Search

Endowing titanium surfaces with multifunctional properties can reduce implant-related infections and enhance osseointegration. In this study, titanium dioxide nanotubes with strontium doping (STN) were first created on the titanium surface using anodic oxidation and hydrothermal synthesis techniques. Next, casein phosphopeptide (CCP) and an antimicrobial peptide (HHC36) were loaded into the STN with the aid of vacuum physical adsorption (STN-CP-H), giving the titanium surface a dual function of "antimicrobial-osteogenic". The surface of STN-CP-H has a suitable roughness and good hydrophilicity, which is conducive to osteoblasts. STN-CP-H had a 99 % antibacterial rate against S. aureus and E. coli and effectively prevented the growth of bacterial biofilm. Meanwhile, the antibacterial mechanism of STN-CP-H was initially explored with the help of transcriptome sequencing technology. STN-CP-H could greatly increase osteoblast adhesion, proliferation, and expression of osteogenic markers (alkaline phosphatase, runt-related transcription) when CCP and Sr worked together synergistically. In vivo, the STN-CP-H coating could effectively promote new osteogenesis around titanium implant bone and had no toxic effects on heart, liver, spleen, lung and kidney tissues. A potential anti-infection bone healing material, STN-CP-H bifunctional coating developed in this work efficiently inhibited bacterial infection of titanium implants and encouraged early osseointegration., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Chitosan, as a biological macromolecule with excellent biocompatibility, has great potential for application in immobilized metal affinity chromatography (IMAC) strategies. In-depth analysis of low-abundance phosphopeptides in organisms can help reveal the pathological mechanisms of diseases. Here, we developed an IMAC material based on a biomimetic honeycomb chitosan membrane. The material demonstrates excellent biocompatibility, good hydrophilicity, and strong metal chelating capacity, which collectively confer outstanding enrichment properties. The material has high sensitivity (0.05 fmol), great selectivity (1:2000), excellent cycling stability (at least 10 cycles) and acid-base stability. In addition, the material was employed in human serum, successfully enriching 129 phosphopeptides from the serum of gastric cancer patients and 146 phosphopeptides from healthy controls. Sequence logo suggests a potential association between gastric cancer and glutamine. Ultimately, an in-depth gene ontology analysis was carried out on the phosphopeptides that were enriched in the serum samples. Compared to normal controls, our results demonstrated dysregulated expression of biological process, cellular component, and molecular function in gastric cancer patients. This suggests that the disease involves, such as blood coagulation pathways, cholesterol metabolism, and heparin binding. All experimental outcomes converge to demonstrate the substantial promise of the material for applications within proteomics research., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Affinity-based protein depletion and TiO 2 enrichment methods play a crucial role in detection of low-abundant proteins and phosphopeptides enrichment, respectively. Here, we assessed the effectiveness of HSA/IgG (HU2) and Human 7 (HU7) depletion methods and their impact on phosphopeptides coverage through comparative proteome analysis, utilizing in-solution digestion and nano-LC-Orbitrap mass spectrometry (MS). Our results demonstrated that both HU2 and HU7 affinity depletion significantly decreased high-abundant proteins by 1.5-7.8-fold (p < 0.001). A total of 1491 proteins were identified, with 48 proteins showing significant expression in the depleted groups. Notably, cadherin-13, neutrophil defensin 1, APM1, and desmoplakin variant protein were exclusively detected in the HU2/HU7-depleted groups. Furthermore, study on effect of depletion on phosphopeptides revealed an increase in tandem MS spectral counts with notable decrease (∼50%) in peptide spectrum matching in depleted groups, which was attributed to significant reduction in protein counts. Our post translation modification workflow for phosphoproteomics detected 42 phosphorylated peptides, corresponding to 12 phosphoproteins with unique peptide match ≥2 (high false discovery rates confidence). Among them, 10 phosphorylated proteins are highly expressed in depleted groups. Overall, these findings offer valuable insights in selection of protein depletion methods for comprehensive plasma proteomics analysis., (© 2024 Wiley‐VCH GmbH.)

Ferritin, a vital protein required to store iron in a cage-like structure, is critical for maintaining iron balance. Ferritin can be attacked by free radicals during iron reduction and release, thereby leading to oxidative damage. Whether other biomacromolecules such as casein phosphopeptides (CPP) could influence the ferritin's function in iron oxidation and release and affect the ferritin stability remains unclear. This study aims to investigate the effect of CPP on the ferritin‑iron ion interaction, thereby focusing on role of CPP on ferritin stability. Results showed that CPP weakened the iron oxidation activity of ferritin but promoted iron release. Moreover, CPP could effectively chelate iron, capture hydroxyl radicals, and reduce the degradation of ferritin. This study highlights the role of CPP in the ferritin‑iron relationship, and lays a foundation for understanding the interaction between ferritin, peptides, and metal ions., Competing Interests: Declaration of competing interest There are no conflicts of interest to declare., (Copyright © 2024. Published by Elsevier Ltd.)

Cells contain intricate protein nanostructures, but replicating them outside of cells presents challenges. One such example is the vertical fibronectin pillars observed in embryos. Here, we demonstrate the creation of cell-free vertical fibronectin pillar mimics using nonequilibrium self-assembly. Our approach utilizes enzyme-responsive phosphopeptides that assemble into nanotubes. Enzyme action triggers shape changes in peptide assemblies, driving the vertical growth of protein nanopillars into bundles. These bundles, with peptide nanotubes serving as a template to remodel fibronectin, can then recruit collagen, which forms aggregates or bundles depending on their types. Nanopillar formation relies on enzyme-catalyzed nonequilibrium self-assembly and is governed by the concentrations of enzyme, protein, peptide, the structure of the peptide, and peptide assembly morphologies. Cryo-EM reveals unexpected nanotube thinning and packing after dephosphorylation, indicating a complex sculpting process during assembly. Our study demonstrates a cell-free method for constructing intricate, multiprotein nanostructures with directionality and composition.

