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Data-independent acquisition (DIA) has improved the identification and quantitation coverage of peptides and proteins in liquid chromatography-tandem mass spectrometry-based proteomics. However, different DIA data-processing tools can produce very different identification and quantitation results for the same data set. Currently, benchmarking studies of DIA tools are predominantly focused on comparing the identification results, while the quantitative accuracy of DIA measurements is acknowledged to be important but insufficiently investigated, and the absence of suitable metrics for comparing quantitative accuracy is one of the reasons. A new metric is proposed for the evaluation of quantitative accuracy to avoid the influence of differences in false discovery rate control stringency. The part of the quantitation results with high reliability was acquired from each DIA tool first, and the quantitative accuracy was evaluated by comparing quantification error rates at the same number of accurate ratios. From the results of four benchmark data sets, the proposed metric was shown to be more sensitive to discriminating the quantitative performance of DIA tools. Moreover, the DIA tools with advantages in quantitative accuracy were consistently revealed by this metric. The proposed metric can also help researchers in optimizing algorithms of the same DIA tool and sample preprocessing methods to enhance quantitative accuracy.
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Numerical modeling of Taylor dispersion analysis (TDA) was performed using COMSOL Multiphysics to facilitate better and faster optimization of the experimental conditions. Parameters, such as pressure, electric field, diameter, and length of capillary on the TDA conditions, were examined for particles with hydrodynamic radius (Rh) of 2.5-250 Å. The simulations were conducted using 25, 50, and 100 cm length tubes with diameters of 25, 50, and 100 µm. It was shown that particles with larger diffusion coefficients gave more accurate results at higher velocities, and in longer and wider columns; particles with smaller diffusion coefficients gave more accurate results at smaller velocities, and in shorter and thinner columns. Moreover, the effect of electric field on the validity and the applicability of TDA was studied using TDA in conjunction with capillary electrophoresis. Diffusion coefficients were obtained using a pressure and the TDA equation and compared with those obtained with a pressure in combination of an electric field for fluorescein, FD4, FD20, FD70, and FD500. We found that TDA can be used with the presence of moderate electrophoretic migration and electroosmotic flow, when appropriate conditions were met.
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Electroforesis Capilar , Electroforesis Capilar/métodos , Simulación por Computador , Difusión , Modelos Teóricos , Electroósmosis/métodos , Electricidad , Hidrodinámica , Tamaño de la Partícula , PresiónRESUMEN
RATIONALE: In clinical diagnosis of liver injury, which is an important health concern, serum aminotransferase assays have been the go-to method used worldwide. However, the measurement of serum enzyme activity has limitations, including inadequate disease specificity and enzyme specificity. METHODS: With the high selectivity and specificity provided by nano liquid chromatography-tandem mass spectrometry (LC/MS/MS), this work describes a method for the simultaneous determination of six proteins in liver that can be potentially used as biomarkers for liver injury: glutamic-pyruvic transaminase 1 (GPT1), glutamic oxaloacetic transaminase 1 (GOT1), methionine adenosyl transferase 1A (MAT1A), glutathione peroxidase 1 (GPX1), cytokeratin 18 (KRT18) and apolipoprotein E (APOE). RESULTS: In validation, the method was shown to have good selectivity and sensitivity (limits of detection at pg/mL level). The analytical method revealed that, compared with normal mice, in carbon tetrachloride-induced acute liver injury mice, liver MAT1A and GPX1 were significantly lower (p < 0.01 and p < 0.05, respectively), KRT18 was significantly higher (p < 0.05) and APOE and GPT1 were marginally significantly lower (p between 0.05 and 0.1). This is the first work reporting the absolute contents of GPT1, GOT1, MAT1A, GPX1 and KRT18 proteins based on LC/MS. CONCLUSIONS: The proposed method provides a basis for establishing more specific diagnostic indicators of liver injury.
