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Improving glycan isomeric separation via metal ion incorporation for drift tube ion mobility-mass spectrometry
Talanta 211 (2020) 120719
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Improving glycan isomeric separation via metal ion incorporation for drift tube ion mobility-mass spectrometry
T
Chengyi Xiea,b, Qidi Wua,b, Shulei Zhanga,b, Chenlu Wangb,c, Wenqing Gaob,c, Jiancheng Yua,b,∗∗, Keqi Tangb,c,∗ a
Faculty of Electrical Engineering and Computer Science, Ningbo University, Ningbo, 315211, PR China Institute of Mass Spectrometry, Ningbo University, Ningbo, 315211, PR China c School of Material Science and Chemical Engineering, Ningbo University, Ningbo, 315211, PR China b
ARTICLE INFO
ABSTRACT
Keywords: Ion mobility spectrometry Isomeric glycan separation Glycan-metal ion complex Collision cross section Trendline
Glycosylated proteins are an essential class of molecules playing critical roles in complex biological systems. Understanding their biological functions remains extremely difficult due to the extremely broad compositions and structure variations of glycans. Although the combination of ion mobility spectrometry and mass spectrometry (IMS-MS) has become a promising technique in glycan structure characterization and composition identification, the insufficient resolving power of most IMS-MS instruments has limited its utility in performing the comprehensive structure characterization of glycans. To mitigate the low IMS resolving power, metal ion incorporation has been employed to enhance the separation of isomeric glycans. Here, we present a systematic investigation of many different glycan-metal ion complexes in an attempt to optimize the IMS separation of different isomeric glycans. By selecting optimum glycan-metal ion complexes, partial IMS separation was realized for all the 21 isomeric glycan pairs used in the experimental study. Baseline IMS separation was achieved for 76% of these isomeric glycan pairs. The best IMS separation of isomeric glycans was achieved in some cases by incorporating multiple ions with a glycan, such as the complex [glycan + Ca + Cl]+. In addition, the wellknown IMS-MS measurement trendlines, often used to identify specific compound classes, were preserved for glycans even for all the 270 glycan-metal ion complexes observed in IMS-MS spectra.
1. Introduction Glycosylation plays critical roles in many biological processes, including the control and regulation of cell signaling pathways, intrinsic and extrinsic recognition, and disease pathogenesis [1–5]. Due to their extreme diversities of chemical compositions and structure variations, it is rather difficult to understand the basic biological functions of glycans [3,6,7]. The combination of changing compositions, different connectivity and diverse structural configurations has also made the identification and quantification of glycans in biological specimen extremely challenging. Nuclear magnetic resonance (NMR) has often been used to obtain glycan structure information [8]. The method typically requires a large amount of sample and the measurement is also time-consuming. In parallel, liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has also been developed to perform more sensitive and higher throughput glycomics analysis in which hundreds to
∗
thousands of glycans could be identified in a single analysis [9]. Different dissociation techniques (CID, ExD and UVPD, etc.) [10–16] have been used for the sequence identification and structure characterization of glycans. However, the tandem MS step cannot easily discriminate many isomers, which has limited its utility in accurate structural identification of glycans. The measurements by NMR and LC-MS/MS techniques can also be used together to increase the confidence of glycan identification and structure determination. Recently, ion mobility spectrometry (IMS) has emerged as a promising alternative technique [17–24] for effective glycan analysis. IMS is a post-ionization separation method in gas phase, where ions are driven by a constant electric field [25,26] or the combination of a changing electric field and a constant gas flow field [27] and separated/ fractionated based on their mobilities. The combination of IMS and mass spectrometry (MS) has been demonstrated for discerning glycan isomers efficiently [28,29]. A recent development of the Waters cyclic IMS system has increased the resolving power of as much as 750
Corresponding author. Institute of Mass Spectrometry, Ningbo University, Ningbo, 315211, PR China Corresponding author. Faculty of Electrical Engineering and Computer Science, Ningbo University, Ningbo, 315211, PR China E-mail addresses: [email protected] (J. Yu), [email protected] (K. Tang).
∗∗
https://doi.org/10.1016/j.talanta.2020.120719 Received 22 November 2019; Received in revised form 29 December 2019; Accepted 3 January 2020 Available online 07 January 2020 0039-9140/ © 2020 Elsevier B.V. All rights reserved.
