Oxygen-Vacancy-Engineered W18 O49-x Nanobrush with a Suitable Band Structure for Highly Efficient Sonodynamic Therapy.

With the rapid development of external minimally invasive or noninvasive therapeutic modalities, ultrasound-based sonodynamic therapy (SDT) is a new alternative for treating deep tumors. However, inadequate sonosensitizer efficiency and poor biosecurity limit clinical applications. In this study, we prepared an oxygen-vacancy-engineered W18 O49-x nanobrush with a band gap of 2.79 eV for highly efficient SDT using a simple solvothermal method. The suitable band structures of the W18 O49-x nanobrush endows it with the potential to simultaneously produce singlet oxygen (1 O2 ), superoxide anions (⋅O2 - ), and hydroxyl radicals (⋅OH) under ultrasound irradiation. Additionally, abundant oxygen vacancies that serve as further charge traps that inhibit electron-hole recombination are incidentally introduced through one-step thermal reduction. Collectively, the in vitro and in vivo results demonstrate that the oxygen-vacancy-engineered W18 O49-x nanobrush delivers highly efficient reactive oxygen species (ROS) for SDT in a very biosafe manner. Overall, this study provides a new avenue for discovering and designing inorganic nanosonosensitizers with enhanced therapeutic efficiencies for use in SDT.

Updating the dual C and O isotope-Gas-exchange model: A concept to understand plant responses to the environment and its implications for tree rings.

The combined study of carbon (C) and oxygen (O) isotopes in plant organic matter has emerged as a powerful tool for understanding plant functional responses to environmental change. The approach relies on established relationships between leaf gas exchange and isotopic fractionation to derive a series of model scenarios that can be used to infer changes in photosynthetic assimilation and stomatal conductance driven by changes in environmental parameters (CO2 , water availability, air humidity, temperature, nutrients). We review the mechanistic basis for a conceptual model, in light of recently published research, and discuss where isotopic observations do not match our current understanding of plant physiological response to the environment. We demonstrate that (1) the model was applied successfully in many, but not all studies; (2) although originally conceived for leaf isotopes, the model has been applied extensively to tree-ring isotopes in the context of tree physiology and dendrochronology. Where isotopic observations deviate from physiologically plausible conclusions, this mismatch between gas exchange and isotope response provides valuable insights into underlying physiological processes. Overall, we found that isotope responses can be grouped into situations of increasing resource limitation versus higher resource availability. The dual-isotope model helps to interpret plant responses to a multitude of environmental factors.

MONTE CARLO SIMULATION OF THE RADIATION SOURCE TERM FROM [18O]H2O CYCLOTRON TARGET BOMBARDMENT WITH PROTONS OF 16.5 MEV.

PET cyclotrons are widely used for producing medical diagnostic radionuclides. The main radionuclide produced in these facilities is the 18F, which is obtained from the [18O (p,n)18F] reaction when 18O-enriched water is bombarded with the proton beams. This work aimed to estimate the radiation source term from the bombardment of an 18O-enriched water target with protons of 16.5 MeV to determine the radiation neutron field around the accelerator.

Hydrological Controls on Dissolved Organic Matter Composition throughout the Aquatic Continuum of the Watershed of Selin Co, the Largest Lake on the Tibetan Plateau.

Alpine river and lake systems on the Tibetan Plateau are highly sensitive indicators and amplifiers of global climate change and important components of the carbon cycle. Dissolved organic matter (DOM) encompasses organic carbon in aquatic systems, yet knowledge about DOM variation throughout the river-lake aquatic continuum within alpine regions is limited. We used optical spectroscopy, ultrahigh-resolution mass spectrometry (Fourier transform ion cyclotron resonance mass spectrometry), and stable water isotopic measurements to evaluate linkages between DOM composition and hydrological connection. We investigated glacial influences on DOM composition throughout the watershed of Selin Co, including upstream glacier-fed rivers and downstream-linked lakes. We found that the dissolved organic carbon concentration increased, whereas specific ultraviolet absorbance (SUVA254) decreased along the river-lake continuum. Relative to rivers, the downstream lakes had low relative abundances of polyphenolic and condensed aromatic compounds and humic-like substances but increased relative abundances of aliphatics and protein-like compounds. SUVA254 decreased while protein-like components increased with enriched stable water isotope δ2H-H2O, indicating that DOM aromaticity declined while autochthonous production increased along the flow paths. Glacier meltwater contributed to elevated relative abundances of aliphatic and protein-like compounds in headwater streams, while increased relative abundances of aromatics and humic-like DOM were found in glacier-fed lakes than downstream lakes. We conclude that changes in hydrological conditions, including glacier melt driven by a warming climate, will significantly alter DOM composition and potentially their biogeochemical function in surface waters on the Tibetan Plateau.

