Dissociation of Mannose-Rich Glycans Using Collision-Based and Electron-Based Ion Activation Methods.

Three dissociation methods, including collision-induced dissociation (CID), electron capture dissociation (ECD), and electronic excitation dissociation (EED), were evaluated for the dissociation of doubly charged glycans using sodium or magnesium ions as charge carriers. CID produced mainly glycosidic cleavages, although more cross-ring fragment ions could be obtained at higher intensities when magnesium ions were used as charge carriers [M + Mg]2+. The 0,2A3, 0,3A3, and 0,4A3 ions provided structural information on the 3 → 1 and 6 → 1 linkages of the mannoses. Some internal fragment ions, such as 2,4A5_Y3ß, were also produced in high abundance, thus providing additional information on the glycan structure. ECD produced limited fragments compared to other dissociation methods when either of the metal ions were used as charge carriers. Cross-ring fragments were obtained in relatively high abundance, with the charge mainly retained on the nonreducing end. EED produced extensive glycosidic and cross-ring cleavages when either metal charge carrier was used. A higher fragmentation efficiency was achieved and more structural-specific fragments were produced when Na+ was used as the charge carrier. Of the 31 possible cross-ring cleavages, including 0,2-, 0,4-, 1,5-, 2,4-, and 3,5-cleavages, 25 were found, thus providing extensive linkage information. A wide range of fragment ions could be obtained in all dissociation methods when Mg2+ was used as the charge carrier. Two specific analytical approaches were found to produce extensively structural-specific information on the glycans studied, namely CID of magnesiated glycans and EED of sodiated glycans. These two methods were selected to further analyze the larger mannose-rich glycans Man6GlcNAc2 and Man8GlcNAc2 and generated extensive structural information.

Development of an All-in-One Protein Digestion Platform Using Sorbent-Attached Membrane Funnel-Based Spray Ionization Mass Spectrometry.

In this work, the sorbent-attached microfunnels used in funnel-based spray ionization mass spectrometry were evaluated for the all-in-one digestion of proteins. Sorbent materials, including C18 and TiO2 powders, were used as substrates to support in-funnel digestion and subsequent solid-phase extraction and purification of the digested products. In-funnel digestion protocols with and without reductive alkylation were developed for the analysis of proteins with and without disulfide linkages. Compared with in-solution digestion of the same loadings, the sequence coverage of in-funnel digestion of ovalbumin (with one disulfide bond) and ovocystatin (with two disulfide bonds) increased from 36% to 65% and from 21% to 81%, respectively. Loading 100 fmol of ovalbumin was sufficient to generate detectable tryptic fragments on C18-attached funnels. Notably, some phosphorylated digestion fragments were solely detected on C18-attached funnels and some nonphosphorylated digestion fragments were detected only on TiO2-attached funnels. Complex biological protein mixtures (i.e., bovine milk) and mouse liver protein extract could also be digested on C18- and TiO2-attached funnels. Using this platform, 30 samples were digested at the same time with enhanced digestion efficiency and were analyzed by funnel-based spray ionization mass spectrometry. This approach is potentially useful for sensitive and high-throughput bottom-up proteomic studies of complex biological samples.

Rapid Differentiation of Asian and American Ginseng by Differential Ion Mobility Spectrometry-Tandem Mass Spectrometry Using Stepwise Modulation of Gas Modifier Concentration.

This study reports a rapid and robust method for the differentiation of Asian and American ginseng samples based on differential ion mobility spectrometry-tandem mass spectrometry (DMS-MS/MS). Groups of bioactive ginsenoside/pseudo-ginsenoside isomers, including Rf/Rg1/F11, Rb2/Rb3/Rc, and Rd/Re, in the ginseng extracts were sequentially separated using DMS with stepwise changes in the gas modifier concentration prior to MS analysis. The identities of the spatially separated ginsenoside/pseudo-ginsenoside isomers were confirmed by their characteristic compensation voltages at specific modifier loading and MS/MS product ions. As expected, Asian ginseng samples contained some Rf and an insignificant amount of F11, whereas American ginseng samples had a high level of F11 but no Rf. The origin of the whole and sliced ginseng could further be confirmed using the quantitative ratios of three sets of ginsenoside markers, namely, Rg1/Re, Rb1/Rg1, and Rb2/Rc. Based on our results, new benchmark ratios of Rg1/Re < 0.15, Rb1/Rg1 > 2.15, and Rb2/Rc < 0.26 were proposed for American ginseng (as opposed to Asian ginseng).

