One-step fabrication of dipeptide-based bifunctional polymer for individual enrichment of glycopeptides and phosphopeptides from serum.

A dipeptide-based bifunctional material immobilized with Ti4+ (denoted as APE-MBA-VPA-Ti4+) 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, ß-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.

A Rapid One-Pot Workflow for Sensitive Microscale Phosphoproteomics.

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 TiO2 beads in a single-tube format. By reducing surface adsorptive losses via utilizing n-dodecyl ß-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.

Exploitation of porphyrin-based titanium-rich porous organic polymers for targeted phosphopeptide enrichment from the serum of colorectal cancer individuals.

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

Ti4+ functionalized zirconium metal-organic frameworks with polymer brushes for specific identification of phosphopeptides in human serum and skimmed milk.

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

µPhos: a scalable and sensitive platform for high-dimensional phosphoproteomics.

Mass spectrometry has revolutionized cell signaling research by vastly simplifying the analysis of many thousands of phosphorylation sites in the human proteome. Defining the cellular response to perturbations is crucial for further illuminating the functionality of the phosphoproteome. Here we describe µPhos ('microPhos'), an accessible phosphoproteomics platform that permits phosphopeptide enrichment from 96-well cell culture and small tissue amounts in <8 h total processing time. By greatly minimizing transfer steps and liquid volumes, we demonstrate increased sensitivity, >90% selectivity, and excellent quantitative reproducibility. Employing highly sensitive trapped ion mobility mass spectrometry, we quantify ~17,000 Class I phosphosites in a human cancer cell line using 20 µg starting material, and confidently localize ~6200 phosphosites from 1 µg. This depth covers key signaling pathways, rendering sample-limited applications and perturbation experiments with hundreds of samples viable. We employ µPhos to study drug- and time-dependent response signatures in a leukemia cell line, and by quantifying 30,000 Class I phosphosites in the mouse brain we reveal distinct spatial kinase activities in subregions of the hippocampal formation.

Identification of phosphorylated small ORF-encoded peptides in Hep3B cells by LC/MS/MS.

Small ORF-encoded peptides (SEPs) are a class of low molecular weight proteins and peptides comprising <100 amino acids with important functions in various life activities. Although the sequence length is short, SEPs might also have post-translational modification (PTM). Phosphorylation is one of the most essential PTMs of proteins. In this work, we enriched phosphopeptides with IMAC and TiO2 materials and analyzed the phosphorylated SEPs in Hep3B cells. A total of 24 phosphorylated SEPs were identified, and 11 SEPs were coded by ncRNA. For the sequence analysis, we found that the general characteristics of phosphorylated SEPs are roughly the same as canonical proteins. Besides, two phosphorylation SEPs have the Stathmin family signature 2 motif, which can regulate the microtubule cytoskeleton. Some SEPs have domains or signal peptides, indicating their specific functions and subcellular locations. Kinase network analysis found a small number of kinases that may be a clue to the specific functions of some SEPs. However, only one-fifth of the predicted phosphorylation sites were identified by LC/MS/MS, indicating that many SEP PTMs are hidden in the dark, waiting to be uncovered and verified. This study helps expand our understanding of SEP and provides information for further SEP function investigation. SIGNIFICANCE: Small ORF-encoded peptides (SEPs) are important in various life activities. Although the sequence length is short (<100AA), SEPs might also have post-translational modification (PTM). Phosphorylation is one of the most essential PTMs of proteins. We enriched phosphopeptides and analyzed the phosphorylated SEPs in Hep3B cells. That is the first time to explore the PTM of SPEs systematically. Kinase network analysis found a small number of kinases that may be a clue to the specific functions of SEPs. More SEP PTMs are hidden in the dark and waiting to be uncovered and verified. This study helps expand our understanding of SEP and provides information for further SEP function investigation.

[Preparation of magnetic carbon nitride composite toward phosphopeptide enrichment].

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 ß-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, ß-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.

Real-time monitoring of dephosphorylation process of phosphopeptide and rapid assay of PTP1B activity based on a 100 MHz QCM biosensing platform.