A dipeptide-based bifunctional material immobilized with Ti 4+ (denoted as APE-MBA-VPA-Ti 4+ ) was developed using precipitation polymerization. This polymer combines hydrophilic interaction liquid chromatography (HILIC) and immobilized metal affinity chromatography (IMAC) enrichment strategies, allowing for the individual and simultaneous enrichment of glycopeptides and phosphopeptides. It demonstrated high sensitivity (0.1 fmol μL -1 for glycopeptides, 0.005 fmol μL -1 for phosphopeptides), strong selectivity (molar ratio HRP: BSA = 1:1000, β-casein: BSA = 1:2500), consistent reusability (10 cycles) and satisfactory recovery rate (93.5 ± 1.8 % for glycopeptides, 91.6 ± 0.6 % for phosphopeptides) in the individual enrichment. Utilizing nano LC-MS/MS technology, the serum of liver cancer patients was analyzed after enrichment individually, resulting in the successful capture of 333 glycopeptides covering 262 glycosylation sites, corresponding to 131 glycoproteins, as well as 67 phosphopeptides covering 57 phosphorylation sites, related to 48 phosphoproteins. In comparison, the serum of normal healthy individuals yielded a total of 283 glycopeptides covering 244 glycosylation sites corresponding to 126 glycoproteins, as well as 66 phosphopeptides covering 56 phosphorylation sites related to 37 phosphoproteins. Label-free quantification identified 10 differentially expressed glycoproteins and 8 differentially expressed phosphoproteins in the serum of liver cancer patients. Among them, glycoproteins (HP, BCHE, AGT, C3, and PROC) and phosphoproteins (ZYX, GOLM1, GP1BB, CLU, and TNXB) showed upregulation and displayed potential as biomarkers for liver cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Compared to advancements in single-cell proteomics, phosphoproteomics sensitivity has lagged behind due to low abundance, complex sample preparation, and substantial sample input requirements. We present a simple and rapid one-pot phosphoproteomics workflow (SOP-Phos) integrated with data-independent acquisition mass spectrometry (DIA-MS) for microscale phosphoproteomic analysis. SOP-Phos adapts sodium deoxycholate based one-step lysis, reduction/alkylation, direct trypsinization, and phosphopeptide enrichment by TiO 2 beads in a single-tube format. By reducing surface adsorptive losses via utilizing n -dodecyl β-d-maltoside precoated tubes and shortening the digestion time, SOP-Phos is completed within 3-4 h with a 1.4-fold higher identification coverage. SOP-Phos coupled with DIA demonstrated >90% specificity, enhanced sensitivity, lower missing values (<1%), and improved reproducibility (8%-10% CV). With a sample size-comparable spectral library, SOP-Phos-DIA identified 33,787 ± 670 to 22,070 ± 861 phosphopeptides from 5 to 0.5 μg cell lysate and 30,433 ± 284 to 6,548 ± 21 phosphopeptides from 50,000 to 2,500 cells. Such sensitivity enabled mapping key lung cancer signaling sites, such as EGFR autophosphorylation sites Y1197/Y1172 and drug targets. The feasibility of SOP-Phos-DIA was demonstrated on EGFR-TKI sensitive and resistant cells, revealing the interplay of multipathway Hippo-EGFR-ERBB signaling cascades underlying the mechanistic insight into EGFR-TKI resistance. Overall, SOP-Phos-DIA is an efficient and robust protocol that can be easily adapted in the community for microscale phosphoproteomic analysis.

In the present study, click chemistry and Schiff base reactions were simultaneously applied to prepare polymer brush (PEG)-functionalized MOF materials (UiO-66-NH 2 ) and immobilized with Ti 4+ (MOF-Brush-THBA-Ti 4+ ) 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., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

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 (Fe 3 O 4 @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. Fe 3 O 4 @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, Fe 3 O 4 @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 Fe 3 O 4 @MIL-125-POM for efficient identification of low-abundance phosphopeptides., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

This research aimed to investigate the characteristics of casein phosphopeptides in Chinese human milk, and their potential relationship to infant growth. Using the liquid chromatography-Orbitrap-mass spectrometry technique, a total of 15 casein phosphopeptides were identified from 200 human milk samples. Also, our results indicate that casein phosphopeptides were phosphorylated with only one phosphate. The relative concentrations of casein phosphopeptides at 6 months postpartum were increased compared with milk at 2 months (FDR < 0.05). Significantly positive correlations were observed between casein phosphopeptides and infant growth, as shown by four casein phosphopeptides were positively correlated with the infants' weight-for-age Z-scores (r s range from 0.20 to 0.29), and three casein phosphopeptides were positively correlated with the infants' length-for-age Z-scores (r s range from 0.19 to 0.27). This study is the first to reveal the phosphorylated level and composition of casein phosphopeptides in Chinese human milk, and their potential relationship with infant growth., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Calcium is the most abundant mineral in the human body and is involved in critical physiological and cellular processes. It is essential for the development, maintenance, and integrity of bone tissue throughout life. Identifying new natural food-grade chelating agents to improve calcium uptake is of increasing interest. Casein phosphopeptides (CPPs), highly phosphorylated peptides obtained after enzymatic hydrolysis of caseins, represent promising calcium-chelating candidates. The aim of this study was to investigate, using cell culture models, the ability of a digested milk matrix enriched in CPPs to regulate calcium transport through the intestinal barrier and elucidate the involved mechanisms. To this end, a CPP-preparation underwent in vitro static digestion and was subsequently incubated with an intestinal barrier model to monitor calcium uptake and transport. Our results demonstrated that the digested CPP preparation enhanced the trans -epithelial calcium transport via paracellular pathways and that CPPs, identified by peptidomics, crossed the intestinal barrier in the same time.

A porphyrin-based titanium-rich porous organic polymer (Th-PPOPs@Ti 4+ ) 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@Ti 4+ 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@Ti 4+ 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., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

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.

For mass spectrometry (MS)-based phosphoproteomics studies, sample pretreatment is an essential step for efficient identification of low-abundance phosphopeptides. Herein, a cobalt phthalocyanine-modified magnetic metal-organic framework (MOF) (Fe 3 O 4 @MIL-101-CoPc) was prepared and applied to enrich phosphopeptides before MS analysis. Fe 3 O 4 @MIL-101-CoPc exhibited an excellent magnetic response (74.98 emu g -1 ) and good hydrophilicity (7.75°), which were favorable for the enrichment. Fe 3 O 4 @MIL-101-CoPc showed good enrichment performance with high selectivity (1:1:5000), sensitivity (0.1 fmol), reusability (10 circles), and recovery (91.3%). Additionally, the Fe 3 O 4 @MIL-101-CoPc-based MS method was able to successfully detect 827 phosphopeptides from the A549 cell lysate, demonstrating a high enrichment efficiency (89.3%). This study promotes the application of postfunctionalized MOFs for phosphoproteomics analysis.

Protein phosphorylation plays an important role in cellular signaling and disease development. Advances in mass spectrometry-based proteomics have enabled qualitative and quantitative phosphorylation studies as well as in-depth biological explorations for biomarker discovery and signaling pathway analysis. However, the dynamic changes that occur during phosphorylation and the low abundance of target analytes render direct analysis difficult because mass spectral detection offers no selectivity, unlike immunoassays such as Western blot and enzyme-linked immunosorbent assay (ELISA). The present study aimed to solve one of the key problems in the specific and efficient isolation of phosphorylated peptides. A method based on a magnetic carbon nitride composite coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was developed for the enrichment and analysis of phosphopeptides with low abundance in complex samples. Magnetic carbon nitride composite was synthesized and characterized by electron microscopy, infrared spectroscopy, and X-ray diffractometry. The composite showed a well-distributed two-dimensional layered structure and functional groups with excellent paramagnetic performance. Two classical phosphoproteins, namely, α - and β -caseins, were selected as model phosphorylated samples to assess the performance of the proposed enrichment technique. The magnetic carbon nitride composite exhibited high selectivity and sensitivity for phosphopeptide enrichment. The limit of detection was determined by MALDI-TOF-MS analysis to be 0.1 fmol. The selectivity of the method was investigated using the digest mixtures of α -casein, β -casein, and bovine serum albumin (BSA) with different mass ratios (1∶1∶1000, 1∶1∶2000, and 1∶1∶5000). Direct analysis of the samples revealed the dominance of spectral signals from the abundant peptides in BSA. After enrichment with the magnetic carbon nitride composite, the high concentration of background proteins was washed away and only the signals of the phosphopeptides were captured. The signals from the casein proteins were clearly observed with little background noise, indicating the high selectivity of the composite material. The robustness of the method was tested by assessing the reusability of the same batch of magnetic carbon nitride materials over 20 cycles of enrichment. The composite showed nearly the same enrichment ability even after several cycles of reuse, demonstrating its potential applicability for a large number of clinical samples. Finally, the method was applied to the analysis of phosphopeptides from several commonly used phosphoprotein-containing samples, including skimmed milk digest, human serum, and human saliva; these samples are significant in the analysis of food quality, disease biomarkers, and liquid biopsies for cancer. Without enrichment, no phosphopeptide was detected because of the high abundance of nonphosphopeptide materials dominating the spectral signals obtained. After pretreatment with the developed magnetic carbon nitride composite, most of the phosphosites were identified with high selectivity and sensitivity via MALDI-TOF-MS. These results revealed the practicality of the developed approach for clinical applications. In addition, our method may potentially be employed for phosphoproteomics with real complex biological samples.