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Hígado , Espectrometría de Masas en Tándem , Animales , Ratones , Espectrometría de Masas en Tándem/métodos , Cromatografía Liquida/métodos , Hígado/metabolismo , Apolipoproteínas E/metabolismoRESUMEN
Carbohydrates play critically important roles in energy supply and biological functions in living systems. However, it has been a great challenge to identify saccharides and distinguish their isomers because they have highly similar structures and many possible positions for glycosidic linkages. In this work, an ambient ionization tandem mass spectrometry method was developed to characterize disaccharide structural isomers with in situ methylation. The direct analysis in real time ion source can be used to facilitate the methylation reaction of disaccharides with tetramethylammonium hydroxide. The hydroxyl groups of disaccharides can be methylated instantaneously, and the products can be ionized at the same time. The methylated product ions from full scan mass spectrometry (MS) and tandem MS can be used to distinguish a variety of disaccharide structural isomers with different glycosidic linkages, compositions, and configurations. Characteristic marker ions were discovered, and they can be used for the assignment of linkage type and identification of specific isomeric forms. The method was used for the direct identification of disaccharide isomers from real commercial products such as honey, wine, and milk without complex sample pretreatment or chromatographic separation.
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Disacáridos , Espectrometría de Masas en Tándem , Disacáridos/química , Espectrometría de Masas en Tándem/métodos , Metilación , Carbohidratos , Iones , Isomerismo , Glicósidos , Espectrometría de Masa por Ionización de Electrospray/métodosRESUMEN
Proteomics provides molecular bases of biology and disease, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a platform widely used for bottom-up proteomics. Data-independent acquisition (DIA) improves the run-to-run reproducibility of LC-MS/MS in proteomics research. However, the existing DIA data processing tools sometimes produce large deviations from true values for the peptides and proteins in quantification. Peak-picking error and incorrect ion selection are the two main causes of the deviations. We present a cross-run ion selection and peak-picking (CRISP) tool that utilizes the important advantage of run-to-run consistency of DIA and simultaneously examines the DIA data from the whole set of runs to filter out the interfering signals, instead of only looking at a single run at a time. Eight datasets acquired by mass spectrometers from different vendors with different types of mass analyzers were used to benchmark our CRISP-DIA against other currently available DIA tools. In the benchmark datasets, for analytes with large content variation among samples, CRISP-DIA generally resulted in 20 to 50% relative decrease in error rates compared to other DIA tools, at both the peptide precursor level and the protein level. CRISP-DIA detected differentially expressed proteins more efficiently, with 3.3 to 90.3% increases in the numbers of true positives and 12.3 to 35.3% decreases in the false positive rates, in some cases. In the real biological datasets, CRISP-DIA showed better consistencies of the quantification results. The advantages of assimilating DIA data in multiple runs for quantitative proteomics were demonstrated, which can significantly improve the quantification accuracy.
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Proteómica , Espectrometría de Masas en Tándem , Cromatografía Liquida/métodos , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Reproducibilidad de los Resultados , Proteínas/análisis , Péptidos/química , Programas Informáticos , Proteoma/análisisRESUMEN
The thermodynamic properties of molecular recognition in host-guest inclusion complexes can be studied by Taylor dispersion analysis (TDA). Host-guest inclusion complexes have modest size, and it is possible to get convergent results fast, achieving greater certainty for the obtained thermodynamic properties. Cyclodextrins (CDs) and their derivatives can be used as drug carriers that can boost stability, solubility, and bioavailability of physiologically active substances. A simple and effective approach for assessing the binding properties of CD complexes that are critical in the early stages of drug and formulation development is needed to fully understand the process of CD and guest molecules' complex formation. In this work, TDA was successfully used to rapidly determine interaction parameters, including binding constant and stoichiometry, between ß-CD and folic acid (FA) along with the diffusivities of the free FA and its complex with ß-CD. Additionally, the FA diffusion coefficient obtained by TDA was compared to the results previously obtained by nuclear magnetic resonance. Affinity capillary electrophoresis (ACE) was also used to compare the binding constants obtained by different methods. The results showed that the binding constants obtained by ACE were somewhat lower than those obtained by the two TDA procedures.
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Ciclodextrinas , beta-Ciclodextrinas , beta-Ciclodextrinas/química , Ciclodextrinas/química , Termodinámica , Espectroscopía de Resonancia Magnética , Electroforesis Capilar/métodosRESUMEN
To systematically study the influence of host-guest interactions on the analytical performance of direct analysis in real time mass spectrometry (DART-MS), the interactions between cyclodextrins (CDs) and different Sudan dyes were investigated. The results showed that the host-guest interaction between CDs and Sudan dyes did not affect qualitative analysis of the target compounds, but led to a lower signal intensity for Sudan dyes, thus affecting quantitative analysis of the target compounds. The stronger the host-guest interaction, the weaker the signal intensity of target compound on DART-MS. The results also show that both in solution and in solid-phase microextraction (SPME), the addition of organic solvents can weaken the host-guest interaction between CDs and Sudan dyes, thus improving the signal intensity in DART-MS. In SPME, adding organic solvents has a certain practical value and can improve the efficiency of Sudan dye analysis. This study suggests that appropriate sample pretreatment is needed to weaken noncovalent interactions prior to DART-MS analysis to obtain more accurate quantitative results. The data provide some insight into the effects of other noncovalent interactions on the efficiency of DART-MS as an analytical tool, as well as the potential to study intermolecular interactions with DART-MS.