Performance Enhancements in Differential Ion Mobility Spectrometry-Mass Spectrometry (DMS-MS) by Using a Modified CaptiveSpray Source.

Differential ion mobility spectrometry (DMS) spatially separates ions in the gas phase using the mobility differences of the ions under applied low and high electric fields. The use of DMS as an ion filter (or ion selector) prior to mass spectrometry analysis has been compromised by the limited ion transmission efficiency. This paper reports enhancement of the DMS-MS sensitivity and signal stability using a modified CaptiveSpray™ source. In terms of the ion sampling and transmission efficiency, the modified CaptiveSpray source swept ~ 89% of the ions generated by the tapered capillary through the DMS device (compared to ~ 10% with a conventional microspray source). The signal fluctuation improved from 11.7% (relative standard deviation, RSD) with microspray DMS-MS to 3.6% using CaptiveSpray-DMS-MS. Coupling of LC to DMS-MS via the modified CaptiveSpray source was simple and robust. Using DMS as a noise-filtering device, LC-DMS-MS performed better than conventional LC-MS for analyzing a BSA digest standard. Although LC-DMS-MS had a lower sequence coverage (55%), a higher Mascot score (283) was obtained compared to those of LC-MS (sequence coverage 65%; Mascot score 192) under the same elution conditions. The improvement in the confidence of the search result was attributed to the preferential elimination of noise ions. Graphical Abstract ᅟ.

Electron-ion reaction-based dissociation: A powerful ion activation method for the elucidation of natural product structures.

The structural elucidation of natural products (NPs) remains a challenge due to their structurally diversities and unpredictable functionalities, motifs, and scaffolds. Tandem mass spectrometry (MS/MS) is an effective method that assists the full elucidation of complicated NP structures. Ion activation methods play a key role in determining the fragmentation pathways and the structural information obtained from MS/MS. Electron-ion reaction-based dissociation (ExD) methods, including electron capture dissociation (ECD), electron transfer dissociation (ETD), electron-induced dissociation (EID), and electron detachment dissociation (EDD), can induce the breakage of specific chemical bonds and the generation of distinct fragment ions. This review article provides an overview of the mechanisms, instrumentation, and typical applications related to ExD MS/MS in the structural elucidation of NPs, primarly including lipids, oligosaccharides, glycoconjugates, metabolites, and pharmaceutical drugs. This work aims to reveal the capacity and potential of ExD mass spectrometry in analyzing NPs and consequently helping the NP communities to utilize the modern capabilities of MS/MS in the discovery and evaluation of novel NPs.

Structural Characterization of Intact Glycoconjugates by Tandem Mass Spectrometry Using Electron-Induced Dissociation.

Characterizing the structures of glycoconjungates is important because of glycan heterogeneity and structural complexity of aglycon. The presence of relatively weak glycosidic linkages leads to preferential cleavages that limit the acquisition of structural information under typical mass spectrometry dissociation conditions, such as collision-induced dissociation (CID) and infrared multiphoton dissociation. In this paper, we explored the dissociation behaviors of different members of glycoconjugates, including glycopeptides, glycoalkaloids, and glycolipids, under electron-induced dissociation (EID) conditions. Using CID spectra as references, we found that EID is not only a complementary method to CID, but also a method that can generate extensive fragment ions for the structural characterization of all intact glycoconjugates studied. Furthermore, isomeric ganglioside species can be differentiated, and the double bond location in the ceramide moiety of the gangliosides can be identified through the MS3 approach involving sequential CID and EID processes.

Generation and Characterization of Gas-Phase Doubly Charged Biradical Peptide Ions (M2+••).