Detection of endogenous peptides, especially those with modifications (such as phosphorylation) in biofluids, can serve as an indicator of intracellular pathophysiology. Although great progress has been made in phosphoproteomics in recent years, endogenous phosphopeptidomics has largely lagged behind. One main hurdle in endogenous phosphopeptidomics analysis is the coexistence of proteins and highly abundant nonmodified peptides in complex matrices. In this study, we developed an approach using zirconium(IV)-grafted mesoporous beads to enrich phosphopeptides, followed by analysis with a high resolution nanoRPLC-MS/MS system. The bifunctional material was first tested with digests of standard phosphoproteins and HeLa cell lysates, with excellent enrichment performance achieved. Given the size exclusion nature, the beads were directly applied for endogenous phosphopeptidomic analysis of serum samples from pancreatic ductal adenocarcinoma (PDAC) patients and controls. In total, 329 endogenous phosphopeptides (containing 113 high confidence sites) were identified across samples, by far the largest endogenous phosphopeptide data set cataloged to date. In addition, the method was readily applied for phosphoproteomics of the same set of samples, with 172 phosphopeptides identified and significant changes in dozens of phosphopeptides observed. Given the simplicity and robustness of the proposed method, we envision that it can be readily used for comprehensive phosphorylation studies of serum and other biofluid samples.

The specific enrichment of multi-phosphopeptides in the presence of non-phosphopeptides and mono-phosphopeptides was still a challenge for phosphoproteomics research. Most of these enrichment materials relied on Zn, Ti, Sn, and other rare precious metals as the bonding center to enrich multi-phosphopeptides while ignoring the use of common metal elements. The addition of rare metals increased the cost of the experiment, which was not conducive to their large-scale application in biomedical proteomics laboratories. In addition, multiple high-speed centrifugation steps also resulted in the loss of low-abundance multi-phosphopeptides in the treatment procedure of biological samples. This study proposed the use of calcium, a common element, as the central bonding agent for synthesizing magnetic calcium phosphate materials (designated as CaP-Fe 3 O 4 ). These materials aim to capture multi-phosphopeptides and identifying phosphorylation sites. The current results demonstrate that CaP-Fe 3 O 4 exhibited excellent selection specificity, high sensitivity, and stability in the enrichment of multi-phosphopeptides and the identification of phosphorylation sites. Additionally, the introduction of magnetic separation not only reduced the time required for multi-phosphopeptides enrichment but also prevented the loss of these peptides during high-speed centrifugation. These findings contribute to the widespread application and advancement of phosphoproteomics research., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Phosphorylation is a major constituent of the CTD code, which describes the set of post-translational modifications on 52 repeats of a YSPTSPS consensus heptad that orchestrates the binding of regulatory proteins to the C-terminal domain (CTD) of RNA polymerase II. Phospho-specific antibodies are used to detect CTD phosphorylation patterns. However, their recognition repertoire is underexplored due to limitations in the synthesis of long multiphosphorylated peptides. Herein, we describe the development of a synthesis strategy that provides access to multiphosphorylated CTD peptides in high purity without HPLC purification for immobilization onto microtiter plates. Native chemical ligation was used to assemble 12 heptad repeats in various phosphoforms. The synthesis of >60 CTD peptides, 48-90 amino acids in length and containing up to 6 phosphosites, enabled a detailed and rapid analysis of the binding characteristics of different anti-pSer2 antibodies. The three antibodies tested showed positional selectivity with marked differences in the affinity of the antibodies for pSer2-containing peptides. Furthermore, the length of the phosphopeptides allowed a systematic analysis of the multivalent chelate-type interactions. The absence of multivalency-induced binding enhancements is probably due to the high flexibility of the CTD scaffold. The effect of clustered phosphorylation proved to be more complex. Recognition of pSer2 by anti-pSer2-antibodies can be prevented and, perhaps surprisingly, enhanced by the phosphorylation of "bystander" amino acids in the vicinity. The results have relevance for functional analysis of the CTD in cell biological experiments.

Mass-spectrometry-based methods have made significant progress in the characterization of post-translational modifications (PTMs) in peptides and proteins; however, room remains to improve fragmentation methods. Ideal MS/MS methods are expected to simultaneously provide extensive sequence information and localization of PTM sites and retain labile PTM groups. This collection of criteria is difficult to meet, and the various activation methods available today offer different capabilities. In order to examine the specific case of phosphorylation on peptides, we investigate electron transfer dissociation (ETD), electron-activated dissociation (EAD), and 193 nm ultraviolet photodissociation (UVPD) and compare all three methods with classical collision-induced dissociation (CID). EAD and UVPD show extensive backbone fragmentation, comparable in scope to that of CID. These methods provide diverse backbone fragmentation, producing a / x , b / y , and c / z ions with substantial sequence coverages. EAD displays a high retention efficiency of the phosphate modification, attributed to its electron-mediated fragmentation mechanisms, as observed in ETD. UVPD offers reasonable retention efficiency, also allowing localization of the PTM site. EAD experiments were also performed in an LC-MS/MS workflow by analyzing phosphopeptides spiked in human plasma, and spectra allow accurate identification of the modified sites and discrimination of isomers. Based on the overall performance, EAD and 193 nm UVPD offer alternative options to CID and ETD for phosphoproteomics.

Highly selective extraction of phosphopeptides is necessary before mass spectrometry (MS) analysis. Herein, zirconium phthalocyanine-modified magnetic nanoparticles were prepared through a simple method. The Fe-O groups on Fe 3 O 4 and the zirconium ions on phthalocyanine had a strong affinity for phosphopeptides based on immobilized metal ion affinity chromatography (IMAC). The enrichment platform exhibited low detection limit (0.01 fmol), high selectivity (α-/β-casein/bovine serum albumin, 1/1/5000), good reusability (10 circles), and recovery (91.1 ± 1.1%) toward phosphopeptides. Nonfat milk, human serum, saliva, and A549 cell lysate were employed as actual samples to assess the applicability of the enrichment protocol. Metallo-phthalocyanine will be a competitive compound for designing highly efficient adsorbents and offers a new approach to phosphopeptide analysis.