Imidazo[1,2-a]quinazolines as novel, potent EGFR-TK inhibitors: Design, synthesis, bioactivity evaluation, and in silico studies.

Tyrosine protein kinases (TKs) have been proved to play substantial roles on many cellular processes and their overexpression tend to be found in various types of cancers. Therefore, over recent decades, numerous tyrosine protein kinase inhibitors particularly epidermal growth factor receptor (EGFR) inhibitors have been introduced to treat cancer. Present study describes a novel series of imidazo[1,2-a]quinazolines 18 as potential -inhibitors. These imidazoquinazolines (18a and 18o, in particular) had great anti-proliferative activities with IC50 values in the micromolar (µM) range against PC3, HepG2, HeLa, and MDA-MB-231 comparing with Erlotinib as reference marketed drug. Further evaluations on some derivatives revealed their potential to induce apoptotic cell death and cell growth arrest at G0 phase of the cell cycle. Afterwards, the kinase assay on the most potent compounds 18a and 18o demonstrated their inhibitory potencies and selectivity toward EGFR (with EGFR-IC50 values of 82.0 µM and 12.3 µM, respectively). Additionally, western blot analysis on these compounds 18a and 18o exhibited that they inhibited the phosphorylation of EGFR and its downstream molecule extracellular signal-regulated kinase (ERK1/2). However, the level of B-Actin phosphorylation was not changed. Finally, density functional theory calculations, docking study, and independent gradient model (IGM) were performed to illustrate the structure-activity relationship (SAR) and to assess the interactions between proteins and ligands. The results of molecular docking studies had great agreement with the obtained EGFR inhibitory results through in vitro evaluations.

Progress in high-resolution isotope-ratio analysis of tree rings using laser ablation.

Stable isotope ratio analysis of tree rings has been widely and successfully applied in recent decades for climatic and environmental reconstructions. These studies were mostly conducted at an annual resolution, considering one measurement per tree ring, often focusing on latewood. However, much more information could be retrieved with high-resolution intra-annual isotope studies, based on the fact that the wood cells and the corresponding organic matter are continuously laid down during the growing season. Such studies are still relatively rare, but have a unique potential for reconstructing seasonal climate variations or short-term changes in physiological plant properties, like water-use efficiency. The reason for this research gap is mostly technical, as on the one hand sub-annual, manual splitting of rings is very tedious, while on the other hand automated laser ablation for high-resolution analyses is not yet well established and available. Here, we give an update on the current status of laser ablation research for analysis of the carbon isotope ratio (δ13C) of wood, describe an easy-to-use laser ablation system, its operation and discuss practical issues related to tree core preparation, including cellulose extraction. The results show that routine analysis with up to 100 laser shot-derived δ13C-values daily and good precision and accuracy (ca. 0.1‰) comparable to conventional combustion in an elemental analyzer are possible. Measurements on resin-extracted wood is recommended as most efficient, but laser ablation is also possible on cellulose extracted wood pieces. Considering the straightforward sample preparation, the technique is therefore ripe for wide-spread application. With this work, we hope to stimulate future progress in the promising field of high-resolution environmental reconstruction using laser ablation.

A retrospective assessment of the clinical efficacy of different internal fixation methods in the treatment of distal fibula fractures in the elderly.

An ankle fracture is a fracture of the distal tibia or fibula that forms the ankle joint, usually associated with ligament and soft tissue injury, and is a common type of lower limb fracture and one of the most common types of fracture in the elderly. Although ankle fractures are one of the most common injuries seen by orthopedic trauma surgeons, there is no uniform protocol for the diagnosis and treatment of ankle fractures in the elderly, and there are many controversial indications for surgery. The aim of this study is to assess the clinical efficacy of different internal fixation methods in the treatment of distal fibular fractures in the elderly, in an effort to improve the rational selection and application of clinical acts. A retrospective analysis was performed on 68 cases of patients who suffered an ankle fracture and were treated with different internal fixation methods according to the fracture types and individual differences in distal fibula fractures. The postoperative therapeutic effect assessment was performed in terms of clinical examination, imaging evaluation, and AOFAS ankle-hind foot function scoring. There was no unhealed bone, ankle instability and loose/fractured internal fixation. Fracture healing time was 2.7 to 4.0 months (average 3.2 months). AOFAS score was 88.3 ± 6.2, of which, 34 excellent cases, 30 good cases, and 4 fair cases. Ankle activity dorsiflexion 6º~18º, average 15º; plantar flexion 26º~47º, average 37º. A good clinical efficacy could be achieved from the most appropriate individualized internal fixation for distal fibula fractures of elderly patients.