The misregulation of protein phosphatases is a key factor in the development of many human diseases, notably cancers. Here, based on a 100 MHz quartz crystal microbalance (QCM) biosensing platform, the dephosphorylation process of phosphopeptide (P-peptide) caused by protein tyrosine phosphatase 1B (PTP1B) was monitored in real time for the first time and PTP1B activity was assayed rapidly and sensitively. The QCM chip, coated with a gold (Au) film, was used to immobilized thiol-labeled single-stranded 5'-phosphate-DNAs (P-DNA) through Au-S bond. The P-peptide, specific to PTP1B, was then connected to the P-DNA via chelation between Zr4+ and phosphate groups. When PTP1B was injected into the QCM flow cell where the P-peptide/Zr4+/MCH/P-DNA/Au chip was placed, the P-peptide was dephosphorylated and released from the Au chip surface, resulting in an increase in the frequency of the QCM Au chip. This allowed the real-time monitoring of the P-peptide dephosphorylation process and sensitive detection of PTP1B activity within 6 min with a linear detection range of 0.01-100 pM and a detection limit of 0.008 pM. In addition, the maximum inhibitory ratios of inhibitors were evaluated using this proposed 100 MHz QCM biosensor. The developed 100 MHz QCM biosensing platform shows immense potential for early diagnosis of diseases related to protein phosphatases and the development of drugs targeting protein phosphatases.

Fully Automated Workflow for Integrated Sample Digestion and Evotip Loading Enabling High-Throughput Clinical Proteomics.

Protein identification and quantification is an important tool for biomarker discovery. With the increased sensitivity and speed of modern mass spectrometers, sample preparation remains a bottleneck for studying large cohorts. To address this issue, we prepared and evaluated a simple and efficient workflow on the Opentrons OT-2 robot that combines sample digestion, cleanup, and loading on Evotips in a fully automated manner, allowing the processing of up to 192 samples in 6 h. Analysis of 192 automated HeLa cell sample preparations consistently identified ∼8000 protein groups and ∼130,000 peptide precursors with an 11.5 min active liquid chromatography gradient with the Evosep One and narrow-window data-independent acquisition (nDIA) with the Orbitrap Astral mass spectrometer providing a throughput of 100 samples per day. Our results demonstrate a highly sensitive workflow yielding both reproducibility and stability at low sample inputs. The workflow is optimized for minimal sample starting amount to reduce the costs for reagents needed for sample preparation, which is critical when analyzing large biological cohorts. Building on the digesting workflow, we incorporated an automated phosphopeptide enrichment step using magnetic titanium-immobilized metal ion affinity chromatography beads. This allows for a fully automated proteome and phosphoproteome sample preparation in a single step with high sensitivity. Using the integrated digestion and Evotip loading workflow, we evaluated the effects of cancer immune therapy on the plasma proteome in metastatic melanoma patients.

In-Depth Endogenous Phosphopeptidomics of Serum with Zirconium(IV)-Grafted Mesoporous Silica Enrichment.

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.

Zirconium-rich magnetic polyoxometalate-based metal-organic framework: Tailored for phosphopeptide analysis from lung cancer A549 cells.

Polyoxometalate-based metal-organic frameworks (POMOFs) have become a promising affinity material for separation and enrichment. The analysis of protein phosphorylation represents a challenge for the development of efficient enrichment materials. Here, a novel zirconium-rich magnetic POMOF was successfully designed and prepared for the enrichment of phosphopeptides. The binding affinity of the nanomaterial partly came from Fe-O clusters in the MOF. The Lewis acid-base interactions between V-O clusters and zirconium ions in V10O28-Zr4+ and phosphate groups in phosphopeptides further strengthened the enrichment ability. The zirconium-rich magnetic POMOF was employed to capture phosphopeptides from non-fat milk, human saliva, and serum. Additionally, 748 unique phosphopeptide peaks were detected from the tryptic digests of lung cancer A549 cell proteins with a high specificity (86.9 %). POMOFs will become an active competitor for the design of protein affinity materials and will provide a new approach for phosphopeptide analysis.

Assessment of the Enzymatic Dephosphorylation Kinetics in the Assemblies of a Phosphopentapeptide that Forms Intranuclear Nanoribbons.