The gas-phase chemistry of peptide radical ions is attracting considerable interest in the fields of biology and mass spectrometry owing to its capability to provide sequence information on peptides and proteins. In this study, we observed that doubly charged peptide ions (M2+) can be produced from the collision-induced dissociation (CID) of Hg(II)-adducted peptide ions. The chemical nature and, thus, the dissociation pathways of this hydrogen-deficient biradical M2+ species is intriguing. We investigated the generation and dissociation behavior of this M2+ species under electron-capture dissociation (ECD) and CID conditions. The side-chain loss in the CID of the charge-reduced M+• ions formed by single-electron capture suggested that M2+ existed as a biradical ion. This ion underwent the combination of the two radical sites and conversion to hydrogen surplus species through structural rearrangement with increased energies. This study demonstrated a promising method to generate reactive doubly charged biradical precursor ions and, thus, help characterize novel biomolecules.

Magnetic metal-organic framework-titanium dioxide nanocomposite as adsorbent in the magnetic solid-phase extraction of fungicides from environmental water samples.

In this work, a core-shell Fe3O4@SiO2@MOF/TiO2 nanocomposite was synthesized and used to as adsorbent for magnetic solid-phase extraction (MSPE) of triazole fungicides from environmental water samples. Five triazole fungicides, namely, triadimenol, hexaconazole, diniconazole, myclobutanil, and tebuconazole, were selected as target analytes for MSPE. These analytes were quantitatively adsorbed on microspheres, and the sorbents were separated from the solution by using a magnet. The analytes were desorbed by methanol and determined through liquid-chromatography coupled with tandem mass spectrometry. The extraction parameters affecting the extraction efficiency were optimized through response surface methodology. The limits of detection and limits of quantification for the selected fungicides were 0.19-1.20ngL(-1) and 0.61-3.62ngL(-1), respectively. The proposed method was applied to determine the concentration of fungicides in actual environmental water samples. The accuracy of the proposed method was evaluated by measuring the recovery of the spiked samples. The satisfying recoveries of the four water samples ranged from 90.2% to 104.2%. Therefore, the magnetic metal-organic framework/TiO2 nanocomposite based MSPE is a potential approach to analyze fungicides in actual water samples.

Metal-Organic Framework@Microporous Organic Network as Adsorbent for Solid-Phase Microextraction.

The practical applications of moisture sensitive metal-organic frameworks (MOFs) in the extraction technique are faced with avoided challenges related to competitive adsorption and hydrostability. The target analytes cannot be effectively extracted under humid conditions because of the competitive moisture adsorption and/or framework structure collapse of MOFs. In this Letter, metal-organic framework (MOF)@microporous organic network (MON) hybrid materials were explored for the first time as fiber coatings for solid-phase microextraction (SPME). Microporous materials with a hydrophobic surface was formed by coating the MOFs (MIL-101 and MOF-5) with MON through a sonogashira coupling reaction. MON acted as a hydrophobic "shield" to hinder the competitive moisture adsorption and improve moisture resistance and stability of the fiber. The sorbent exhibited higher enrichment factors (1215-3805) toward PAHs than other analytes in the water samples. An SPME method using MOF@MON-based fiber was developed to quantitatively determine PAHs. The proposed method was successfully applied to analyze PAHs in environmental water, particulate matter (PM2.5), and food samples. A successful technique is proposed to chemically control MOF for applications in solid-phase sorption-based extraction techniques.

C18-attached membrane funnel-based spray ionization mass spectrometry for quantification of anti-diabetic drug from human plasma.

In this work, sorbent-attached membrane funnel-based spray ionization mass spectrometry was explored for quantitative analysis of anti-diabetic drugs spiked in human plasma. C18-attached membrane funnel was fabricated for in situ extraction and clean-up to alleviate matrix suppression effect in the ionization process. Repaglinide was used as a target analyte of anti-diabetic drugs. Under optimal working conditions, good linearity (R(2) > 0.99) was obtained in the concentration range of 1-100 ng mL(-1). The method detection limit of target drugs spiked in the human plasma was around 0.30 ng mL(-1). Through the application of an isotope-labeled internal standard, the signal fluctuation caused by residual background matrices was largely alleviated and the precision of measurement (RSD) was below 15%. The recovery of repaglinide for 5, 25, and 100 ng mL(-1) of spiked human plasma matrixes ranged from 87% to 112%. The developed method was successfully applied to determine repaglinide in plasma volunteers who orally received a dose of drug association. Our results demonstrated that membrane funnel-based spray is a simple and sensitive method for rapid screening analysis of complex biological samples.