Role of Dust and Iron Solubility in Sulfate Formation during the Long-Range Transport in East Asia Evidenced by 17O-Excess Signatures.

Numerical models have been developed to elucidate air pollution caused by sulfate aerosols (SO42-). However, typical models generally underestimate SO42-, and oxidation processes have not been validated. This study improves the modeling of SO42- formation processes using the mass-independent oxygen isotopic composition [17O-excess; Δ17O(SO42-)], which reflects pathways from sulfur dioxide (SO2) to SO42-, at the background site in Japan throughout 2015. The standard setting in the Community Multiscale Air Quality (CMAQ) model captured SO42- concentration, whereas Δ17O(SO42-) was underestimated, suggesting that oxidation processes were not correctly represented. The dust inline calculation improved Δ17O(SO42-) because dust-derived increases in cloud-water pH promoted acidity-driven SO42- production, but Δ17O(SO42-) was still overestimated during winter as a result. Increasing solubilities of the transition-metal ions, such as iron, which are a highly uncertain modeling parameter, decreased the overestimated Δ17O(SO42-) in winter. Thus, dust and high metal solubility are essential factors for SO42- formation in the region downstream of China. It was estimated that the remaining mismatch of Δ17O(SO42-) between the observation and model can be explained by the proposed SO42- formation mechanisms in Chinese pollution. These accurately modeled SO42- formation mechanisms validated by Δ17O(SO42-) will contribute to emission regulation strategies required for better air quality and precise climate change predictions over East Asia.

Design, synthesis, and biological evaluation of novel dual inhibitors of heat shock protein 90/mammalian target of rapamycin (Hsp90/mTOR) against bladder cancer cells.

In this study, a novel class of thieno [2,3-d] pyrimidine derivatives containing resorcinol and morpholine fragments as Hsp90/mTOR dual inhibitors was designed, synthesized, and evaluated. In vitro anti-tumor assay results: the obtained compounds demonstrated effectiveness in suppressing the enzymatic activities of the Hsp90 and mTOR and inhibiting the proliferation of J82, T24, and SW780 cancer cell lines. Among these dual inhibitors, the most potent compound 17o, confirmed remarkable inhibitory activities on Hsp90, mTOR, and SW780 cell. Furthermore, the molecular dynamics simulation and a panel of mechanism studies revealed that inhibitor 17o suppressed the proliferation of SW780 cells through the over-activation of the PI3K/AKT/mTOR pathway regulated by mTOR inhibition and apoptosis regulated by the mitochondrial pathway. In subcutaneous J82 xenograft models, the compound 17o also presented considerable in vivo anti-tumor activity. Therefore, our investigations highlight that a new-found dual Hsp90/mTOR inhibitor by rational drug design strategies could be a promising lead compound for targeted bladder cancer therapy and deserves further studies.

Oxygen Isotope Exchange between Carbon Dioxide and Iron Oxides on Mars' Surface.

An investigation of the fundamental processes leading to the incorporation of 18O isotopes in carbon dioxide and in iron oxides is critical to understanding the atmospheric evolution and geochemistry of Mars. Whereas signatures of 18O have been observed by the Phoenix Lander and the sample analysis at Mars for carbon dioxide, the underlying isotopic exchange pathways with minerals of the crust of Mars are still elusive. Here, we reveal that reactions of gaseous 18O-carbon dioxide over goethite (FeO(OH)) and hematite (Fe2O3) lead to an 18O transfer from the atmosphere that enriches the 18O content of the iron oxides in the absence of water and light. This proof-of-concept study shows that isotopic enrichment processes on Mars not only are limited to the atmosphere but also proceed via chemical interaction with dry iron oxides. These processes are decisive to comprehending the 18O cycle between the atmosphere and the surface on the planetary scale.

Helium-flushed sheathed nickel tube reactor for continuous flow oxygen stable isotope compound-specific analysis.

