Rare ginsenosides, compounds O and Mc1, were produced from the major ginsenosides Rb2 and Rc by β-glucosidase from Esteya vermicola via the pathways of Rb2–compound O and Rc–compound Mc1. The biotransformation conditions for the compounds using E. vermicola CBS 115803 were established with a transformation temperature and time of 26 °C and seven days, respectively, and a pH value of 5.5 at 130 rpm. The structure of the key metabolite was confirmed by MS analysis. The biotransformation yields for the two compounds were 96.7 and 95.2%, respectively. A feasible method to recover the products was also developed, in which the products were purified from the fermentation broth, and the unreacted substrates were recovered from the mycelia. The present study provides a practical and potentially valuable approach that can be applied in the preparation of compounds O and Mc1 without byproducts.

Monitoring the in vivo dynamics of neurochemicals is very important for studies on learning and memory impairment. However, drawbacks in conventional sampling and off-line analysis methods require the employment of a more satisfying system. We present an online microdialysis coupled with liquid chromatography-tandem mass spectrometry method (LC-MS/MS) to determine eight neurochemicals simultaneously, including glutamate, aspartic acid, γ-aminobutyric acid, serine, taurine and acetylcholine, as well as dopamine and serotonin. A self-assembled automated injector with a high-pressure-resisting six-port valve was utilized to control the switch of the two states between loading and injection, which potentially simplified the sample preparation procedures and avoided the restrictions offered by small sample volume. Good linearity (R2 > 0.997) was observed in each analyte dynamic range. The inter-day and intra-day precisions (RSD) were <15%, and the accuracy (RE) was from −13.84% to 11.16%. The overall matrix effect was within an acceptable range, from 87.79% to 111.71%. Morris water maze (MWM) test was performed to evaluate the differences of cognitive abilities among normal control, diabetes mellitus (DM) model and Rhizoma coptidis (R. coptidis)-treated DM group rats. Moreover, the developed method was applied for the further exploration of the R. coptidis efficacies on the eight analytes changes in the hippocampus of diabetic rats with mild cognitive impairment. It was found that an improvement in neurochemicals imbalance was observed in diabetic rats after treatment with R. coptidis. In conclusion, the developed online analysis method is selective, sensitive and accurate, which is suitable for in vivo neurochemicals monitoring and could be extended to pharmacology studies.

Cu, Zn-superoxide dismutase (SOD1) is a homodimeric enzyme of approximately 32 kDa. Each monomer contains one Cu2+ and one Zn2+ ion, which play catalytic and structural roles in the enzyme. Dimer formation is also essential to its functionality. The spatial structure of this metalloenzyme is also closely related to its bioactivities. Here we investigate the structural and conformational changes of SOD1 in the gas phase by electrospray ionization mass spectrometry (ESI-MS) and ion-mobility (IM) separation combined with tandem mass spectrometry (MS/MS). First, the composition and forms of SOD1 were analyzed by ESI-MS. The dimer, monomer, and apomonomer were observed under different solvent conditions. The dimer was found to be stable, and could retain its native structure in neutral buffer. Ion-mobility separation combined with MS/MS was used to reveal the conformational changes and dissociation process of SOD1 when it was activated in the gas phase. Three different dimeric and two monomeric conformers were observed; three unfolding and dissociation pathways were also identified. The results from this study demonstrate that IM-MS/MS could be used to obtain spatial structural information on SOD1 and that the technique could therefore be employed to investigate the conformational changes in mutant SOD1, which is related to amyotrophic lateral sclerosis and other neurodegenerative disorders.

•An UPLC–ESI–QTOF method has been established in positive and negative ion mode.•74 components in WTD were identified or presumed.•Rationalized fragmentation patterns of components in WTD were analyzed.Wu-tou decoction (WTD), a traditional Chinese medicine (TCM) formula, is composed of Aconiti Radix Cocta, Ephedrae Herba, Paeoniae Radix Alba, Astragali Radix and Glycyrrhiza Radix Preparata, and it has been used for more than a thousand years to treat rheumatic arthritis, rheumatoid arthritis and pain of joints, while the active constitutions of WTD are unclear. In this research, an ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC–Q-TOF-MS) method in both positive and negative ion mode was established to investigate the major constitutions in WTD. A Waters ACQUITY UPLC BEH C18 column was used to separate the aqueous extract of WTD. Acetonitrile and 0.1% aqueous formic acid (v/v) were used as the mobile phase. 74 components including alkaloids, monoterpene glycosides, triterpene saponins, flavones and flavone glycosides were identified or tentatively characterized in WTD based on the accurate mass within 15 ppm error and tandem MS behavior. All the constitutions were also detected in the corresponding individual herbs. These results will provide a basis for further study in vivo of WTD and the information of potential new drug structure for treating rheumatic arthritis and rheumatoid arthritis.