One of the most crucial and prevalent post-translational modifications is the phosphorylation of proteins. The study and examination of protein phosphorylation hold immense importance in comprehending disease mechanisms and discovering novel biomarkers. However, the inherent low abundance, low ionization efficiency, and coexistence with non phosphopeptides seriously affect the direct analysis of phosphopeptides by mass spectrometry. In order to tackle these problems, it is necessary to carry out selective enrichment of phosphopeptides prior to conducting mass spectrometry analysis. Herein, magnetic chitosan nanoparticles were developed by incorporating arginine, and were then utilized for phosphopeptide enrichment. A tryptic digest of β-casein was chosen as the standard substance. After enrichment, combined with matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), the detection limit of the method was 0.4 fmol. The synthesized magnetic material demonstrated great potential in the detection of phosphopeptides in complex samples, as proven by its successful application in detecting phosphopeptides in skim milk and human saliva samples.

A facile and mild method based on self-assembled lysozyme (LYZ) to fabricate bifunctional MNPs@UIO-66-Arg core-shell-satellite nanocomposites (CSSNCs) is reported for the high-efficiency enrichment of phosphopeptides. Under physiological conditions, LYZ rapidly self-assembled into a robust coating on Fe 3 O 4 @SiO 2 magnetic nanoparticles (MNPs) with abundant surface functional groups, which effectively mediate heterogeneous nucleation and growth of UIO-66 nanocrystals. Well-defined MNPs@UIO-66 CSSNCs with stacked pores, showing high specific surface area (333.65 m 2 g - 1 ) and low mass transfer resistance, were successfully fabricated by fine-tuning of the reaction conditions including reaction time and acetic acid content. Furthermore, the UIO-66 shells were further modified with arginine to obtain bifunctional MNPs@UIO-66-Arg CSSNCs. Thanks to the unique morphology and synergistic effect of Zr-O clusters and guanidine groups, the bifunctional MNPs@UIO-66-Arg CSSNCs exhibited outstanding enrichment performance for phosphopeptides, delivering a low limit of detection (0.1 fmol), high selectivity (β-casein/BSA, mass ratio 1:2000), and good capture capacity (120 mg g - 1 ). The mechanism for phosphopeptides capture may attribute to the hydrogen bonds, electrostatic interactions, and Zr-O-P bonds between phosphate groups in peptides and guanidyl/Zr-O clusters on bifunctional MNPs@UIO-66-Arg CSSNCs. In addition, the small stacking pores on the core-shell-satellite architecture may selectively capture phosphopeptides with low molecular weight, eliminating interference of other large molecular proteins in complex biological samples., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Reasonable design and construction of functionalized materials are of great importance for the enrichment of global phosphopeptides. In this work, Ti 4+ 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-Ti 4+ ) 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-Ti 4+ 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., (© 2024 Wiley-VCH GmbH.)

The comprehensive investigation of protein phosphorylation and glycosylation aids in the discovery of novel biomarkers as well as the understanding of the pathophysiology of illness. In this work, a nitrogen/titanium-rich porous organic polymer was developed by copolymerizing carbohydrazide (CH) and 2,3-dihydroxyterephthalaldehyde (2,3-Dha) and modifying with Ti 4+ (CH-Dha-Ti 4+ ). The adequate nitrogen contributes to the enrichment of glycopeptides via HILIC, while titanium benefits from capturing phosphopeptides through IMAC. The proposed method exhibits excellent selectivity (1 : 1000, both for glycopeptides and phosphopeptides), LOD (for glycopeptides: 0.05 fmol μL -1 , for phosphopeptides: 0.2 fmol), loading capacity (for glycopeptides: 100 mg g -1 , for phosphopeptides: 125 mg g -1 ) and size-exclusion effect (1 : 10 000, both for glycopeptides and phosphopeptides). Furthermore, CH-Dha-Ti 4+ was applied to capture glycopeptides and phosphopeptides from human serum; 205 glycopeptides and 45 phosphopeptides were detected in the serum of normal controls; and 294 glycopeptides and 63 phosphopeptides were found in the serum of uremia patients after being analyzed by nano LC-MS/MS. The discovered glycopeptides and phosphopeptides were involved in several molecular biological processes and activities, according to a gene ontology study.

Background: Calcium is important in the formation of bones and teeth, cell metabolism, and other physiological activities. In this work, casein phosphopeptide-calcium chelate (CPP-Ca) was synthesized and the optimal process parameters for the chelation reaction were obtained. The bioavailability of calcium in CPP-Ca was investigated by in vitro gastrointestinal simulated digestion. The existence of phytic acid and oxalic acid in the digestion system was evaluated to clarify the calcium holding ability of casein phosphopeptide (CPP). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify oligopeptides from CPP-Ca., Results: The optimal process parameters for the chelation reaction were: peptide concentration 7.76 mgmL -1 , pH 8.54, and reaction temperature 43.3 °C. The digestion in vitro results indicated that the calcium release rate of CPP-Ca in the stomach for 2 h reached 85%, and about 50% of the ionized calcium was re-chelated with CPP in the intestine. Phytic acid and oxalic acid could lead to a sharp decrease in soluble calcium but around 50% of the calcium was still retained in the form of chelates in the presence of CPP. The LC-MS/MS identified 19 casein-derived oligopeptides after digestion, and calcium modifications were found on eight peptides derived from β-casein and α s2 -casein., Conclusions: This study clarified the excellent calcium holding capacity of CPP in the presence of phytic acid and oxalic acid. Liquid chromatography-tandem mass spectrometry also revealed peptide changes, and identified peptides that chelate with calcium. These findings provided significant insights that could be relevant to the further utilization and product development of peptide-calcium chelate in the food industry. © 2023 Society of Chemical Industry., (© 2023 Society of Chemical Industry.)

Antibodies play a crucial role in monitoring post-translational modifications, like phosphorylation, which regulates protein activity and location; however, commercial polyclonal and monoclonal antibodies have limitations in renewability and engineering compared to recombinant affinity reagents. A scaffold based on the Forkhead-associated domain (FHA) has potential as a selective affinity reagent for this post-translational modification. Engineered FHA domains, termed phosphothreonine-binding domains (pTBDs), with limited cross-reactivity were isolated from an M13 bacteriophage display library by affinity selection with phosphopeptides corresponding to human mTOR, Chk2, 53BP1, and Akt1 proteins. To determine the specificity of the representative pTBDs, we focused on binders to the pT543 phosphopeptide (536-IDEDGENpTQIEDTEP-551) of the DNA repair protein 53BP1. ELISA and western blot experiments have demonstrated the pTBDs are specific to phosphothreonine, demonstrating the potential utility of pTBDs for monitoring the phosphorylation of specific threonine residues in clinically relevant human proteins., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Charging of analytes is a prerequisite for performing mass spectrometry analysis. In proteomics, electrospray ionization is the dominant technique for this process. Although the observation of differences in the peptide charge state distribution (CSD) is well-known among experimentalists, its analytical value remains underexplored. To investigate the utility of this dimension, we analyzed several public data sets, comprising over 250,000 peptide CSD profiles from the human proteome. We found that the dimensions of the CSD demonstrate high reproducibility across multiple laboratories, mass analyzers, and extensive time intervals. The general observation was that the CSD enabled effective partitioning of the peptide property space, resulting in enhanced discrimination between sequence and constitutional peptide isomers. Next, by evaluating the CSD values of phosphorylated peptides, we were able to differentiate between phosphopeptides that indicate the formation of intramolecular structures in the gas phase and those that do not. The reproducibility of the CSD values (mean cosine similarity above 0.97 for most of the experiments) qualified CSD data suitable to train a deep-learning model capable of accurately predicting CSD values (mean cosine similarity - 0.98). When we applied the CSD dimension to MS1- and MS2-based proteomics experiments, we consistently observed around a 5% increase in protein and peptide identification rate. Even though the CSD dimension is not as effective a discriminator as the widely used retention time dimension, it still holds the potential for application in direct infusion proteomics.

