A magnetic calcium phosphate for selective capture of multi-phosphopeptides.

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-Fe3O4). These materials aim to capture multi-phosphopeptides and identifying phosphorylation sites. The current results demonstrate that CaP-Fe3O4 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.

Characterization of Phosphorylated Peptides by Electron-Activated and Ultraviolet Dissociation Mass Spectrometry: A Comparative Study with Collision-Induced Dissociation.

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.

Bifunctional MNPs@UIO-66-Arg core-shell-satellite nanocomposites for enrichment of phosphopeptides.

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 Fe3O4@SiO2 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 m2 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 (ß-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.

Enrichment of Phosphopeptides Based on Zirconium Phthalocyanine-Modified Magnetic Nanoparticles.

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 Fe3O4 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-/ß-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.

Characteristics of casein phosphopeptides in Chinese human milk and its correlation with infant growth: A cross-sectional study.

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 (rs range from 0.20 to 0.29), and three casein phosphopeptides were positively correlated with the infants' length-for-age Z-scores (rs 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.

Combination of click chemistry and Schiff base reaction: Post-synthesis of covalent organic frameworks as an immobilized metal ion affinity chromatography platform for efficient capture of global phosphopeptides in serum with chronic obstructive pulmonary disease.

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.

Enrichment of phosphopeptides by arginine-functionalized magnetic chitosan nanoparticles.

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 ß-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.

Exploiting Charge State Distribution To Probe Intramolecular Interactions in Gas-Phase Phosphopeptides and Enhance Proteomics Analyses.

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.

Facile preparation of nitrogen/titanium-rich porous organic polymers for specific enrichment of N-glycopeptides and phosphopeptides.

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 Ti4+ (CH-Dha-Ti4+). 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-Ti4+ 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.

GreenPhos, a universal method for in-depth measurement of plant phosphoproteomes with high quantitative reproducibility.

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.

Biobased Tannic Acid-Chitosan Composite Membranes as Reusable Adsorbents for Effective Enrichment of Phosphopeptides.

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 Ti4+ 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 ß-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.

Casein phosphopeptide calcium chelation: preparation optimization, in vitro gastrointestinal simulated digestion, and peptide fragment exploration.

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 ß-casein and αs2 -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.

Quantitative Phosphoproteomic Analysis Provides Insights into the Sodium Bicarbonate Responsiveness of Glycine max.

Sodium bicarbonate stress caused by NaHCO3 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 NaHCO3 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.

Cotton Ti-IMAC: Developing Phosphorylated Cotton as a Novel Platform for Phosphopeptide Enrichment.

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/ß-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.

Crystal Structures of Plk1 Polo-Box Domain Bound to the Human Papillomavirus Minor Capsid Protein L2-Derived Peptide.

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.

Data-independent acquisition-based global phosphoproteomics reveal the diverse roles of casein kinase 1 in plant development.

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.

Magnetic resin composites for the enrichment of proteins, peptides and phosphopeptides.

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@Fe3O4 and FPA90CL@Fe3O4, and assessed their applications as adsorbents for enriching proteins, peptides and phosphopeptides. With the combination of Fe3+-IMAC interaction (MTS9200) or electrostatic attraction (FPA90CL) of resins and the adsorption of Fe3O4, the prepared composites exhibited higher capacities for adsorbing a protein (bovine serum albumin, at 195.71 and 135.03 mg g-1 for MTS9200@Fe3O4 and FPA90CL@Fe3O4, respectively) than MTS9200, FPA90CL and Fe3O4. In addition, due to the contributions of the hydrophobic skeleton of resins and Fe3O4, the magnetic resin composites allowed for efficient enrichment of peptides. Moreover, through Fe3+-IMAC interaction or electrostatic attraction of resins and Fe-O MOAC interaction of Fe3O4 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@Fe3O4 and MTS9200@Fe3O4, respectively. Together, our work revealed the great potential of magnetic resin composites as enrichment materials for proteomics and phosphoproteomics analysis.

Suspension Trapping-Based Sample Preparation Workflow for In-Depth Plant Phosphoproteomics.

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.

Facile preparation of porphyrin-based porous organic polymers for specific enrichment and isolation of phosphopeptides and phosphorylated exosomes.

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): ß-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.

An easy and straightforward synthesized nano calcium phosphate for highly capture of multiply phosphorylated peptides.

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 (CaCl2·2H2O and Na2HPO4·12H2O) 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.