RATIONALE: Compound-specific oxygen stable isotope analysis has been commercially available for about 20 years but the technique has been infrequently applied and practical operating knowledge is limited. Existing reactor designs and chemistry fail to fully exclude contaminating oxygen, are chromatographically flawed, and are generally fabricated from expensive platinum tubing. METHODS: As a new interface between the gas chromatograph and isotope ratio mass spectrometer, we used a nickel tube reactor (containing no platinum wires) in a ceramic sheath with countercurrent helium flushing, operated at 1250°C. We analyzed suites of four long-chain alcohols (LCAs), defining the least and most 18 O-enriched LCAs as internal standards. The other two LCAs, co-injected in varying amounts, were treated as unknowns and amplitude-corrected during data reduction. RESULTS: With this design, signals from non-oxygenated compounds are minimized with a hydrogen trickle, while excessive graphitization is counterproductive. A balance of hydrogen and graphite is required to maintain a proper, stable redox state in the reactor. With low extraneous oxygen, the exact hydrogen flow has little effect on final δ18 O data quality. Overall average δ18 O values precisions and errors for the unknowns were better than 0.4 ‰ for injections between 2 and 7 nmol per component. CONCLUSIONS: Our actively flushed sheath reduces oxygen infiltration to negligible levels and results in a chromatographic flow devoid of dead or poorly swept volumes. The improved design allows measurement of LCAs with accuracy and precision sufficient to enable investigating real processes, and may also have wider applications to other isotopes.

Operative Treatment of Severe Scoliosis in Symptomatic Adults: Multicenter Assessment of Outcomes and Complications With Minimum 2-Year Follow-up.

BACKGROUND: Few reports focus on adults with severe scoliosis. OBJECTIVE: To report surgical outcomes and complications for adults with severe scoliosis. METHODS: A multicenter, retrospective review was performed on operatively treated adults with severe scoliosis (minimum coronal Cobb: thoracic [TH] ≥ 75°, thoracolumbar [TL] ≥ 50°, lumbar [L] ≥ 50°). RESULTS: Of 178 consecutive patients, 146 (82%; TH = 8, TL = 88, L = 50) achieved minimum 2-yr follow-up (mean age = 53.9 ± 13.2 yr, 92% women). Operative details included posterior-only (58%), 3-column osteotomy (14%), iliac fixation (72%), and mean posterior fusion = 13.2 ± 3.7 levels. Global coronal alignment (3.8 to 2.8 cm, P = .001) and maximum coronal Cobb improved significantly (P ≤.020): TH (84º to 57º; correction = 32%), TL (67º to 35º; correction = 48%), L (61º to 29º; correction = 53%). Sagittal alignment improved significantly (P < .001), most notably for L: C7-sagittal vertical axis 6.7 to 2.5 cm, pelvic incidence-lumbar lordosis mismatch 18º to 3º. Health-related quality-of-life (HRQL) improved significantly (P < .001), most notably for L: Oswestry Disability Index (44.4 ± 20.5 to 26.1 ± 18.3), Short Form-36 Physical Component Summary (30.2 ± 10.8 to 39.9 ± 9.8), and Scoliosis Research Society-22r Total (2.9 ± 0.7 to 3.8 ± 0.7). Minimal clinically important difference and substantial clinical benefit thresholds were achieved in 36% to 75% and 29% to 51%, respectively. Ninety-four (64%) patients had ≥1 complication (total = 191, 92 minor/99 major, most common = rod fracture [13.0%]). Fifty-seven reoperations were performed in 37 (25.3%) patients, with most common indications deep wound infection (11) and rod fracture (10). CONCLUSION: Although results demonstrated high rates of complications, operative treatment of adults with severe scoliosis was associated with significant improvements in mean HRQL outcome measures for the study cohort at minimum 2-yr follow-up.

Leaf scale quantification of the effect of photosynthetic gas exchange on Δ47 of CO2.