•A novel method for measuring superoxide anion radical scavenging activity was established.•The method is simple, rapid and sensitive.•The O2− scavenging activities of tissue homogenates, serum and herbal extracts were determined.•The method can be used for screening superoxide anion radical scavengers.Superoxide anion radical (O2−) plays an important role in several human diseases. The xanthine/xanthine oxidase system is frequently utilized to produce O2−. However, false positive results are easily got by using this system. The common spectrophotometric probes for O2− are nitroblue tetrazolium (NBT) and cytochrome c. Nevertheless, the application of NBT method is limited because of the water-insolubility of NBT formazan and the assay using cytochrome c lacks sensitivity and is not suitable for microplate measurement. We overcome these problems by using 1,2,3-trihydroxybenzene (pyrogallol) as O2−-generating system and a highly water-soluble tetrazolium salt, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium sodium salt (WST-1) which can be reduced by superoxide anion radical to a stable water-soluble formazan with a high absorbance at 450 nm. The method is simple, rapid and sensitive. Moreover, it can be adapted to microplate format. In this study, the O2− scavenging activities of superoxide dismutase (SOD), l-ascorbic acid, N-acetyl-l-cysteine (NAC), albumin from human serum, flavonoids and herbal extracts were assessed by using this method. Meanwhile, the activities of tissue homogenates and serum were determined by using this validated method. This method, applicable to tissue homogenates, serum and herbal extracts, proved to be efficient for measuring O2− scavenging activities of biological and abiological samples.

Xanthine oxidase (XOD) inhibitors play an important role in the treatment of gout and many other diseases related to the superoxide anion metabolism. In this study, an ultrafiltration-liquid chromatography–mass spectrometry (UF-LC–MS) method was developed for the screening and identification of potential XOD inhibitors from Radix Salviae Miltiorrhizae extract. Eleven lipophilic diterpenoid quinines were identified as XOD inhibitors from the extract. The relationship between the structure and activity of the detected compounds was estimated on the basis of the UF-LC–MS data. The results demonstrate that the 1,2-naphthoquinone group is necessary for the XOD inhibitory activity of the compounds, and that furan and hydroxyl on the alicyclic ring could enhance the activity of the compounds at different levels. These results may explain and support the medical use of the extract of Radix S. Miltiorrhizae for the prevention and treatment of hyperuricemia and gout.Highlights► UF-LC–MS method for screening XOD inhibitors was developed. ► Method is suited for rapid screening and characterization of XOD inhibitors from TCM. ► UF-LC–MS is a rapid, sensitivity and high throughput tool to screen XOD inhibitors. ► Eleven XOD inhibitors were screened from the Radix Salvia Miltiorrhizae extract. ► The relationship between the structure and activity of inhibitors was estimated.

A procedure involving acetonitrile-based extraction combined with dispersive liquid–liquid microextraction (DLLME) and detection by ultra high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) was used for determination of 39 pesticides in ginseng. The extraction of pesticide residues in ginseng was performed with acetonitrile, applying QuEChERS methodology, and the extract was further disposed by DLLME method before analyzed by UHPLC–MS/MS. The average recoveries ranged from 70 to 120% for 82% of the analytes with RSD lower than 15%. The calibration curves obtained with blank matrices were linear with a correlation coefficient of over 0.99. The limits of detection were between 0.01 and 1.0 μg/kg. Matrix effects were studied by comparing solvent calibration curves and matrix-matched calibration curves. The results indicate the feasibility of this method for the determination of 39 pesticides in ginseng.Highlights► A QuEChERS-DLLME-UPLC-MS/MS has been developed for rapid determination of multiclass pesticide residues in ginseng. ► The procedure to increase the sensitivity was concentration by DLLME method instead of evaporation by nitrogen. ► This method was simple, rapid and cheap.