High-performance reusable materials from renewable resources are rare and urgently required in bioseparation. Herein, a series of tannic acid-chitosan composite membranes for the enrichment of phosphopeptides were fabricated by the freeze casting method. First, a tannic acid-chitosan composite membrane was acquired via the multiple hydrogen bonds between tannic acid and chitosan, which had a long-range aligned three-dimensional microstructure. Second, a covalent-hydrogen bond hybrid composite was also fabricated, with stable and aligned honeycomb-like microstructures that formed by the synergy of covalence and hydrogen bonding. Besides, a ternary composite membrane was "one-pot" synthesized by the copolymerization of tannic acid, chitosan, and Ti 4+ ions, indicating the feasibility of involving metal ions in the composition of the polymer skeleton in place of additional modification steps. The as-prepared chitosan composite membranes exhibited excellent performance in the enrichment of phosphopeptides from β-casein tryptic digest and human serum. Benefitting from the long-range aligned honeycomb-like structure coordinated by hydrogen bonds and covalent bonds, and a large number of pyrogallol functional groups provided by tannic acid, the covalent-hydrogen bond hybrid membrane showed excellent reusability and could be reused up to 16 times in phosphopeptide enrichment, as far as we know, which is the best reported result to date.

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., (Copyright © 2023 The Author. Published by Elsevier Inc. All rights reserved.)

Hydrogen-bonded organic frameworks (HOFs) have emerged as a promising class of materials for applications of separation and enrichment. Utilizing multiple-ligands to construct HOFs is a promising avenue towards the development of structurally stable and functionally diverse frameworks, offering opportunities to create customized binding sites for selective recognition of biomolecules. In recent years, due to the crucial role that protein post-translational modifications (PTMs) play in maintaining protein function and regulating signaling pathways, and the growing recognition of the extensive cross-talk that can occur between PTMs, simultaneous analysis of different types of PTMs represents a requirement of a new generation of enrichment materials. Here, for the first attempt, we report a dual-ligand HOF constructed from borate anion and guanidinium cation for the simultaneous identification of glycopeptides and phosphopeptides, especially mono-phosphopeptides. According to theoretical calculations, the HOF functional sites display a synergistic "matching" effect with mono-phosphopeptides, resulting in a stronger enrichment effect for mono-phosphopeptides as compared to multi-phosphopeptides. Also, due to its high hydrophilicity and boronate affinity, this material can efficiently capture glycoproteins. HOF is set to become an active research direction in the development of highly efficient simultaneous protein enrichment materials, and offers a new approach for comprehensive PTMs analysis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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.

Exosomes, which can be used to investigate various disease processes, are novel disease markers that have been extensively studied in recent years. In this work, zirconium-rich porphyrin-based porous organic polymers (Imi-Pops-Zr) were synthesized by a facile and low-cost strategy for specific enrichment and isolation of phosphorylated peptides and exosomes. The proposed material demonstrates a low detection limit (0.5 fmol), a high selectivity (bovine serum albumin (BSA): β-casein = 1000:1), and a loading capability of 100 mg/g for phosphopeptides. For complex practical samples, after enrichment with Imi-Pops-Zr, 4 characteristic phosphopeptides from human serum, 20 and 12 phosphopeptides from human saliva and defatted milk were detected, respectively. Besides, 74 phosphorylated peptides with 67 phosphorylation sites belonging to 61 phosphoproteins and 67 phosphorylated peptides with 63 phosphorylation sites belonging to 65 phosphoproteins were detected from the serum of normal controls and uremic patients, respectively. Biological processes, cellular components and molecular functions revealed that interleukin-6, tumor necrosis factor, high density lipoprotein and proteases binding may be associated with uremia. Furthermore, Imi-Pops-Zr was successfully used to enrich and isolate exosomes from human serum. The experimental results show that Imi-Pops-Zr has promising application in the specific enrichment of phosphorylated peptides and exosomes in complex bio-samples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Protein phosphorylation is an important post-translational modification (PTM), which is involved in many important cellular functions. Understanding protein phosphorylation at the molecular level is critical to deciphering its relevant biological processes and signaling networks. Mass spectrometry (MS) has become a powerful tool for the comprehensive profiling of protein phosphorylation. Yet the low ionization efficiency and low abundance of phosphopeptides among complex biological samples make its MS analysis challenging; an enrichment strategy with high efficiency and selectivity is always necessary prior to MS analysis. In this study, we developed a phosphorylated cotton-fiber-based Ti(IV)-IMAC material (termed as Cotton Ti-IMAC) that can serve as a novel platform for phosphopeptide enrichment. The cotton fiber can be effectively grafted with phosphate groups covalently in a single step, where the titanium ions can then be immobilized to enable capturing phosphopeptides. The material can be prepared using cost-effective reagents within only 4 h. Benefiting from the flexibility and filterability of cotton fibers, the material can be easily packed as a spin-tip and make the enrichment process convenient. Cotton Ti-IMAC successfully enriched phosphopeptides from protein standard digests and exhibited a high selectivity (BSA/β-casein = 1000:1) and excellent sensitivity (0.1 fmol/μL). Moreover, 2354 phosphopeptides were profiled in one LC-MS/MS injection after enriching from only 100 μg of HeLa cell digests with an enrichment specificity of up to 97.51%. Taken together, we believe that Cotton Ti-IMAC can serve as a widely applicable and robust platform for achieving large-scale phosphopeptide enrichment and expanding our knowledge of phosphoproteomics in complex biological systems.