The clumped isotope composition (Δ47, the anomaly of the mass 47 isotopologue relative to the abundance expected from a random isotope distribution) of CO2 has been suggested as an additional tracer for gross CO2 fluxes. However, the effect of photosynthetic gas exchange on Δ47 has not been directly determined and two indirect/conceptual studies reported contradicting results. In this study, we quantify the effect of photosynthetic gas exchange on Δ47 of CO2 using leaf cuvette experiments with one C4 and two C3 plants. The experimental results are supported by calculations with a leaf cuvette model. Our results demonstrate the important roles of the Δ47 value of CO2 entering the leaf, kinetic fractionation as CO2 diffuses into, and out of the leaf and CO2-H2O isotope exchange with leaf water. We experimentally confirm the previously suggested dependence of Δ47 of CO2 in the air surrounding a leaf on the stomatal conductance and back-diffusion flux. Gas exchange can enrich or deplete the Δ47 of CO2 depending on the Δ47 of CO2 entering the leaf and the fraction of CO2 exchanged with leaf water and diffused back to the atmosphere, but under typical ambient conditions, it will lead to a decrease in Δ47.

The effects on isotopic composition of leaf water and transpiration of adding a gas-exchange cuvette.

An expression was earlier derived for the non-steady state isotopic composition of a leaf when the composition of the water entering the leaf was not necessarily the same as that of the water being transpired (Farquhar and Cernusak 2005). This was relevant to natural conditions because the associated time constant is typically sufficiently long to ensure that the leaf water composition and fluxes of the isotopologues are rarely steady. With the advent of laser-based measurements of isotopologues, leaves have been enclosed in cuvettes and time courses of fluxes recorded. The enclosure modifies the time constant by effectively increasing the resistance to the one-way gross flux out of the stomata because transpiration increases the vapour concentration within the chamber. The resistance is increased from stomatal and boundary layer in series, to stomata, boundary layer and chamber resistance, where the latter is given by the ratio of leaf area to the flow rate out of the chamber. An apparent change in concept from one-way to net flux, introduced by Song, Simonin, Loucos and Barbour (2015) is resolved, and shown to be unnecessary, but the value of their data is reinforced.

18O Isotope Labeling Combined with 31P Nuclear Magnetic Resonance Spectroscopy for Accurate Quantification of Hydrolyzable Phosphorus Species in Environmental Samples.

31P nuclear magnetic resonance (NMR) spectra can be biased due to the hydrolysis of labile P species during sample treatment and NMR analysis. This paper offers an approach to circumvent this problem by performing sample preparation and analysis in 18O-enriched medium. Heavy 18O isotope atoms were introduced into the resulting artificial hydrolysis products. The NMR signal of 18O-labeled P was shifted upfield relative to the unlabeled P nuclei in natural metabolites. This isotope shift enabled an immediate differentiation of artificial hydrolysis products from natural metabolites. Moreover, the hydrolysis products could be accurately quantified. Our data suggest that the extent to which artificial hydrolysis alters NMR spectra varies among different types of environmental samples. For instance, 72-84% of the detected monoesters in the organic soils of this study were actually artificially hydrolyzed diesters. By contrast, artificial hydrolysis products in the mineral soils used for this study accounted for less than 6% of the total monoesters. Polyphosphate was also hydrolyzed to yield 18O-labeled products in algal biomass.

Comparison of total and activity energy expenditure estimates from physical activity questionnaires and doubly labelled water: a systematic review and meta-analysis.

Physical activity questionnaires (PAQ) could be suitable tools in free-living people for measures of physical activity, total and activity energy expenditure (TEE and AEE). This meta-analysis was performed to determine valid PAQ for estimating TEE and AEE using doubly labelled water (DLW). We identified data from relevant studies by searching Google Scholar, PubMed and Scopus databases. This revealed thirty-eight studies that had validated PAQ with DLW and reported the mean differences between PAQ and DLW measures of TEE (TEEDLW - TEEPAQ) and AEE (AEEDLW - AEEPAQ). We assessed seventy-eight PAQ consisting of fifty-nine PAQ that assessed TEE and thirty-five PAQ that examined AEE. There was no significant difference between TEEPAQ and TEEDLW with a weighted mean difference of -243·3 and a range of -841·4 to 354·6 kJ/d, and a significant weighted mean difference of AEEDLW - AEE PAQ 414·6 and a range of 78·7-750·5. To determine whether any PAQ was a valid tool for estimating TEE and AEE, we carried out a subgroup analysis by type of PAQ. Only Active-Q, administered in two seasons, and 3-d PA diaries were correlated with TEE by DLW at the population level; however, these two PAQ did not demonstrate an acceptable limit of agreement at individual level. For AEE, no PAQ was correlated with DLW either at the population or at the individual levels. Active-Q and 3-d PA diaries were identified as the only valid PAQ for TEE estimation. Further well-designed studies are needed to verify this result and identify additional valid PAQ.