Xanthine oxidase (XOD) inhibitors and superoxide anion scavengers play an important role in the treatment of gout and the inhibition of many diseases related to superoxide anion. The respective quantitation of uric acid and superoxide anion by traditional spectroscopic methods is routine in XOD inhibitors and superoxide anion scavengers screening at laboratories worldwide. In the present study, we established an ultrahigh performance liquid chromatography and triple quadrupole mass spectrometry (UHPLC–TQ-MS) method of higher accuracy and speed that combines screening of superoxide anion scavenger and XOD inhibitor in a single analysis by adding WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium sodium salt) to the enzymatic reaction. We applied the established method to determine the XOD inhibitory activities and superoxide scavenging activities of some herbal extracts and compounds from natural products, which could be classified into six groups based on the results of the assay. Our innovative protocol is fast, accurate and robust. Moreover, it can eliminate false positive and false negative results which may occur in the traditional spectroscopic methods.Graphical abstractIt is a novel method which combines screening of superoxide anion scavenger and xanthine oxidase inhibitor in a single analysis by adding WST-1 to the enzymatic reaction. This method can accurately quantify the amount of analytes by using multiple reaction monitoring (MRM) function which is through monitoring the ratio of mass and charge (m/z) of parent ions and diagnostic fragment ions, therefore, it can eliminate the false positive and false negative results.Highlights► A novel method to screen superoxide anion scavenger and xanthine oxidase inhibitor. ► This method can be used to detect and quantify superoxide anion. ► It is a fast, accurate and robust method. ► It can eliminate false positive and negative results.

An ultra-high-performance liquid chromatographic–tandem mass spectrometry (UHPLC–MS/MS) method for the simultaneous quantification and identification of 116 pesticide residues which were most widely used in plants used in Traditional Chinese Medicine (TCM) in 15 min has been developed and validated. Samples were extracted and cleaned up with modified QuEChERS method and detected by UHPLC–MS/MS under multiple reactions monitoring mode, and quantified by matrix-match calibration. The validation study was carried out on five different matrixes following DG SANCO/2007/3131 of the European Quality Control Guidelines. The linearity of the calibration was good between 5 and 100 ng ml−1 concentration ranges, and the limits of quantification (LOQs) less than 0.01 mg/kg for most pesticides. The mean recoveries of almost all pesticides were in the range from 70% to 120% at three concentration levels ranging from 0.01 mg/kg to 0.1 mg/kg with relative standard deviations (RSD) better than 15%. The method was applied on 138 real samples from 102 different kinds of Chinese herbal medicine. 95 positive samples were detected. This method is fast, robust, accurate, selective, sensitive and easy to operate.

Various kinds of Fructus schisandrae were studied by surface desorption atmospheric pressure chemical ionization mass spectrometry (DAPCI-MS) without any sample pretreatment. The volatile components in F. schisandrae were detected in the ambient environment and the analytical time for each sample was only 30 s. F. schisandrae are produced mainly in 5 different geographical regions (Elunchun, Mudanjiang, Tonghua, Tieling and Shangluo), and they could be successfully differentiated according to their chemical markers by Principal Component Analysis (PCA). A total of 8 components which gave more contribution for PCA analysis were unambiguously identified by comparison of the MS2 data of chemical markers to the data of reference compounds as reported in the literature. Similarly, wild grown and cultivatable species of F. schisandrae were well separated by the above-mentioned method. In addition, raw and processed cultivatable F. schisandrae (steamed by water, alcohol, vinegar, or honey, and fried by honey) were found to be clustered at different location, respectively. Furthermore, the clustered degree of differently processed products was correlated with their clinical effects. Our results demonstrated that DAPCI-MS in combination with PCA was a feasible technique for high-throughput differentiation of various kinds of F. schisandrae. It is also possible that DAPCI-MS could become a powerful technology in the studies of traditional Chinese medicine studies and in situ analysis of Chinese herbs.Graphical abstractVarious kinds of Fructus schisandrae were studied by surface desorption atmospheric pressure chemical ionization mass spectrometry (DAPCI-MS) for the first time and the schematic diagram was shown in Fig. 1. The results demonstrated that DAPCI-MS in combination with PCA was a feasible technique for high-throughput differentiation of various kinds of F. schisandrae. It is also possible that DAPCI-MS could become a powerful technology in the studies of traditional Chinese medicine studies and in situ analysis of Chinese herbs.Highlights► The method is a convenient technique for differentiation of Fructus schisandrae. ► The analysis was operated in the ambient environment without sample pretreatment. ► The analytical time for each sample was only 30 s.