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Colorantes , Microextracción en Fase Sólida , Espectrometría de Masas/métodos , Microextracción en Fase Sólida/métodos , Solventes/químicaRESUMEN
Characterization of structural isomers of bioactive molecules is important for recognizing their functions, but it has been challenging due to their highly similar structures. As the main bioactive constituents of Panax ginseng, ginsenosides have different structural isomers attributed to the aglycone structure and glycosylation sites as well as stereochemistry of sugar groups attached. This work demonstrated a simple and robust in situ methylation reaction with tetramethylammonium hydroxide (TMAH) using ambient ionization source of direct analysis in real time (DART) to characterize saponin structural isomers. The DART ion source provides favorable conditions to methylate hydroxyl groups of ginsenoside instantaneously with TMAH, and it can ionize the methylated products at the same time. Methylated ginsenoside stereoisomers even with subtle structure differences generated very different mass signals from full-scan MS and tandem MS. High-resolution mass spectrometry aided the assignment of molecular structures of the various precursor and fragment ions from different ginsenosides, which provided structural information for both the aglycone skeleton and the sugar moieties in ginsenosides. The presented method was successfully used for the identification of ginsenosides in Panax ginseng, and saponin isomers were characterized without the need for chromatographic separation and/or tedious offline sample pretreatment.
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Ginsenósidos , Panax , Saponinas , Espectrometría de Masas en Tándem , Ginsenósidos/análisis , Metilación , Cromatografía Líquida de Alta Presión/métodos , Panax/química , AzúcaresRESUMEN
RATIONALE: Exhaled breath contains many substances that are closely related to human metabolism. Analysis of its composition is important for human health, but it is difficult. Since the volatile molecules in breath samples are exhaled instantaneously, easily diffused and modified, and at low level of presence, they are difficult to identify and quantify. METHODS: A modified direct analysis in real time ion source was used for high-resolution mass spectrometry measurement of human metabolites in exhaled breath through online monitoring and offline analysis, in both positive and negative ion modes. The improved system enabled the breath volatiles as well as condensates to be directly sampled, rapidly transmitted and efficiently ionized in a confined region, and then detected using an Orbitrap mass analyzer. RESULTS: The molecular features with online and offline analysis of exhaled breath were demonstrated with obvious differences. A total of about 65 metabolites in positive ion mode and about 55 metabolites in negative ion mode were identified based on accurate m/z values. Exhalome profile and the composition proportion of different classes of compounds were obtained. The relative contents of metabolites from breath varied during different time periods throughout a day. CONCLUSIONS: A more complete picture of the human breath metabolome was provided combining the results obtained from both online and offline analysis. The developed method allows analysis of breath samples with different status rapidly and directly, and it features simple operation and metabolite identification, requiring little or no sample preparation.
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Pruebas Respiratorias , Espiración , Humanos , Pruebas Respiratorias/métodos , Espectrometría de Masas/métodos , MetabolomaRESUMEN
Ambient ionization mass spectrometry (AIMS) is simple to operate for analytes adsorbed on the surface of various shaped probes. However, gaseous substances or liquids that are easy to evaporate, diffuse, and escape in the atmosphere are harder to capture. In this work, a Tee-shaped sample introduction device coupled with direct analysis in real time mass spectrometry (DART-MS) is developed. The Tee-shaped device is placed between the DART ion source and the MS inlet with a heated sample transfer tube. Gaseous samples from either a Tedlar sampling bag or liquids evaporated from a graduated syringe were tested. The Tee-shaped device was used for several volatile organic compounds with a wide range of boiling points, and detection limits of ng/mL to fg/mL were obtained. To test the device for real-life samples, puff-by-puff analysis of a complex gaseous mainstream smoke was performed. Individual puffs can be analyzed rapidly, and there is no cross contamination between consecutive puffs. The dynamic changes of chemical components among different puffs for different types of cigarettes can be observed. This work provides a universal Tee-shaped sampling device to enhance AIMS for the analysis of volatile compounds and gases, which is adapted to different sampling modules applicable for various forms of samples. The device enables direct exploration of chemical components in complex gaseous samples without tedious sample preparation and time-consuming LC or GC separation.
