Forkhead-associated phosphopeptide binding domain 1 (FHAD1) deficiency impaired murine sperm motility.

Background: Genetic knockout-based studies conducted in mice provide a powerful means of assessing the significance of a gene for fertility. Forkhead-associated phosphopeptide binding domain 1 (FHAD1) contains a conserved FHA domain, that is present in many proteins with phospho-threonine reader activity. How FHAD1 functions in male fertility, however, remains uncertain. Methods: Fhad1-/- mice were generated by CRISPR/Cas9-mediated knockout, after which qPCR was used to evaluate changes in gene expression, with subsequent analyses of spermatogenesis and fertility. The testis phenotypes were also examined using immunofluorescence and histological staining, while sperm concentrations and motility were quantified via computer-aided sperm analysis. Cellular apoptosis was assessed using a TUNEL staining assay. Results: The Fhad1-/-mice did not exhibit any abnormal changes in fertility or testicular morphology compared to wild-type littermates. Histological analyses confirmed that the testicular morphology of both Fhad1-/-and Fhad1+/+ mice was normal, with both exhibiting intact seminiferous tubules. Relative to Fhad1+/+ mice, however, Fhad1-/-did exhibit reductions in the total and progressive motility of epididymal sperm. Analyses of meiotic division in Fhad1-/-mice also revealed higher levels of apoptotic death during the first wave of spermatogenesis. Discussion: The findings suggest that FHAD1 is involved in both meiosis and the modulation of sperm motility.

The Chronic Effects of a Single Low-Intensity Blast Exposure on Phosphoproteome Networks and Cognitive Function Influenced by Mutant Tau Overexpression.

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

Enrichment of Phosphopeptides 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.

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 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.

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.

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.

Facile fabrication of Ti4+-immobilized magnetic nanoparticles by phase-transitioned lysozyme nanofilms for enrichment of phosphopeptides.

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

Facile synthesis of Fe3+ immobilized magnetic polydopamine-polyethyleneimine composites for phosphopeptide enrichment.

As one of the most common post-translational modification of proteins, protein phosphorylation plays a vital role in many physiological processes. The enrichment of phosphopeptides is highly important before the mass spectrometry detection since phosphopeptides are susceptible to interferences from high-abundance non-phosphopeptides. In this study, we designed a novel magnetic composite (Fe3O4@PDA-PEI-Fe3+) for phosphopeptide enrichment with a facile protocol. The developed Fe3O4@PDA-PEI-Fe3+ is a marvelous material with multiple functional groups, and can effectively enrich phosphopeptides through the synergistic effect of three mechanisms, i.e., immobilized metal ion affinity chromatography raised form Fe3+, electrostatic interaction between amine and phosphate groups, and hydrogen bond between the hydrogen atoms of amine groups and oxygen atoms of phosphate groups. Combined with mass spectrometry, the material shows excellent enrichment performance, high sensitivity (0.4 fmol), good selectivity (ß-casein:BSA= 1:500, w:w), and stable reusability (at least 5 cycles). In addition, the material was successfully applied to enrich phosphopeptides from skim milk and human saliva samples, implying that it is an ideal adsorbent for the phosphopeptide enrichment in complex biological samples and provides valuable insights into the field of phosphopeptide analysis.

The effect of photobiomodulation therapy associated with casein phosphopeptide-amorphous calcium phosphate fluoride paste on the treatment of posthome whitening tooth sensitivity and color change: A randomized clinical trial.