Although the formation of peptide assemblies catalyzed by alkaline phosphatase (ALP) has received increasing attention in inhibiting cancer cells, the detailed enzyme kinetics of the dephosphorylation of the corresponding phosphopeptide assemblies have yet to be determined. We recently discovered that assemblies from a phosphopentapeptide can form intracellular nanoribbons that kill induced pluripotent stem cells or osteosarcoma cells, but the kinetics of enzymatic dephosphorylation remain unknown. Thus, we chose to examine the enzyme kinetics of the dephosphorylation of the phosphopentapeptide [NBD-LLLLpY (1)] from concentrations below to above its critical micelle concentration (CMC). Our results show that the phosphopeptide exhibits a CMC of 75 µM in phosphate saline buffer, and the apparent Vmax and Km values of alkaline phosphatase catalyzed dephosphorylation are approximately 0.24 µM/s and 5.67 mM, respectively. Despite dephosphorylation remaining incomplete at 60 min in all the concentrations tested, dephosphorylation of the phosphopeptide at concentrations of 200 µM or above mainly results in nanoribbons, dephosphorylation at concentrations of CMC largely produces nanofibers, and dephosphorylation below the CMC largely generates nanoparticles. Moreover, the formation of nanoribbons correlates with the intranuclear accumulation of the pentapeptide. By providing the first examination of the enzymatic kinetics of phosphopeptide assemblies, this work further supports the notion that the assemblies of phosphopentapeptides can act as a new functional entity for controlling cell fates.

Ti3C2@UiO-TCPP Schottky junction photoelectrochemical sensor for detecting alkaline phosphatase through the steric hindrance effect of phosphopeptide.

Alkaline phosphatase (ALP) is a major biomarker for clinical diagnosis, but detection methods of ALP are limited in sensitivity and selectivity. In this paper, a novel method for ALP determination is proposed. A photoelectrochemical (PEC) sensor was prepared by growing UiO-tetratopic tetrakis (4-carbox-yphenyl) porphyrin (TCPP) in situ between layered Ti3C2 through a one-pot hydrothermal method. The obtained Schottky heterojunction photoelectric material Ti3C2@UiO-TCPP not only has a large light absorption range but also greatly improves the efficiency of photogenerated electron hole separation and thereby enhances sensitivity for PEC detection. The phosphate group on the phosphorylated polypeptide was utilized to form a Zr-O-P bond with the zirconium ion on UiO-66, and then photocurrent decreases due to the steric hindrance effect of phosphorylated polypeptides, that is, the hindrance of electron transfer between the photoelectric material and a solution. The specific interaction between ALP and phosphorylated polypeptides shears the bond between phosphate and zirconium ion on UiO-66 in the peptides then weakens the hindrance effect and increases the photocurrent, thus realizing ALP detection. The linear range of ALP is 0.03-10,000 U·L-1, and the detection limit is 0.012 U·L-1. The method is highly sensitive and selective, and has been applied in detection of ALP in serum 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.

Influence of casein phosphopeptide-amorphous calcium phosphate on the surface topography and composition of nickel-titanium archwires during orthodontic treatment with fixed appliances.

PURPOSE: To investigate the surface topography and nickel content of nickel-titanium (NiTi) archwires exposed to either routine oral hygiene or a prophylactic regimen with casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) during orthodontic treatment. METHODS: This in vivo study involved 40 orthodontic patients with fixed appliances, who were randomly assigned to either a routine oral hygiene group or a CPP-ACP supplementary regimen group. Twenty new NiTi archwires served as controls. All archwires underwent scanning electron microscopy and energy-dispersive spectroscopy to evaluate their surface topography and elemental composition. The nickel content was quantified as a percentage of total weight and the Ni/Ti ratio, and statistical comparisons were made using pairwise tests. RESULTS: Wires exposed to fluoride toothpaste showed signs of pitting corrosion, deep grooves, and corrosion debris. In contrast, wires exposed to supplementary CPP-ACP exhibited smooth surface areas interspersed with microdefects and deposits. Statistically significant differences in nickel content were found between the new and retrieved archwires, as well as between wires exposed to routine oral hygiene and CPP-ACP (P < 0.001). The archwires exposed to CPP-ACP had the lowest nickel content (P < 0.001). CONCLUSION: The use of CPP-ACP holds promise for application as a safe anticariogenic agent with possible protective properties during orthodontic treatment.