Sodium bicarbonate stress caused by NaHCO 3 is one of the most severe abiotic stresses affecting agricultural production worldwide. However, little attention has been given to the molecular mechanisms underlying plant responses to sodium bicarbonate stress. To understand phosphorylation events in signaling pathways triggered by sodium bicarbonate stress, TMT-labeling-based quantitative phosphoproteomic analyses were performed on soybean leaf and root tissues under 50 mM NaHCO 3 treatment. In the present study, a total of 7856 phosphopeptides were identified from cultivated soybeans ( Glycine max L. Merr.), representing 3468 phosphoprotein groups, in which 2427 phosphoprotein groups were newly identified. These phosphoprotein groups contained 6326 unique high-probability phosphosites (UHPs), of which 77.2% were newly identified, increasing the current soybean phosphosite database size by 43.4%. Among the phosphopeptides found in this study, we determined 67 phosphopeptides (representing 63 phosphoprotein groups) from leaf tissue and 554 phosphopeptides (representing 487 phosphoprotein groups) from root tissue that showed significant changes in phosphorylation levels under sodium bicarbonate stress (fold change >1.2 or <0.83, respectively; p < 0.05). Localization prediction showed that most phosphoproteins localized in the nucleus for both leaf and root tissues. GO and KEGG enrichment analyses showed quite different enriched functional terms between leaf and root tissues, and more pathways were enriched in the root tissue than in the leaf tissue. Moreover, a total of 53 different protein kinases and 7 protein phosphatases were identified from the differentially expressed phosphoproteins (DEPs). A protein kinase/phosphatase interactor analysis showed that the interacting proteins were mainly involved in/with transporters/membrane trafficking, transcriptional level regulation, protein level regulation, signaling/stress response, and miscellaneous functions. The results presented in this study reveal insights into the function of post-translational modification in plant responses to sodium bicarbonate stress.

Casein kinase 1 (CK1) is serine/threonine protein kinase highly conserved among eukaryotes, and regulates multiple developmental and signaling events through phosphorylation of target proteins. Arabidopsis early flowering 1 (EL1)-like (AELs) are plant-specific CK1s with varied functions, but identification and validation of their substrates is a major bottleneck in elucidating their physiological roles. Here, we conducted a quantitative phosphoproteomic analysis in data-independent acquisition mode to systematically identify CK1 substrates. We extracted proteins from seedlings overexpressing individual AEL genes (AEL1/2/3/4-OE) or lacking AEL function (all ael single mutants and two triple mutants) to identify the high-confidence phosphopeptides with significantly altered abundance compared to wild-type Col-0. Among these, we selected 3985 phosphopeptides with higher abundance in AEL-OE lines or lower abundance in ael mutants compared with Col-0 as AEL-upregulated phosphopeptides, and defined 1032 phosphoproteins. Eight CK1s substrate motifs were enriched among AEL-upregulated phosphopeptides and verified, which allowed us to predict additional candidate substrates and functions of CK1s. We functionally characterized a newly identified substrate C3H17, a CCCH-type zinc finger transcription factor, through biochemical and genetic analyses, revealing a role for AEL-promoted C3H17 protein stability and transactivation activity in regulating embryogenesis. As CK1s are highly conserved across eukaryotes, we searched the rice, mouse, and human protein databases using newly identified CK1 substrate motifs, yielding many more candidate substrates than currently known, largely expanding our understanding of the common and distinct functions exerted by CK1s in Arabidopsis and humans, facilitating future mechanistic studies of CK1-mediated phosphorylation in different species., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest., (Copyright © 2023 Science China Press. Published by Elsevier B.V. All rights reserved.)

Plant phosphoproteomics provides a global view of phosphorylation-mediated signaling in plants; however, it demands high-throughput methods with sensitive detection and accurate quantification. Despite the widespread use of protein precipitation for removing contaminants and improving sample purity, it limits the sensitivity and throughput of plant phosphoproteomic analysis. The multiple handling steps involved in protein precipitation lead to sample loss and process variability. Herein, we developed an approach based on suspension trapping (S-Trap), termed tandem S-Trap-IMAC (immobilized metal ion affinity chromatography), by integrating an S-Trap micro-column with a Fe-IMAC tip. Compared with a precipitation-based workflow, the tandem S-Trap-IMAC method deepened the coverage of the Arabidopsis ( Arabidopsis thaliana ) phosphoproteome by more than 30%, with improved number of multiply phosphorylated peptides, quantification accuracy, and short sample processing time. We applied the tandem S-Trap-IMAC method for studying abscisic acid (ABA) signaling in Arabidopsis seedlings. We thus discovered that a significant proportion of the phosphopeptides induced by ABA are multiply phosphorylated peptides, indicating their importance in early ABA signaling and quantified several key phosphorylation sites on core ABA signaling components across four time points. Our results show that the optimized workflow aids high-throughput phosphoproteome profiling of low-input plant samples.

There is growing interest in the development of materials for enriching proteins and phosphoproteins from complex sample matrices for mass spectrometric analysis. Herein, we designed and synthesized two types of magnetic resin composites, i.e. , MTS9200@Fe 3 O 4 and FPA90CL@Fe 3 O 4 , and assessed their applications as adsorbents for enriching proteins, peptides and phosphopeptides. With the combination of Fe 3+ -IMAC interaction (MTS9200) or electrostatic attraction (FPA90CL) of resins and the adsorption of Fe 3 O 4 , the prepared composites exhibited higher capacities for adsorbing a protein (bovine serum albumin, at 195.71 and 135.03 mg g -1 for MTS9200@Fe 3 O 4 and FPA90CL@Fe 3 O 4 , respectively) than MTS9200, FPA90CL and Fe 3 O 4 . In addition, due to the contributions of the hydrophobic skeleton of resins and Fe 3 O 4 , the magnetic resin composites allowed for efficient enrichment of peptides. Moreover, through Fe 3+ -IMAC interaction or electrostatic attraction of resins and Fe-O MOAC interaction of Fe 3 O 4 with phosphate groups, phosphopeptides could also be captured. Furthermore, we employed the prepared composites for enriching proteins and phosphopeptides from human serum, where 466 and 506 proteins, and 434 and 356 phosphorylation sites, were detected from human serum after being processed with FPA90CL@Fe 3 O 4 and MTS9200@Fe 3 O 4 , respectively. Together, our work revealed the great potential of magnetic resin composites as enrichment materials for proteomics and phosphoproteomics analysis.

Human papillomaviruses (HPVs) can increase the proliferation of infected cells during HPV-driven abnormalities, such as cervical cancer or benign warts. To date, more than 200 HPV genotypes have been identified, most of which are classified into three major genera: Alphapapillomavirus, Betapapillomavirus, and Gammapapillomavirus. HPV genomes commonly encode two structural (L1 and L2) and seven functional (E1, E2, E4-E7, and E8) proteins. L2, the minor structural protein of HPVs, not only serves as a viral capsid component but also interacts with various human proteins during viral infection. A recent report revealed that L2 of HPV16 recruits polo-like kinase 1 (Plk1), a master regulator of eukaryotic mitosis and cell cycle progression, for the delivery of viral DNA to mitotic chromatin during HPV16 infection. In this study, we verified the direct and potent interactions between the polo-box domain (PBD) of Plk1 and PBD-binding motif (S-S-pT-P)-containing phosphopeptides derived from L2 of HPV16/HPV18 (high-risk alphapapillomaviruses), HPV5b (low-risk betapapillomavirus), and HPV4 (low-risk gammapapillomavirus). Subsequent structural determination of the Plk1 PBD bound to the HPV18 or HPV4 L2-derived phosphopeptide demonstrated that they interact with each other in a canonical manner, in which electrostatic interactions and hydrogen bonds play key roles in sustaining the complex. Therefore, our structural and biochemical data imply that Plk1 is a broad binding target of L2 of various HPV genotypes belonging to the Alpha-, Beta-, and Gammapapillomavirus genera., (© 2023. The Author(s), under exclusive licence to Microbiological Society of Korea.)