OBJECTIVE: This study assessed whether combining photobiomodulation therapy (PBMT) with casein phosphopeptide-amorphous calcium phosphate fluoride (CPP-ACPF) paste can effectively reduce post-home whitening tooth sensitivity (TS) without compromising shade change. METHODS: Fifty participants were selected and assigned to one of four groups: (1) PLACEBO group-received a placebo paste and PBMT simulation; (2) PBMT group-received a placebo paste + PBMT; (3) CPP-ACPF group-received CPP-ACPF paste and PBMT simulation; (4) CPP-ACPF + PBMT group-received both CPP-ACPF paste and PBMT. The participants used whitening trays containing 22% carbamide peroxide for 2 h a day for 21 days. TS was measured daily using a visual analog scale, while shade change was assessed using a spectrophotometer: before bleaching treatment (T0), after the first (T1), second (T2), and third (T3) weeks of treatment, and 30 days (T4) after completing the whitening treatment. RESULTS: Intragroup analysis revealed that the PLACEBO group had the highest increase in sensitivity during the whitening treatment. The CPP-ACPF and PBMT groups showed no significant difference tooth whitening (TW) between weeks regarding aesthetic change. The CPP-ACPF and PBMT group exhibited a significant reduction in TS between the first and third and between the second and third weeks TW, but not between the first and second. Conversely, the PLACEBO group showed a higher sensitivity than the other groups (p < .05). The CPP-ACPF and PBMT groups did not differ from each other. Furthermore, the CPP-ACPF and PBMT group showed a greater decrease in sensitivity than the PLACEBO group at T1, T2, and T3 (p < .01), and was significantly differed from CPP-ACPF and PBMT groups only at T2 and T3. All groups confirmed TW effectiveness. Student's and paired t-test did not reveal any significant difference between groups (p > .05). CONCLUSION: Therefore, PBMT associated with CPP-ACPF paste can reduce TS without compromising the efficacy of TW.

Stable isotope labeling-based two-step derivatization strategy for analysis of Phosphopeptides.

Investigating site-specific protein phosphorylation remains a challenging task. The present study introduces a two-step chemical derivatization method for accurate identification of phosphopeptides. Methylamine neutralizes carboxyl groups, thus reducing the adsorption of non-phosphorylated peptides during enrichment, while dimethylamine offers a cost-effective reagent for stable isotope labeling of phosphorylation sites. The derivatization improves the mass spectra obtained through liquid chromatography-tandem mass spectrometry. The product ions at m/z 58.07 and 64.10 Da, resulting from dimethylamine-d0 and dimethylamine-d6 labeled phosphorylation sites respectively, can serve as report ions. Derivatized phosphopeptides from casein demonstrate enhanced ionization and formation of product ions, yielding a significant increase in the number of identifiable peptides. When using the parallel reaction monitoring technique, it is possible to distinguish isomeric phosphopeptides with the same amino acid sequence but different phosphorylation sites. By employing a proteomic software and screening the report ions, we identified 29 endogenous phosphopeptides in 10 µL of human saliva with high reliability. These findings indicate that the two-step derivatization strategy has great potential in site-specific phosphorylation and large-scale phosphoproteomics research. SIGNIFICANCE: There is a significant need to improve the accuracy of identifying phosphoproteins and phosphopeptides and analyzing them quantitatively. Several chemical derivatization techniques have been developed to label phosphorylation sites, thus enabling the identification and relative quantification of phosphopeptides. Nevertheless, these methods have limitations, such as incomplete conversion or the need for costly isotopic reagents. Building upon previous contributions, our study moves the field forward due to high efficiency in site-specific labeling, cost-effectiveness, improved sensitivity, and comprehensive product ion coverage. Using the two-step derivatization approach, we successfully identified 29 endogenous phosphopeptides in 10 µL of human saliva with high reliability. The outcomes underscore the possibility of the method for site-specific phosphorylation and large-scale phosphoproteomics investigations.

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.

Effect of casein phosphopeptide-amorphous calcium phosphate, Riva Star, and Moringa oleifera-based nano-silver fluoride on caries affected dentin remineralization bonded to composite resin.