Deep Learning Prediction Boosts Phosphoproteomics-Based Discoveries Through Improved Phosphopeptide Identification.

Shotgun phosphoproteomics enables high-throughput analysis of phosphopeptides in biological samples. One of the primary challenges associated with this technology is the relatively low rate of phosphopeptide identification during data analysis. This limitation hampers the full realization of the potential offered by shotgun phosphoproteomics. Here we present DeepRescore2, a computational workflow that leverages deep learning-based retention time and fragment ion intensity predictions to improve phosphopeptide identification and phosphosite localization. Using a state-of-the-art computational workflow as a benchmark, DeepRescore2 increases the number of correctly identified peptide-spectrum matches by 17% in a synthetic dataset and identifies 19% to 46% more phosphopeptides in biological datasets. In a liver cancer dataset, 30% of the significantly altered phosphosites between tumor and normal tissues and 60% of the prognosis-associated phosphosites identified from DeepRescore2-processed data could not be identified based on the state-of-the-art workflow. Notably, DeepRescore2-processed data uniquely identifies EGFR hyperactivation as a new target in poor-prognosis liver cancer, which is validated experimentally. Integration of deep learning prediction in DeepRescore2 improves phosphopeptide identification and facilitates biological discoveries.

Preparation of high-efficiency titanium ion immobilized magnetic graphite nitride nanocomposite for phosphopeptide enrichment.

BACKGROUND: Protein phosphorylation has been implicated in life processes including molecular interaction, protein structure transformation, and malignant disease. An in-depth study of protein phosphorylation may provide vital information for the discovery of early biomarkers. Mass spectrometry (MS)-based techniques have become an important method for phosphopeptide identification. Nevertheless, direct detection remains challenging because of the low ionization efficiency of phosphopeptides and serious interference from non-phosphopeptides. There is a great need for an efficient enrichment strategy to analyze protein phosphorylation prior to MS analysis. RESULTS: In this study, a novel nanocomposite was prepared by introducing titanium ions into two-dimensional magnetic graphite nitride. The nanocomposite was combined with immobilized metal ion affinity chromatography (IMAC) and anion-exchange chromatography mechanisms for phosphoproteome research. The nanocomposite had the advantages of a large specific surface (412.9 m2 g-1), positive electricity (175.44 mV), and excellent magnetic property (35.7 emu g-1). Moreover, it presented satisfactory selectivity (α-casein:ß-casein:bovine serum albumin = 1:1:5000), a low detection limit (0.02 fmol), great recyclability (10 cycles), and high recovery (92.8%). The nanocomposite demonstrated great practicability for phosphopeptides from non-fat milk, human serum, and saliva. Further, the nanocomposite was applied to enrich phosphopeptides from a more complicated specimen, A549 cell lysate. A total of 890 phosphopeptides mapping to 564 phosphoproteins were successfully detected with nano LC-MS. SIGNIFICANCE: We successfully designed and developed an efficient analysis platform for phosphopeptides, which includes protein digestion, phosphopeptide enrichment, and MS detection. The MS-based enrichment platform was further used to analyze phosphopeptides from complicated bio-samples. This work paves the way for the design and preparation of graphite nitride-based IMAC materials for phosphoproteome analysis.

Effect of the phosphorylation structure in casein phosphopeptides on the proliferation, differentiation, and mineralization of osteoblasts and its mechanism.

