides mapped to 18 phosphoproteins were identified from 5 μL milk, and the limit of detection is 10 fmol. It is expected that Ti4+-Brush@MAR would be utilized to enrich both multi-phosphopeptides and mono-phosphopeptides in additional biological or food samples. [ABSTRACT FROM AUTHOR]

Quantitative phosphoproteomic data has mostly been reported from experiments comparing relative phosphopeptides intensities in two or more different conditions, while the ideal parameter to compare is phosphopeptides occupancies. This term is scarcely used and therefore barely implemented in phosphoproteomics studies, and this should be of concern for the scientific journals. In order to demonstrate the relevance of this issue, here we show how the method of choice affects the interpretation of the data. The phosphoproteomic profile modulated in two AML cell lines after CK2 inhibition with CIGB-300 or CX-4945 is shown. Following the downstream action of CK2 the phosphosite intensity and occupancy results were compared to validate the best approach for quantitative phosphoproteomic studies. Even when the total number of quantified phosphopeptides was higher by using the intensity calculation, in all the cases the percent of CK2 consensus sequences which were down-regulated in response to CK2 inhibition was higher using the phosphosite occupancy quantification. To note, a high number of CK2 consensus sequences was found down-regulated with at least a 10% or 15% of phosphosite occupancy variation illustrating that low thresholds of occupancy modulation might be indicative of biological effect. Additionally, several biological processes only appear significantly over-represented in the phosphoproteome quantified by occupancy. The functional enrichment analysis per ranges of occupancy variations also illustrated clear differences among AML cell lines subjected to CK2 inhibition by CX-4945. A low overlap between the phosphoproteomes quantified by intensity and occupancy was obtained illustrating that new developments in proteomics techniques are needed to improve the performance of the occupancy approach. Even in such context, results indicate that occupancy quantification performs better than phosphorylation quantification based on intensity reinforcing the importance of such quantification approach to describe phosphoproteomic data. [ABSTRACT FROM AUTHOR]