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Gases , Compuestos Orgánicos Volátiles , Espectrometría de Masas , Tereftalatos Polietilenos , Manejo de EspecímenesRESUMEN
Streaming potential is created when an electrolyte solution is forced to flow pass a charged surface. For an uncoated fused silica capillary, the streaming potential is measured between the inlet and outlet vials while applying a pressure across the capillary. The changes in streaming potential can be used to characterize the properties of the capillary inner surface. In this work, HCl, NaCl, and NaOH solutions ranging from 0.4 to 6 mM were used as the background electrolyte (BGE) at temperatures of 15 to 35 °C for the mesurements. The streaming potential decreases with the increase in BGE concentration, and the trend is amplified at higher temperatures. When buffer solutions in the pH range of 1.5 to 12.7 were used as the BGE, streaming potential was shown to be sensitive to changes in pH but reaches a maximum at around 9.5. At pH < 3.3, no streaming potentials were observed. The pH of zero surface charge (streaming potential equals 0) changes with temperature, and is measured to be 3.3 to 3.1 when the temperature is changed from 15 to 35°C. Zeta potentials can be calculated from the measured streaming potential, conductivity, and the solution viscosity. Surface charge densities were calculated in this work using the zeta potentials obtained. We demonstrated that capillary surface conditions can significantly change the streaming potential, and with three different solutions, we showed that analyte-dependent adsorption can be monitored and mitigated to improve the peak symmetry and migration times reproducibility.
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Electrólitos , Dióxido de Silicio , Adsorción , Reproducibilidad de los Resultados , TemperaturaRESUMEN
Concentration sensitivity is a key performance indicator for analytical techniques including for capillary electrophoresis-mass spectrometry (CE-MS) with electrospray ionization (ESI). In this study, a flow-through microvial interface was used to couple CE with MS and improve the ESI stability and detection sensitivity. By infusing a peptide mixture through the interface into an MS detector at a typical flow rate for CE-MS analysis, the spatial region near the interface was mapped for MS signal intensity. When the sprayer tip was within a 6 × 6.5 × 5 mm region in front of the MS inlet, the ESI was stable with no significant loss of signal intensity for ions with m/z 239. Finite element simulations showed that the average electric field strength at the emitter tip did not change significantly with minor changes in emitter tip location. Experiments were conducted with four different mass spectrometer platforms coupled to CE via the flow-through microvial interface. Key performance indicators, that is, limit of detection (LOD) and linearity of calibration curves were measured for nine amino acids and five peptides. Inter- and intraday reproducibility were also tested. The results were shown to be suitable for quantification when internal standards were used.
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Electroforesis Capilar/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Aminoácidos/análisis , Diseño de Equipo , Límite de Detección , Modelos Lineales , Péptidos/análisis , Péptidos/química , Reproducibilidad de los Resultados , Espectrometría de Masa por Ionización de Electrospray/instrumentaciónRESUMEN
A quantitative method was developed for the direct identity confirmation and quantification of alendronate using CE-MS combined with a pH-assisted focusing technique, dynamic pH barrage junction focusing. A pH-induced variation in electrophoretic mobility led to online focusing of alendronate at the sample/pH barrage boundary, significantly improving the detection sensitivity. In addition, the use of a flow-through microvial CE electrospray interface and the multiple reaction monitoring mode of MS further improved the specificity and quantification capability of this technology. This quantitative method presented a wide linear dynamic range over 8-2000 ng/mL and an LOD of 2 ng/mL. A 460-fold improvement in sensitivity was obtained when pH barrage junction focusing was applied during the CE process, in comparison to when normal CE was conducted without online sample stacking. The superior detection sensitivity over previously reported methods enables direct analysis of bisphosphonate compounds, eliminating tedious pre-column sample enrichment and derivatization. Validation of alendronate content in a commercial drug tablet further proved the reliability and power of this method. This simple method with no sample derivatization, superior sensitivity, and short run time (<8 min) is a promising alternative for accurate quantification of alendronate and other types of bisphosphonate compounds in both drug formulations and plasma samples.