Our previous studies have shown that highly phosphorylated casein phosphopeptides (residues 1-25) P5 could efficiently bind calcium and promote intestinal calcium absorption, and enhanced bone development in rats. The purpose of this study was to investigate the effect of the phosphorylation structure in P5 on the proliferation, differentiation, and mineralization of osteoblasts (MC3T3-E1) and its mechanism. P5 was obtained by high-performance liquid chromatography (HPLC) and non-phosphorylated peptide P5-0 was obtained by chemical synthesis. Compared with the control group, the proliferation rate of MC3T3-E1 cells treated by P5 was 1.10 times that of P5-0 at 200 µg mL-1. P5 caused the cell cycle retention of MC3T3-E1 cells in the G2/M phase, while P5-0 had no significant difference in the G2/M phase. MC3T3-E1 cells incubated with P5 showed stronger alkaline phosphatase (ALP) activity than with P5-0, suggesting a tendency to promote cellular differentiation. Compared to the P5-0 treatment group, the P5 treatment group at concentrations of 10 µg mL-1 showed significant differences in the mineralization rates (p < 0.05). P5 significantly upregulated the expressions of Runx2, ALP, ColIα1, and OCN compared with the control group (p < 0.05). In addition, in silico molecular docking showed that the binding force of the P5-EGFR complex was stronger than that of the P5-0-EGFR complex, which was significantly related to the phosphorylation structure in P5 and might be an important reason for osteoblast proliferation. In conclusion, the phosphorylation structure and amino acid composition in P5 stimulated the osteogenic activity of MC3T3-E1 cells, and could be expected to be a functional food for the prevention of osteoporosis.

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.

The landscape of MHC-presented phosphopeptides yields actionable shared tumor antigens for cancer immunotherapy across multiple HLA alleles.

BACKGROUND: Certain phosphorylated peptides are differentially presented by major histocompatibility complex (MHC) molecules on cancer cells characterized by aberrant phosphorylation. Phosphopeptides presented in complex with the human leukocyte antigen HLA-A*02:01 provide a stability advantage over their non-phosphorylated counterparts. This stability is thought to contribute to enhanced immunogenicity. Whether tumor-associated phosphopeptides presented by other common alleles exhibit immunogenicity and structural characteristics similar to those presented by A*02:01 is unclear. Therefore, we determined the identity, structural features, and immunogenicity of phosphopeptides presented by the prevalent alleles HLA-A*03:01, HLA-A*11:01, HLA-C*07:01, and HLA-C*07:02. METHODS: We isolated peptide-MHC complexes by immunoprecipitation from 11 healthy and neoplastic tissue samples using mass spectrometry, and then combined the resulting data with public immunopeptidomics data sets to assemble a curated set of phosphopeptides presented by 96 samples spanning 20 distinct healthy and neoplastic tissue types. We determined the biochemical features of selected phosphopeptides by in vitro binding assays and in silico docking, and their immunogenicity by analyzing healthy donor T cells for phosphopeptide-specific multimer binding and cytokine production. RESULTS: We identified a subset of phosphopeptides presented by HLA-A*03:01, A*11:01, C*07:01 and C*07:02 on multiple tumor types, particularly lymphomas and leukemias, but not healthy tissues. These phosphopeptides are products of genes essential to lymphoma and leukemia survival. The presented phosphopeptides generally exhibited similar or worse binding to A*03:01 than their non-phosphorylated counterparts. HLA-C*07:01 generally presented phosphopeptides but not their unmodified counterparts. Phosphopeptide binding to HLA-C*07:01 was dependent on B-pocket interactions that were absent in HLA-C*07:02. While HLA-A*02:01 and HLA-A*11:01 phosphopeptide-specific T cells could be readily detected in an autologous setting even when the non-phosphorylated peptide was co-presented, HLA-A*03:01 or HLA-C*07:01 phosphopeptides were repeatedly non-immunogenic, requiring use of allogeneic T cells to induce phosphopeptide-specific T cells. CONCLUSIONS: Phosphopeptides presented by multiple alleles that are differentially expressed on tumors constitute tumor-specific antigens that could be targeted for cancer immunotherapy, but the immunogenicity of such phosphopeptides is not a general feature. In particular, phosphopeptides presented by HLA-A*02:01 and A*11:01 exhibit consistent immunogenicity, while phosphopeptides presented by HLA-A*03:01 and C*07:01, although appropriately presented, are not immunogenic. Thus, to address an expanded patient population, phosphopeptide-targeted immunotherapies should be wary of allele-specific differences.