Four water-soluble polysaccharide fractions (OCAP-2-1, OCAP-2-2, OCAP-3-1 and OCAP-3-3) extracted from the Ornithogalum caudatum Ait, were obtained by DEAE fast flow Sepharose anion-exchange, and Sephadex G-75 gel-permeation chromatography. The monosaccharide components of four polysaccharides were characterized by gas chromatography (GC), and the majority of the monosaccharide components were glucose (30–42.5%) and galactose (23–29%), with low levels of arabinose (6–14%), mannose (2–9%), xylose (4–6%), glucuronic acid (1.0–7.5%), galactose acid (1–6%). The high-performance gel-permeation chromatography (HPGPC) analysis showed that the average molecular weight (Mw) of four polysaccharides were approximately 102.1, 62.3, 46.4 and 22.8 KDa, respectively. The protein contents of four fractions were 2.13%, 2.82%, 3.45% and 3.97%, respectively. All polysaccharide fractions exhibited significantly higher antitumor activity against solid tumor Sarcoma 180 in vivo than did a blank control. Fractions OCAP-2-2, OCAP-3-1 and OCAP-3-2 significantly inhibited the growth of K562 cells in vitro. Results of these studies demonstrated that the polysaccharide had a potential application as natural antitumor drugs.

In vitro α-glucosidase inhibition assays and ultrafiltration liquid chromatography with photodiode array detection coupled to electrospray ionization tandem mass spectrometry (ultrafiltration LC-DAD-ESI-MSn) were combined to screen α-glucosidase inhibitors from hawthorn leaf flavonoids extract (HLFE). As a result, four compounds were identified as α-glucosidase inhibitors in the HLFE, and their structures were confirmed to be quercetin-3-O-rha- (1-4)-glc-rha and C-glycosylflavones (vitexin-2″-O-glucoside, vitexin-2″-O-rhamnoside and vitexin) by high-resolution sustained off resonance irradiation collision-induced dissociation (SORI-CID) data obtained by Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). Several other C-glycosylflavones (vitexin, isovitexin, orientin, isooriention) and their aglycones apigenin and luteolin were evaluated by in vitro assays, and were found to possess strong α-glucosidase inhibitory activities as well. Moreover, the substituent groups on the flavones had a great impact on the enzyme inhibition activity. C-3′-OH of the B-ring of flavones in particular increased the α-glucosidase inhibition activity, whereas C-glycosylations at C-6 or C-8 of the A ring weakened the inhibition activity.

In this report, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used to study the binding interactions between calmodulin and two target peptides (melittin and substance P). Various matrix conditions were tested and the less acidic matrix DHAP and THAP were found to favor the survival of the intact calcium-calmodulin as well as the calmodulin-peptide complexes. However, the application of direct MALDI-MS to detect the intact complexes turned out to be very difficult due to the dissociation of the complexes and the formation of nonspecific aggregates. In contrast, the specific binding of the target peptides to calmodulin could be easily deduced using intensity-fading (IF) MALDI-MS. Compared with the nonbinding control, clear reduction in the ion abundances of the target peptides was observed with the addition of calmodulin. Relative binding affinities of different peptides towards the protein could also be estimated using IF-MALDI-MS. This study may extend the application of IF-MALDI-MS in the analysis of noncovalent complexes and offer a perspective into the utility of MALDI-MS as an alternative approach to study the peptides binding to calmodulin.

