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 a Matrix Sublimation Device with Controllable Crystallization Temperature for MALDI Mass Spectrometry Imaging.

The size and distribution of matrix crystals deposited on the surface of a tissue section play a key role in the performance of MALDI mass spectrometry imaging (MALDI-MSI). In this study, uniform distribution and a restricted size of matrix crystals were achieved via a homemade matrix sublimation device with controllable crystallization temperature. The crystallization temperature was stably controlled at a subzero temperature, and homogeneous matrix crystals with diameters <0.2 µm were generated on the sample surface. Typical MALDI-MSI experiments of endogenous and exogenous components in the tissues of strawberries, kidneys, and mussels were conducted to examine the performance of the sublimator. Good reproducibility of MALDI-MSI was achieved, and the quality of ion images was significantly improved. These results demonstrate that the developed sublimator should have potential in matrix deposition for further high resolution MALDI-MSI application.

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.

Tissue imaging with in situ solid-phase extraction micro-funnel based spray ionization mass spectrometry.

Current imaging mass spectrometry techniques are faced with a major challenge related to ion suppression effect. Data regarding low-abundance components or low-polarity compounds cannot be normally obtained presumably because of the discrimination effect of easily ionized chemical components on desorption/ionization process. In this study, a new method was proposed to obtain images of chemical components in biological tissues or sections through in situ solid-phase extraction in sorbent mounted micro-funnel based spray ionization mass spectrometry. An imprint of a strawberry section was formed by gently pressing against a 2D array of micro-funnels. The sorbent mounted micro-funnels were then subjected to in situ single-pixel solid-phase extraction to alleviate the matrix-related ion suppression effect. The achievable spatial resolution is approximately 250 µm. The imaging of the spatial distribution of low-abundance or low-polarity chemicals in the strawberry imprint could be obtained by using a gradient elution strategy. Results demonstrated that the "not observed" remark does not necessarily indicate that a specific compound is non-existent when traditional imaging mass spectrometry techniques are used. The proposed method can be applied to conduct low-abundance chemical imaging through in situ single-pixel sample pretreatment.

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.

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.

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.