Multisite phosphorylation of proteins regulates various cellular life activities, however, the capture of low abundance multi-phosphopeptides from biosamples and identification of phosphorylation sites are largely limited due to the limited enrichment materials and their unclear interactions with multi-phosphopeptides. Here we propose using two cheap raw materials (CaCl 2 ·2H 2 O and Na 2 HPO 4 ·12H 2 O) in 10 min at room temperature to synthesize the structurally simple Nanometric Calcium Phosphate (CaP) to resolve this challenge. The current results showed that the "simple" CaP has good selection specificity, high sensitivity and stability for multi-phosphopeptides enrichment and the identification of phosphorylation sites, which facilitate the popularization and application of phosphoproteomics research. Further, the interaction of CaP and multi-phosphopeptides were qualitatively characterized at the molecular/atomic level and the high affinity between them was quantified by the isothermal titration microcalorimeter based on the laws of thermodynamics. The results indicated that the interaction was a spontaneous (ΔG < 0) exothermic reaction with enthalpy reduction (ΔH < 0) and driven mainly by hydrogen bond and electrostatic interaction process., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Calcium has limited bioavailability because of the formation of calcium phosphate deposits in the gastrointestinal tract. In this study, we prepared a dextran-casein phosphopeptide (CPP)-Ca 2+ delivery system and evaluated for Ca 2+ binding mechanism, structure, stability, and sustained release of Ca 2+ and assessed inhibition of calcium phosphate precipitation. The results revealed that Ca 2+ binds to dextran-CPP through the phosphate, carboxyl, and amino groups and forms crystal clusters. Furthermore, compared with single polymer CPP-Ca 2+ conjugates, copolymer dextran-CPP-Ca 2+ conjugates exhibited improved stability at various conditions (pH, temperature, and coexisting food), efficiently reduced the calcium phosphate precipitation, and improved sustained-release of Ca 2+ . Collectively, dextran-CPP-Ca 2+ conjugates can be an efficient Ca 2+ delivery system., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

We have successfully developed a bioinertized nanoflow LC/MS/MS (nanoLC/MS/MS) system for the highly sensitive analysis of phosphopeptides by depleting metal ions from the mobile phase. We found that not only direct contact of phosphopeptides with metal components, but also indirect contact with nanoLC pumps through the mobile phase causes significant losses during the recovery of phosphopeptides. Moreover, electrospray ionization was adversely affected by the mobile phase containing multiple metal ions as well as by the sample solvents contaminated with metal ions used in immobilized metal ion affinity chromatography for phosphopeptide enrichment. To solve these problems, metal ions were depleted by inserting an online metal ion removal device containing metal-chelating membranes between the gradient mixer and the autosampler. As a result, the peak areas of the identified phosphopeptides increased an average of 9.9-fold overall and 77-fold for multiply phosphorylated peptides with the insertion of the online metal ion removal system. This strategy would be applicable to the highly sensitive analysis of other phosphorylated biomolecules by microscale-LC/MS/MS., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Protein conformational changes with fluctuations are fundamental aspects of protein-protein interactions (PPIs); understanding these motions is required for the rational design of PPI-regulating compounds. Src homology 2 (SH2) domains are commonly found in adapter proteins involved in signal transduction and specifically bind to consensus motifs of proteins containing phosphorylated tyrosine (pY). Here, we analysed the interaction between the N-terminal SH2 domain (nSH2) of the regulatory subunit in phosphoinositide 3-kinase (PI3K) and the cytoplasmic region of the T-cell co-receptor, CD28, using NMR and molecular dynamics (MD) simulations. First, we assigned the backbone signals of nSH2 on 1 H- 15 N heteronuclear single quantum coherence spectra in the absence or presence of the CD28 phosphopeptide, SDpYMNMTPRRPG. Chemical shift perturbation experiments revealed allosteric changes at the BC loop and the C-terminal region of nSH2 upon CD28 binding. NMR relaxation experiments showed a conformational exchange associated with CD28 binding in these regions. The conformational stabilisation of the C-terminal region correlated with the regulation of PI3K catalytic function. Further, using 19 F- and 31 P-labelled CD28 phosphopeptide, we analysed the structural dynamics of CD28 and demonstrated that the aromatic ring of the pY residue fluctuated between multiple conformations upon nSH2 binding. Our MD simulations largely explained the NMR results and the structural dynamics of nSH2 and CD28 in both bound and unbound states. Notably, in addition to its major conformation, we detected a minor conformation of nSH2 in the CD28 bound state that may explain the allosteric conformational change in the BC loop., (© 2022 Federation of European Biochemical Societies.)

MXenes have received lots of attention since discovered and have been applied in various fields. In this work, Ti 3 C 2 -Fe 3 O 4 composites with exposed non-modified Ti 3 C 2 MXene nanosheets were designed and prepared by an in situ growth strategy and then applied in the enrichment of phosphopeptides. The two-dimensional composites could interact with the phosphopeptides through a metal oxide affinity chromatography mechanism provided by Ti-O and Fe-O bonds and a hydrophilic interaction chromatography mechanism by surface hydroxyl groups. This magnetic nanomaterial with a specific surface area of 66.1 m 2 ·g -1 had high sensitivity to phosphopeptides (0.5 nmol·L -1 ) and high selectivity (1:1000 of the molar ratio of β-casein to bovine serum albumin). Non-fat milk was adopted as a real sample to preliminarily examine the applicability of the Ti 3 C 2 -Fe 3 O 4 -based protocol. Subsequently, Qingkailing injection, a kind of traditional Chinese medicine injection, was introduced to further explore the suitability of the nanocomposites for phosphopeptide enrichment from more complex matrices and satisfactory results were obtained.

Here, we present a protocol to identify and quantify phosphopeptides during the dynamic formation of an immunological synapse. We describe steps for mixing isotope-labeled immune and target cells, the stabilization of cell-to-cell conjugates by cross-linking, and their isolation by fluorescence-activated cell sorting. We detail the isolation of phosphopeptides by phosphopeptide enrichment and their subsequent measurement by mass spectrometry. Finally, we describe the analysis of the resulting data to separate cell-specific phosphopeptides using the isotope label and label-free quantification., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Tyrosine kinases and SH2 (phosphotyrosine recognition) domains have binding specificities that depend on the amino acid sequence surrounding the target (phospho)tyrosine residue. Although the preferred recognition motifs of many kinases and SH2 domains are known, we lack a quantitative description of sequence specificity that could guide predictions about signaling pathways or be used to design sequences for biomedical applications. Here, we present a platform that combines genetically encoded peptide libraries and deep sequencing to profile sequence recognition by tyrosine kinases and SH2 domains. We screened several tyrosine kinases against a million-peptide random library and used the resulting profiles to design high-activity sequences. We also screened several kinases against a library containing thousands of human proteome-derived peptides and their naturally-occurring variants. These screens recapitulated independently measured phosphorylation rates and revealed hundreds of phosphosite-proximal mutations that impact phosphosite recognition by tyrosine kinases. We extended this platform to the analysis of SH2 domains and showed that screens could predict relative binding affinities. Finally, we expanded our method to assess the impact of non-canonical and post-translationally modified amino acids on sequence recognition. This specificity profiling platform will shed new light on phosphotyrosine signaling and could readily be adapted to other protein modification/recognition domains., Competing Interests: AL, RV, ML, DG, NS No competing interests declared, (© 2023, Li et al.)