The electrospray ionization ion trap multiple-stage tandem mass spectrometry (ESI-MSn) and electrospray ionization Fourier transform ion cyclotron resonance multiple-stage tandem mass spectrometry (ESI-FT-ICR-MSn) have been applied successfully to the direct investigation of a number of dibenzocyclooctadiene lignan constituents from the methanol extracts of the Fructus Schisandrae in the positive ion mode. The detailed structural characterization of the same skeleton and different peripheral substituents had been studied and the precise elemental compositions of ions at high mass resolution had been obtained. So the fragmentation mechanisms could be clarified. And the lignan components in Schisandra chinensis (Turcz.) Baill. fruits (SCF) and Schisandra sphenanthera Rehd. et Wils. fruits (SSF) were identified by comparing the structural information and fragmentation mechanisms. Then a pair of isobaric compounds was differentiated. Meanwhile these two similar fruits were distinguished. The research results demonstrated that ESI-MSn technique is a sensitive, selective and effective tool for the direct analysis and rapid determination of constituents in complex mixtures from nature products. And these should be useful for the identification of similar compounds and differentiation of similar species from Chinese herbs.

In this study, electrospray ionization mass spectrometry (ESI-MS) was used to investigate the binding interactions of ten flavonoid aglycones and ten flavonoid glycosides with DNA duplexes. Relative binding affinities of the flavonoids toward DNA duplexes were estimated based on the fraction of bound DNA. The results revealed that the 4′-OH group of flavonoid aglycones was essential for their DNA-binding properties. Flavonoid glycosides with sugar chain linked on ring A or ring B showed enhanced binding toward the duplexes over their aglycone counterparts, whereas glycosylation of the flavonol quercetin on ring C exhibited a less pronounced effect. The aglycone skeletons and other hydroxyl substitutions on the aglycone also have an effect on the fractions of bound DNA. Upon collision-induced dissociation, the complexes containing flavonoid aglycones underwent the predominant ejection of a neutral ligand molecule, suggesting an intercalative DNA-binding mode. However, for complexes containing flavonoid glycosides, the loss of nucleobase increased to different extents, indicating a stronger binding or different binding mode. The results may provide not only a deeper insight into the DNA-binding properties of flavonoids but also a useful guideline for the design of efficient DNA-binding agents for chemotherapy.

Doubly charged cluster ions, besides singly charged cluster ions, from sodium and potassium nitrates were produced evidently under normal source capillary temperature of 200 °C in both positive and negative ion electrospray ionization (ESI) ion trap mass spectrometry. The fragmentation pathways for doubly charged cluster ions were studied in detail using ESI tandem mass spectrometry and two pathways were observed depending on the cluster sizes of alkali metal nitrates. In addition, factors that affect the formation of cluster ions were also interrogated.

Eighteen triterpenoidic saponins in crude extracts from leaves of Acanthopanax senticous Harms have been investigated by electrospray ionization multi-stage tandem mass spectrometry and high-resolution mass spectrometry. In ESI-MS spectra, predominant [M + Na]+ ions in the positive ion mode have been observed for molecular mass information. Meanwhile, specific structural correlations between these ions are firstly found. The 18 peaks (ions) can be classified into three groups (group D, E, and F with mass increase) with each group including six peaks. There is a mass difference of 132 Da between group D and E for each corresponding peak in turn (for example peak 1 to peak 7), indicating one more pentose residue was attached to saponins in group E than those corresponding in group D. The mass difference of 146 Da between group E and F implies one more deoxy-hexose attached to saponins in group F than those corresponding in group E. The structural correlations of the corresponding ions are confirmed by tandem mass spectrometry and high-resolution mass spectrometry. These structural features can not only facilitate the rapid characterization of the native known saponins in crude plant extracts, thus avoiding tedious derivation and separation of saponins, but also help find novel compounds of the same type in a specific medicinal plant. The structures of 14 known triterpenoidic saponins and four unknown saponins are thus determined or predicted. This methodology, with a combination of structural correlation of a complex mixture of homologs (which are difficult to separate) and mass spectrometry, has been established as a powerful and practical tool for the profiling of mixtures, especially of crude plant extracts and structural characterization of unknown compounds.