Thermo-responsive polymer tethered metal-organic framework core-shell magnetic microspheres for magnetic solid-phase extraction of alkylphenols from environmental water samples.

In this work, the thermo-responsive polymer PNIPAM tethered to Fe3O4@SiO2@MOF core-shell magnetic microspheres was first synthesized by a surface-selective post-synthetic strategy and underwent highly efficient magnetic solid-phase extraction (MSPE) of alkylphenols from aqueous samples. Alkylphenols, including 4-tert-octylphenol (OP) and 4-n-nonylphenol (NP), were selected as target compounds. The sample quantification was carried out using LC-MS/MS in multiple reaction monitor (MRM) mode. Under optimal working conditions, the developed method showed good linearity in the range of 5-1000ngL(-1), a low limit of detection (1.5ngL(-1)), and good repeatability (relative standard deviation, <8%, n=5) for NP and OP. Owning to the hydrophilic/hydrophobic switchable properties of the nanocomposite, high recoveries (78.7-104.3%) of alkylphenols were obtained under different extraction conditions. The levels of OP and NP in environmental samples collected from local river, lake and pond waters were analyzed using the developed method. It was believed that the synthesized material with the thermo-responsive coating, large surface areas and magnetic properties should have great potential in the extraction and removal of alkylphenols from environmental samples.

Differentiation of Isomeric Ginsenosides by Using Electron-Induced Dissociation Mass Spectrometry.

Current phytochemical research on ginsengs focuses on the structural characterization and isomer differentiation of ginsenosides. In this Letter, electron-induced dissociation (EID) was initially investigated by analyzing isomeric ginsenosides. EID provided more structural information on their differentiation than collision-induced dissociation (CID) did. Glycosyl group migration previously observed in the CID of oligosaccharide ions could also be found in the EID of protonated Rg1. This rearrangement reaction would show substantial ambiguities in differentiating Rg1 from Rf. Although other charge carriers could alleviate this problem, the use of EID in dissociating deprotonated ginsenoside ions was superior to other techniques in terms of eliminating glycosyl group migration and generating diagnostic fragment ions for the differentiation of structural isomers. This study demonstrates a potential method to analyze natural products and thus help discover and evaluate novel compounds.

Suppression of peptide ion dissociation under electron capture: role of backbone amide hydrogen.

RATIONALE: The electron capture dissociation (ECD) of proteins/peptides is affected by the nature and sequence of amino acid residues. Electron capture/transfer with no dissociation is an intriguing phenomenon that has occasionally been observed. We have previously identified that diarginated peptides enriched with glutamic acid residues were found to show suppression of backbone fragmentation. In this paper, we report the effect of geometrical parameters of a peptide, including chain length, conformation and amide hydrogen, on the suppression of ECD fragmentation using synthetic model peptides. METHODS: Glycine containing model polypeptides were used to probe the mechanism. Molecular-mechanics was used to obtain the conformation of the precursor ions. The ECD experiments were performed on a Bruker APEX III 4.7 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. RESULTS: Significant decreases in the intensities of the fragment ions were observed for the 23-mer polypeptide with only one E residue. This implied that the E:R ratio was no longer the sole determining factor for the occurrence of suppression effects. Results of conformational searches showed that there was a hydrogen-bonding 'ladder' formed in the 23-mer polypeptide, which was not found in the 15-mer peptide. Substituting the normal amino acid residues by the corresponding N-methylated amino acid residues in the model peptide, the suppression effect disappeared. CONCLUSIONS: Our results indicate that survival of the intact reduced peptide ion after electron capture depends also on the length of the peptide. The amide hydrogen was critical in forming the resonance structure that suppressed the ECD fragmentation.

Effect of structural parameters on the electron capture dissociation and collision-induced dissociation pathways of copper(II)-peptide complexes.