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Alendronato/análisis , Electroforesis Capilar/métodos , Espectrometría de Masas/métodos , Concentración de Iones de Hidrógeno , Modelos Lineales , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , ComprimidosRESUMEN
A quantification method for imatinib (IM), its major metabolite N-desmethyl imatinib (NDI), and a degradation by-product was developed using CE-MS combined with an online concentration technique. The use of multiple reaction monitoring (MRM)-MS/MS further improved the sensitivity of this technology. Liquid-liquid extraction (LLE) using tertiary butyl methyl ether yielded high recovery and reproducibility for the pretreatment of serum samples. The recovery rate exceeded 83% for all three analytes, and was 90% for IM. To improve quantification results, a conductivity-induced online analyte concentration technique, field-amplified sample stacking (FASS), was used. The S/N ratios were improved at least 10-fold when compared with conventional capillary zone electrophoresis. The detection limits were 0.2 ng/mL for IM, 0.4 ng/mL for NDI, and 4 ng/mL for the degradation by-product. These results are superior to those previously obtained by other reported methods. The new method was validated in terms of its selectivity, intra- and interday repeatability and accuracy, and sample storage stability, following the guidelines issued by the European Medicines Agency. Considering the convenient pretreatment procedure (LLE), superior sensitivity, and fast analysis speed (<15 min), this method can be useful in the determination of imatinib levels in blood.
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Electroforesis Capilar/métodos , Mesilato de Imatinib/análisis , Espectrometría de Masas en Tándem/métodos , Humanos , Mesilato de Imatinib/química , Límite de Detección , Extracción Líquido-Líquido , Modelos Biológicos , Reproducibilidad de los ResultadosRESUMEN
Highly efficient enrichment of target glycopeptides plays a crucial role in glycoproteome research. Owing to their large surface area and excellent hydrophilicity, 2-D Hf-BTB nanosheets showed effective and selective enrichment of 78 glycopeptides derived from 29 glycoproteins with 90 N-glycosylation sites from just 2 µL of human serum.
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Coupling dispersive magnetic solid-phase extraction (DMSPE) to direct analysis in real time mass spectrometry (DART-MS) with a newly developed metal iron probe enables high-throughput, sensitive detection of herbicides such as triazine in environmental waters. Magnetic graphene oxide was used as a dispersive sorbent because it increased adsorption capacity in the DMSPE process. The planar structure and excellent thermal conductivity of graphene oxide facilitated the desorption and ionization of target analytes in DART-MS analysis. The iron probe, which is designed to fit into the moving trail of the DART interface, served as the sorbent collector as well as the support for the magnetic graphene oxide after DMSPE, and was put directly into the DART system. The ratio of magnetic core to graphene oxide in the nanoparticles and other key parameters in DMSPE and DART-MS procedures were systematically investigated and optimized. In addition, the presence of water on the sorbent proves to have a significant effect on DART-MS analysis. No organic solvents are used in this method, and the reusable iron probe is of low cost. Under the optimal conditions, limits of detection were found in the range of 1.6-152.1 ng/L for the triazines. Recovery and reproducibility were found to be in the ranges of 87.5-115.0% and 1.9-10.2%, respectively, for the six herbicides studied. The analytical performance of the DMSPE-DART-MS method indicated that applications for trace analysis of other compounds in liquid samples are also possible.
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Ensayos Analíticos de Alto Rendimiento , Extracción en Fase Sólida , Triazinas/análisis , Contaminantes Químicos del Agua/análisis , Grafito/química , Fenómenos Magnéticos , Espectrometría de Masas , Nanopartículas/química , Factores de TiempoRESUMEN
Capillary isoelectric focusing (cIEF) was online coupled to a Q-TOF MS by a flow-through microvial interface for the analysis of therapeutic mAb. Intact molecular weights obtained from the mass spectrum deconvolution of separated charge variants provided information on the structural heterogeneity of therapeutic mAbs. A sandwich cIEF-MS configuration composed of anolyte, sample, and catholyte segments sequentially injected into a neutrally coated capillary was used for the charge heterogeneity separation of four mAbs. Acetic acid and ammonium hydroxide were used in places of the non-volatile acids and bases commonly used for IEF but are incompatible with online MS detection. Glycerol was added as the anti-convective reagent. A chemical modifier was mixed with the cIEF effluent in the flow-throw microvial to maintain the ESI stability and to mitigate ion suppression from the co-eluted carrier ampholytes and glycerol. Analysis of mAb samples have shown relative populations of two basic variants originating from C-terminal lysine process and acidic variant of deamidation. The lysine clippings, deamidation, and sialic acid modification in oligosaccharide chains were revealed in infliximab. Two lysine clipping variants and a deamidated variant were observed in adalimumab. The duplicate analyses of a reference mAb demonstrated five charge variants separated by cIEF due to some unidentified modifications, as their mass spectra shared close similarities. The mAb analyses demonstrated the feasibility of the cIEF-MS method, and they demonstrated how charge and structural variants and minor differences in therapeutic mAbs are observed with this technology. Online cIEF-MS is an information rich technology with high throughput, demonstrated by the initial data presented here.