OBJECTIVE: This study aims to assess the effects of the most recent remineralizing agents, casein phosphopeptide-amorphous calcium phosphate (CPP-ACP), CO2 laser irradiation + topical fluoride (CO2 laser + TF), and Nanosilver fluoride - M. oleifera (NSF-MOLE), on the shear bond strength (SBS) and bond failure between resin composite and remineralized caries affected dentin (CAD). MATERIALS AND METHODS: Fifty human molars with occlusal caries reaching approximately halfway through the dentin were immersed in a 4% thymol solution. The infected dentin was removed using an excavator and the CAD surface was exposed. The sample was allocated into five groups (n=10) based on the remineralizing agent applied. Group 1: no remineralizing agent, group 2: CPP-ACP, group 3: Riva Star, group 4: NSF:MOLE, and group 5: (CO2 laser + TF). The shear bond testing procedure was conducted utilizing a universal testing machine and a stereo-microscope was used to study the failure pattern. The researchers utilized a one-way analysis of variance. The Tukey post hoc test was conducted for multiple comparison tests. RESULTS: Group 4 (NSF-MOLE) (13.77±1.94 MPa) treated testers recognized the highest bond values of tooth color restoration to the CAD surface. Nonetheless, group 1 test samples with no mineralization unveiled the minimum outcome of bond integrity (9.12±1.14 MPa). Intergroup comparison exploration showed that group 2 (CPP-ACP), group 4 (NSF-MOLE) (13.77±1.94 MPa), and group 5 (CO2 laser + TF) established comparable values of SBS. Furthermore, group 3 (Riva Star) displayed better SBS than group 1 but lower than group 2, group 4, and group 5. CONCLUSIONS: Remineralization of CAD using modern regimes (CPP-ACP, NSF-MOLE, and CO2 laser + TF has the potential to be used to enhance the bond strength of CAD to composite restoration.

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.

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.

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.

Phosphate Migration versus the Loss of Phosphoric Acid in Protonated Phosphopeptides: A Computational Study.

Residue-specific phosphorylation is a protein post-translational modification that regulates cellular functions. Experimental determination of the exact sites of protein phosphorylation provides an understanding of the signaling and processes at work for a given cellular state. Any experimental artifact that involves migration of the phosphate group during measurement is a concern, as the outcome can lead to erroneous conclusions that may confound studies on cellular signal transduction. Herein, we examine computationally the mechanism by which a phosphate group migrates from one serine residue to another serine in monoprotonated pentapeptides [BA-pSer-Gly-Ser-BB + H]+ → [BA-Ser-Gly-pSer-BB + H]+ (where BA and BB are different combinations of the three basic amino acids, histidine, lysine, and arginine). In addition to moving the phosphate group, the overall mechanism involves transferring a proton from the N-terminal amino acid, BA, to the C-terminal amino acid, BB. This is not a synchronous process, and there is a key high-energy intermediate, structure C, that is zwitterionic with both the basic amino acids protonated and the phosphate group attached to both serine residues and carrying a negative charge. The barriers to moving the phosphate group are calculated to be in the range of 219-274 kJ mol-1 at the B3LYP/6-31G(d) level. These barriers are systematically slightly lower and in good agreement with single-point energy calculations at both M06-2X/6-311++G(d,p) and MP2/6-31++G(d,p) levels. The competitive reaction, loss of phosphoric acid from the protonated pentapeptides, has a barrier in the range of 176-202 kJ mol-1 at the B3LYP/6-31G(d) level. Extension of the theory to M06-2X/6-311++G(d,p)//B3LYP/6-31G(d) and MP2/6-31++G(d,p)// B3LYP/6-31G(d) gives higher values for the loss of phosphoric acid, falling in the range of 196-226 kJ mol-1; these are comparable to the barriers against phosphate migration at the same levels of theory. For larger peptides His-pSer-(Gly)n-Ser-His, where n has values from 2 to 5, the barriers against the loss of phosphoric acid are higher than those against the phosphate group migration. This difference is most pronounced and significant when n = 4 and 5 (the differences are approximately 80 kJ mol-1 under the single-point energy calculations at the M06-2X and MP2 levels). Energy differences using two more recent functionals, M08-HX and MN15, on His-pSer-(Gly)n-Ser-His, where n = 1 and 5, are in good agreement with the M06-2X and MP2 calculations. These results provide the mechanistic rationale for phosphate migration versus other competing reactions in the gas phase under tandem mass spectrometry conditions.

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.