GC-MS approach revealing hidden nitrite reactions in hemolysate using 18O-labelled nitrite and cysteine-based thiols.

In erythrocytes, nitric oxide (NO) and its autoxidation product nitrite undergo multiple reactions with hemoglobin species to form nitrate, Fe-nitrosyl hemoglobin (HbFe(II)NO), S-nitrosohemoglobin (HbCysß93SNO), and other intermediates including nitrito-methemoblobin HbFe(III)ONO, nitro-methemoblobin HbFe(III)NO2 and nitrous anhydride (N2O3). Here, we report a stable-isotope dilution GC-MS assay that allows studying reactions of nitrite in hemolysate. The method is based on the use of 18O-labelled nitrite in combination with l-cysteine or N-acetyl-l-cysteine ethyl ester and GC-MS measurement of unlabelled and labelled nitrite and nitrate species. This approach reveals reactions that are hidden at physiological nitrite concentrations.

Isotopic Traceability (13C and 18O) of Greek Olive Oil.

In recent years, isotopic analysis has been proven a valuable tool for the determination of the origin of various materials. In this article, we studied the 18O and 13C isotopic values of 210 olive oil samples that were originated from different regions in Greece in order to verify how these values are affected by the climate regime. We observed that the δ18O isotopic values range from 19.2 ‱ to 25.2 ‱ and the δ13C values range from -32.7 ‱ to -28.3 ‱. These differences between the olive oils' isotopic values depended on the regional temperature, the meteoric water, and the distance from the sea. Furthermore, we studied the 13C isotopic values of biophenolic extracts, and we observed that they have same capability to differentiate the geographic origin. Finally, we compared the isotopic values of Greek olive oils with samples from Italy, and we concluded that there is a great dependence of oxygen isotopes on the climatic characteristics of the different geographical areas.

Real-Time Monitoring of 13C- and 18O-Isotopes of Human Breath CO2 Using a Mid-Infrared Hollow Waveguide Gas Sensor.

Real-time measuring of CO2 isotopes (13CO2, 12CO2, and 18OC16O) in exhaled breath using a mid-infrared hollow waveguide gas sensor incorporating a 2.73 µm distributed feedback laser was proposed and demonstrated for the first time based on calibration-free wavelength modulation spectroscopy. The measurement precisions for δ13C and δ18O were, respectively, 0.26 and 0.57‰ for an integration time of 131 s by Allan variance analysis. These measurement precisions achieved in the present work were at least 3.5 times better than those reported using direct absorption spectroscopy and 1.3 times better than those obtained by calibration-needed wavelength modulation absorption spectroscopy. Continuous measurement of three isotopes in the breathing cycle was performed. Alveolar gas from the expirogram was identified, and the 13C/12C and 18O/16O ratios were found to be almost constant during the alveolar plateau, which enables optimization of breath sampling and provides accurate information on metabolic processes. The 13C/12C and 18O/16O isotope ratios at the alveolar plateau of five breath cycles were averaged, yielding δ13C and δ18O values of (-24.3 ± 3.4) and (-30.7 ± 2.6) ‰, respectively. This study demonstrates the feasibility of real-time analysis of 13C- and 18O-isotopes of human breath CO2 in clinical applications and shows its potential for diagnosing respiratory-related diseases with high sensitivity, selectivity, and specificity.

Detection of Multisite Phosphorylation of Intrinsically Disordered Proteins Using Quantitative Mass-Spectrometry.

Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) within proteins have attracted considerable attention in recent years. Several important biological signaling mechanisms including protein-protein interactions and post-translational modifications can be easily mediated by IDPs and IDRs due to their flexible structure. These regions can encode linear sequences that are indispensable in cell-signaling networks and circuits. For example, the linear multisite phosphorylation networks encoded in disordered protein sequences play a key role in cell-cycle regulation where the phosphorylation of proteins controls the orchestration of all major mechanisms. While elucidating a systems-level understanding of this process and other multisite phosphorylation processes, we extensively used mass-spectrometry and found it to be an ideal tool to identify, characterize, and quantify phosphorylation dynamics within IDPs. Here, we describe a quantitative proteomics method, together with a detailed protocol to analyze dynamic multisite phosphorylation processes within IDPs using an in vitro protein phosphorylation assay with "light" gamma-16O ATP and "heavy" gamma-18O ATP, combined with liquid chromatography mass spectrometry.