The gas-phase dissociation pathways of proteins/peptides are usually affected by the nature of the charge carrier and the sequence of amino acid residues. The effects of peptide structural parameters, including peptide composition, chain length and amide hydrogen, on the gas-phase dissociation of Cu(II)-model peptide complexes were explored in this study. Polyglycine peptides with flexible frames were used as probes to reduce the complexity of the system and illustrate the mechanism. Results revealed that the types of fragment ions generated in the electron capture dissociation (ECD) of Cu(II)-adducted peptides changed according to the basic amino acid residue composition. Charged or neutral tryptophan side-chain losses were observed in the collision-induced dissociation (CID) of Cu(II)-peptide complexes. Internal electron transfer between tryptophan and metal ion within the complex occurred during the CID reaction, leaving the charge-reduced Cu(+) as a closed d-shell stable electron configuration. The choice of the reaction channel was then determined by the gas-phase basicity of the peptide. Amide hydrogen was critical in the formation of metalated b-/y-ions in the ECD process as determined through mutation of the backbone amide group. Increasing the chain length suppressed the ECD of Cu-metalated peptide species. Our results indicate that the structural parameters of peptides play important roles in the gas-phase dissociation processes of Cu-peptide complexes.

Letter: Evaluation and comparison of collision-induced dissociation and electron-capture dissociation for top-down analysis of intact ribonuclease B.

It has been previously reported that the glycosylation site and protein-sequence information could be obtained for ribonuclease B by top-down electron-capture dissociation (ECD) and collision-induced dissociation (CID) mass spectrometry (MS). However, the sequence coverage of ribonuclease B was limited in a single activation, and the structural information on the glycan moiety was not probed successfully in previous experiments. Here, we demonstrate that ECD and CID techniques can be used together as an effective top- down method for the structural characterization of intact glycoprotein. Even without an elaborate pre- or post- ECD activation, a high sequence coverage (<90%) of ribonuclease B could be achieved with substantial amounts of structural information for the glycan moiety. By comparing our work with previous results, it is postulated that the disulfide bond reduction strategy might play a significant role in determining the efficiency of top-down MS.

Membrane funnel-based spray ionization for protein/peptide analysis by Fourier transform ion cyclotron resonance mass spectrometry.

RATIONALE: Samples analyzed in proteomic studies by nanoelectrospray ionization (nanoESI) are extremely limited in quantity requiring careful sample handling to prevent loss upon transfer and to maintain sample concentration. To alleviate the operational process and reduce the cost of nanoESI, it is essential to develop more robust, simple and sensitive analytical variants of the process. Membrane funnel-based spray was developed for analysis of proteins/peptides in this study. METHODS: The membrane funnel was fabricated from thin flexible membrane by a punching method using a homemade device. The performance of the membrane funnel-based spray was demonstrated by analyzing peptides, proteins and trypsin-digested samples in comparison of nanoESI and the Teflon sheet based microfunnel. RESULTS: Compared with the microfunnel, the membrane funnel can be fabricated easily by punching a thin flexible membrane using a sharp needle. Only 50 nL of sample was required for an analysis. The membrane funnel enhanced the spray sensitivity 100-fold. A total of 5 amol of on-spot sample loading was sufficient to provide a measurable signal on a 9.4 Tesla Fourier transform ion cyclotron resonance mass spectrometry system. High-mass proteins (up to 66 kDa) could be analyzed using this funnel-based spray system. Good sequence coverage was obtained for tryptic digested samples. CONCLUSIONS: A rapid, simple and cheap membrane funnel-based sample plate fabrication method was developed. The membrane funnel-based spray is a promising new variant of nanoESI capable of fast and sensitive analysis of peptides/proteins with great potential that could be extended to other applications, including quantitative analysis at high throughput and imaging mass spectrometry.

Development of miniaturized sorbent membrane funnel-based spray platform for biological analysis.

In this work, a miniaturized solid-phase extraction (SPE) platform, called sorbent membrane funnel, which permits in situ cleanup prior to membrane funnel-based spray analysis was developed. The fabrication of funnel and the mounting of SPE sorbent were simple and straightforward by a homemade punching system. Using different sorbents, the SPE sorbent funnel has been successfully applied in spray analysis of drug molecules spiked in human plasma, trypsin digested solution of bovine serum albumin in the presence of high concentration of chaotropic reagents, and phosphopeptides in the tryptic digested solution of casein. The results demonstrated that SPE sorbent attached membrane funnels can be a useful tool in common metabolomic and proteomic applications.