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Anticuerpos Monoclonales/análisis , Electroforesis Capilar/métodos , Focalización Isoeléctrica/métodos , Espectrometría de Masas/métodos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/aislamiento & purificación , Estudios de Factibilidad , Modelos Lineales , Reproducibilidad de los ResultadosRESUMEN
A fast, simple, efficient, and high-throughput analytical protocol using deep eutectic solvents (DES) for mechanochemical extraction (MCE) combined with direct analysis in real time mass spectrometry (DART-MS) was developed to quantify heat-labile bioactive compounds artemisinin (AN), arteannuin B, and artemisinic acid from Aretemisia annua. MCE is performed at room temperature, and target analytes are released into DESs within seconds; this method demonstrated multiple advantages over traditional extraction methods and organic solvents. DART-MS was then used for the structure confirmation and quantification for the three artemisinin major components extracted from plants of five locations. Liquid chromatography (LC) measurements were performed as well for results verification and comparison, and the amounts obtained were consistent between the two techniques. DART-MS showed advantages in simplicity, low limit of detection (5-15 ng mL-1), and superior speed (10-20 s), but with slightly higher relative standard deviation (0.7-10.8%). The entire protocol can be accomplished in a few minutes from raw materials to quantitative results. This study aims to establish a methodology combining high-efficiency sample pretreatment and rapid chemical analysis from complex matrixes, where the time-consuming separation procedure can be eliminated. Additionally, the use of toxic organic solvents needed in the process of chemical extraction and analysis is largely avoided. In general, this investigation provides a robust analytical procedure that can be widely used in many areas of research and industrial activities.
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Artemisininas/análisis , Espectrometría de Masas/métodos , Extracción en Fase Sólida/métodos , Solventes/química , Artemisia annua/química , Artemisininas/aislamiento & purificación , Límite de Detección , Solventes/síntesis químicaRESUMEN
Capturing phosphopeptides from complicated biological samples is essential for the discovery of new post-translational modification sites and disease diagnostics. Although several two-dimensional (2-D) materials have been used for phosphopeptides capturing, metal-organic framework (MOF) nanosheets have not been reported. The Ti-based MOF nanosheets have well-defined 2-D morphology, high density of active sites, large surface area, and an ultrathin structure. Phosphopeptides can be efficiently extracted and superior detection limits of 0.1 fmol µL-1 can be achieved even for an extremely low molar ratio of phosphoprotein/nonphosphoprotein (1:10000) mixtures. The selectivity over nonphosphopeptides can be enhanced further by pretreatment with a 10 mM salt solution (ß-glycerophosphate disodium, NaCl, or KCl). The performance of 2-D Ti-based MOF nanosheets is much better than Zr-based MOF (Zr-BTB) nanosheets or any other Ti-based 3-D MOF counterpart, such as MIL-125 and NH2-MIL-125. The nanosheets were used for in situ isotope labeling for abnormally regulated phosphopeptides analysis from serum samples of type 2 diabetes patients. The relative quantitative results showed that three of the phosphorylated fibrinogen peptides A (FPA, DpSGEGDFLAEGGGV, DpSGEGDFLAEGGGVR, and ADpSGEGDFLAEGGGVR) were down-regulated, while the other isoform (ADpSGEGDFLAEGGGV) was up-regulated in the serum samples of type 2 diabetes patients compared with those of healthy volunteers. Finally, proteomics analysis showed selective enrichment of phosphopeptides with 2-D Ti-based MOF nanosheets from real samples, including tryptic digests of mouse brain neocortex lysate, mouse spinal cord lysate, and mouse testis lysate, followed by LC-MS/MS analysis. Total numbers of 2601, 3208, and 2866 phosphopeptides were successfully identified from the three samples, respectively. The 2-D Ti-based MOF nanosheets significantly improved sample preparation for mass spectrometric analysis in phosphopeptides and phosphoproteomics research.