Phosvitin has excellent calcium binding capacity, related to its phosphopeptides. The phosphopeptides may be used as functional ingredients for improving calcium bioavailability, but the calcium-binding mechanism is unclear. In this study, a novel phosvitin phosphorylated pentapeptide (Glu-Asp-Asp-pSer-pSer, EDDpSpS) was selected to prepare an EDDpSpS calcium complex (EDDpSpS-Ca), and the calcium-binding mechanism and bioavailability investigated. The calcium-binding capacity of EDDpSpS was up to 468 ± 152.80 mg/g. Calcium ions prompted the folding of the EDDpSpS structure to form spherical nanoparticles. The calcium binding sites of EDDpSpS involved peptide bonds, carboxyl, amino, and phosphate groups. Molecular forces involved in these interactions were electrostatic in nature. Moreover, EDDpSpS-Ca had excellent bioavailability when compared to CaCO 3 , calcium lactate, and d-calcium gluconate. This study revealed the calcium-binding mechanism of phosvitin phosphopeptide, and suggested that EDDpSpS-Ca has the potential to be a novel, efficient, and promising calcium supplement., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Alzheimer's disease (AD) is a neurodegenerative disorder of increasing concern. It belongs to diseases termed tauopathies which are characterized by inclusions of abnormally hyperphosphorylated and truncated forms of the protein tau. Studies of tauopathies often focus on detection and characterization of these aberrant tau proteoforms, in particular the phosphorylation sites, which represent a significant analytical challenge for example when several phosphosites can be present on the same peptide. Such isomers can even be difficult to fully separate chromatographically. Since recently introduced cyclic ion mobility-mass spectrometry can offer different selectivity, we have investigated the closely positioned phosphorylation sites S214, T212, and T217 of a tryptic peptide from proline rich region of tau-TPSLPTPPTREPK. The conformational heterogeneity of the isomeric peptides in the gas phase hindered their separation due to their overlapping arrival time distributions. Increasing the resolution of the analysis alone is insufficient to distinguish the peptides in a mixture typical of patient samples. We therefore developed a method based on a combination of collision-induced dissociation, isomeric product ions ( m / z 677) mobility separation and post-mobility dissociation to aid in analyzing the isomeric phosphopeptides of tau in diseased brain extract. For all three isomers (T212, S214, and T217), the ion mobility signal of the ion at m / z 677 was still observable at the concentration of 0.1 nmol/L. This work not only offers insights into the phosphorylation of tau protein in AD but also provides an analytical workflow for the characterization of challenging pathological protein modifications in neurodegenerative diseases.

Identification of specific protein phosphatase-1 (PP1) inhibitors is of special importance regarding the study of its cellular functions and may have therapeutic values in diseases coupled to signaling processes. In this study, we prove that a phosphorylated peptide of the inhibitory region of myosin phosphatase (MP) target subunit (MYPT1), R 690 QSRRS(pT696)QGVTL 701 (P-Thr696-MYPT1 690-701 ), interacts with and inhibits the PP1 catalytic subunit (PP1c, IC 50 = 3.84 µM) and the MP holoenzyme (Flag-MYPT1-PP1c, IC 50 = 3.84 µM). Saturation transfer difference NMR measurements established binding of hydrophobic and basic regions of P-Thr696-MYPT1 690-701 to PP1c, suggesting interactions with the hydrophobic and acidic substrate binding grooves. P-Thr696-MYPT1 690-701 was dephosphorylated by PP1c slowly (t 1/2 = 81.6-87.9 min), which was further impeded (t 1/2 = 103 min) in the presence of the phosphorylated 20 kDa myosin light chain (P-MLC20). In contrast, P-Thr696-MYPT1 690-701 (10-500 µM) slowed down the dephosphorylation of P-MLC20 (t 1/2 = 1.69 min) significantly (t 1/2 = 2.49-10.06 min). These data are compatible with an unfair competition mechanism between the inhibitory phosphopeptide and the phosphosubstrate. Docking simulations of the PP1c-P-MYPT1 690-701 complexes with phosphothreonine (PP1c-P-Thr696-MYPT1 690-701 ) or phosphoserine (PP1c-P-Ser696-MYPT1 690-701 ) suggested their distinct poses on the surface of PP1c. In addition, the arrangements and distances of the surrounding coordinating residues of PP1c around the phosphothreonine or phosphoserine at the active site were distinct, which may account for their different hydrolysis rate. It is presumed that P-Thr696-MYPT1 690-701 binds tightly at the active center but the phosphoester hydrolysis is less preferable compared to P-Ser696-MYPT1 690-701 or phosphoserine substrates. Moreover, the inhibitory phosphopeptide may serve as a template to synthesize cell permeable PP1-specific peptide inhibitors., Competing Interests: The authors declare no conflicts of interest.

The comment regarding a method we used to calculate the binding capacity of casein phosphopeptides (CPPs) with calcium. Dr. Horne stated that we overestimated the concentration of Ca 2+ . Indeed, peptides would bind with more calcium ions with the help of phosphate ions. However, in our statement, the binding capacity of CPPs with calcium we used included the binding with calcium in the presence of phosphate. In our solution, peptides sequester calcium phosphate to form soluble core-shell nanoclusters, preventing calcium phosphate precipitating. Even if the binding capacity of CPPs with calcium are overestimated as stated by Dr. Horne, these could not be a problem in deciphering calcium-binding behaviors. Phosphate was applied only in the optimizing and screening process presented in Fig. 1 and Table 1, other experiments (Fig. 2-5 in the commented article) did not include phosphate in the reaction system. Phosphate could not be a factor of these experiments.

Protein phosphorylation is one of the most ubiquitous post-translational modifications, regulating numerous essential processes in cells. Accordingly, the large-scale annotation of phosphorylation sites continues to provide central insight into the regulation of signaling networks. The global analysis of the phosphoproteome typically relies on mass spectrometry analysis of phosphopeptides, with an enrichment step necessary due to the sub-stoichiometric nature of phosphorylation. Several affinity-based methods and chemical modification strategies have been developed to date, but the choice of enrichment method can have a considerable impact on the results. Here, we show that a biotinylated, photo-cleavable phosphorimidazolide reagent permits the immobilization and subsequent cleavage of phosphopeptides. The method is capable of the capture and release of phosphopeptides of varying characteristics, and this mild and selective strategy expands the current repertoire for phosphopeptide chemical modification with the potential to enrich and identify new phosphorylation sites in the future., (© 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH.)