The liquid chromatography–electrospray ionisation-mass spectrometry (LC–ESI-MS) method was developed for the analyses and identification of saponins in plant extract from the root of Panax notoginseng (Burk.) F.H. Chen. The HPLC experiments were proceeded by means of a reversed-phase C18 column and a binary mobile phase system consisting of 0.2% acetic acid and acetonitrile under gradient elution conditions. Eight major peaks were separated and detected using both evaporative light scattering and MS detectors. The mass spectrometer was operated in the negative ion mode using electrospray ionization. The molecular ions, [M − H]− and the adduct ions [M + AcO]− of saponins were observed, and from which the molecular weights were obtained. A collision-induced dissociation (CID) experiment was carried out to aid the identification of the backbone and glycosidic linkage sites of the saponins. The identification of the saponins (peaks 1–7) in the extract of P. notoginseng was based on matching their retention times, the detection of the saponin molecular ions, and the fragment ions of the molecular ion obtained in the CID experiments with those of the authentic standards and data reported in the literature. The molecular structure of peak 8 was elucidated according to the fragmentation patterns and the literature reports.

In this study, electrospray ionization mass spectrometry (ESI-MS) was used to investigate the binding interactions of ten flavonoid aglycones and ten flavonoid glycosides with DNA duplexes. Relative binding affinities of the flavonoids toward DNA duplexes were estimated based on the fraction of bound DNA. The results revealed that the 4′-OH group of flavonoid aglycones was essential for their DNA-binding properties. Flavonoid glycosides with sugar chain linked on ring A or ring B showed enhanced binding toward the duplexes over their aglycone counterparts, whereas glycosylation of the flavonol quercetin on ring C exhibited a less pronounced effect. The aglycone skeletons and other hydroxyl substitutions on the aglycone also have an effect on the fractions of bound DNA. Upon collision-induced dissociation, the complexes containing flavonoid aglycones underwent the predominant ejection of a neutral ligand molecule, suggesting an intercalative DNA-binding mode. However, for complexes containing flavonoid glycosides, the loss of nucleobase increased to different extents, indicating a stronger binding or different binding mode. The results may provide not only a deeper insight into the DNA-binding properties of flavonoids but also a useful guideline for the design of efficient DNA-binding agents for chemotherapy.

In vitro α-glucosidase inhibition assays and ultrafiltration liquid chromatography with photodiode array detection coupled to electrospray ionization tandem mass spectrometry (ultrafiltration LC-DAD-ESI-MSn) were combined to screen α-glucosidase inhibitors from hawthorn leaf flavonoids extract (HLFE). As a result, four compounds were identified as α-glucosidase inhibitors in the HLFE, and their structures were confirmed to be quercetin-3-O-rha- (1-4)-glc-rha and C-glycosylflavones (vitexin-2“-O-glucoside, vitexin-2”-O-rhamnoside and vitexin) by high-resolution sustained off resonance irradiation collision-induced dissociation (SORI-CID) data obtained by Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). Several other C-glycosylflavones (vitexin, isovitexin, orientin, isooriention) and their aglycones apigenin and luteolin were evaluated by in vitro assays, and were found to possess strong α-glucosidase inhibitory activities as well. Moreover, the substituent groups on the flavones had a great impact on the enzyme inhibition activity. C-3′-OH of the B-ring of flavones in particular increased the α-glucosidase inhibition activity, whereas C-glycosylations at C-6 or C-8 of the A ring weakened the inhibition activity.Screening and Structural Characterization of α-Glucosidase Inhibitors from Hawthorn Leaf Flavonoids Extract by Ultrafiltration LC-DAD-MSn and SORI-CID FTICR MS.Download high-res image (87KB)Download full-size image

In this report, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used to study the binding interactions between calmodulin and two target peptides (melittin and substance P). Various matrix conditions were tested and the less acidic matrix DHAP and THAP were found to favor the survival of the intact calcium–calmodulin as well as the calmodulin–peptide complexes. However, the application of direct MALDI-MS to detect the intact complexes turned out to be very difficult due to the dissociation of the complexes and the formation of nonspecific aggregates. In contrast, the specific binding of the target peptides to calmodulin could be easily deduced using intensity-fading (IF) MALDI-MS. Compared with the nonbinding control, clear reduction in the ion abundances of the target peptides was observed with the addition of calmodulin. Relative binding affinities of different peptides towards the protein could also be estimated using IF-MALDI-MS. This study may extend the application of IF-MALDI-MS in the analysis of noncovalent complexes and offer a perspective into the utility of MALDI-MS as an alternative approach to study the peptides binding to calmodulin.Intensity fading MALDI-MS was demonstrated to be more suitable to study the noncovalent interactions between calmodulin and its target peptides than direct MALDI-MS.Download high-res image (51KB)Download full-size image