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making the IMS significantly more effective in separating glycan isomers [30–32]. However, the resolving power for most commercial IMS instruments has been limited to a couple of hundreds, insufficient to separate glycan isomers in most cases. Alternatively, glycan isomer separation can be effectively improved by the incorporation of metal ions [33–35] and ligands [36,37]. When group I metal ions were used to form glycan-metal ion complexes, the arrival times (tA) of the complexes was shown to increase with the increase of metal ion radius in general [35]. It has also been demonstrated that metal ion incorporation can change the conformation of glycans [34], providing promising opportunities to further optimize the separation of glycan isomers via metal ion incorporation technique. Here, a systematic experimental investigation was performed on the incorporation of different types of metal ions with glycans and its effect on the structural changes of different isomeric glycans in an attempt to optimize the ion mobility separation of isomeric glycans in an IMSQTOF mass spectrometer. While most previous studies mainly focused on the incorporation of single metal ion with a glycan [7,33–35], our experimental study also included the multiple ion incorporation with glycans. The optimum separation of glycan isomers was achieved in some cases by incorporating multiple ions with a glycan, such as [glycan + Ca + Cl]+ ions. As a result, the separation of glycan isomers was further improved by selecting different types of glycan-metal ion complexes. In addition, we also explored the structure similarity of different glycan-metal ion complexes by demonstrating the well-known measurement trendlines based on the collision cross section (CCS) and the mass to charge ratio (m/z) of each glycan-metal ion complex.
Fig. 1. Structures of the glycans used in all experiments. The symbols of constituent monosaccharides and glycosidic bonds were provided at bottom.
2. Materials and method
each tri-, tetra- and pentasaccharide, 16.7 μM of each hexasaccharide. To explore the structure similarity of different glycan-metal ion complexes, the mixture of fifteen glycans (Fig. 1) was prepared with the final concentration at 3.3 μM of each glycan. The salt concentration in all samples was maintained at 50 μM to ensure the complete glycanmetal ion complexation.
2.1. Sample preparation Mannotetraose (MAN4), mannopentaose (MAN5), isomaltohexaose (ISO6) were purchased from Zzstandard Shanghai Zzbio Co., Ltd. (Shanghai, China). Isomaltotetraose (ISO4), isomaltopentaose (ISO5) were purchased from TCI Development Co., Ltd. (Shanghai, China). Raffinose (RAFF), isomaltotriose (ISO3), melezitose (MELE)·H2O, stachyose (STAC)·4H2O, maltopentaose (MAL5), cellopentaose (CEL5), maltohexaose (MAL6), RbCl were purchased from Shanghai Yuanye Biological Technology Co., Ltd. (Shanghai, China). Maltotriose (MAL3), Maltotetraose (MAL4), CsCl were purchased from Solarbio Science and Technology Co. Ltd. (Beijing, China). Cellohexaose (CEL6) was purchased from Toronto Research Chemicals Inc. (North York, ON, Canada). LiCl, NaCl, KCl, CaCl2, SrCl2, BaCl2 were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). MgCl2 was purchased from Aladdin Co. Ltd. (Shanghai, China). Acetonitrile (ACN) was purchased from Merck, Inc. (Darmstadt, Germany). Formic acid (FA) was purchased from Fisher Scientific Inc. (Pittsburgh, PA, USA). An ESI low concentration tune mix was obtained from Agilent Technologies (Santa Clara, CA, USA). The structures of the glycans used in the current experimental study, including four trisaccharide isomers, four tetrasaccharide isomers, four pentasaccharide isomers and three hexasaccharide isomers, was illustrated in Fig. 1 by using the component symbols proposed by Varki et al. [38] and Harvey et al. [39] All the sample solutions were prepared by using deionization water from a Milli-Q water purification system (Millipore Corp., Bedford, MA). Each glycan and salt were individually dissolved into an acetonitrile/water/formic acid solution (49.95:49.95:0.1, v/v/v). For individual glycan analysis, 30 μM or 100 μM of each glycan solution was first prepared and then mixed with 100 μM salt solution at a 1:1 vol ratio. For IMS separation studies, a set of sample solutions containing a mixture of two different glycans at 25 μM each and different salts at 50 μM concentrations were prepared. Four series of glycans mixtures including tri-, tetra-, penta- and hexasaccharides were prepared with the final concentration at 12.5 μM of
2.2. Ion mobility spectrometry and mass spectrometry All glycan analysis experiments were performed on an Agilent 6560 IMS-QTOF MS (Santa Clara, CA) [40], equipped with a dual Agilent Jet Stream electrospray ionization (AJS ESI) source. An Agilent 1290 Infinity liquid chromatography (LC) system was used for flow injection analysis (i.e. direct sample injection without the use of a LC column). Mobile phase A of 0.1% formic acid in 99.9% water (v/v) and Mobile phase B of 0.1% formic acid in 99.9% ACN (v/v) were used in all sample analyses. 5 μL prepared sample was first loaded into the sample loop of the LC system and subsequently injected into the ESI source using 50% B at a flow rate of 0.4 mL/min. A typical extracted ion chromatograms (XIC) of the [MAL3 + Ca + Cl]+ complex (m/z 579.1009, at 0.189 min after the sample injection) was shown in Supplemental Fig. S1. The dual AJS ESI source was operated with a nitrogen sheath gas heated between 300 °C and 350 °C with a flow rate of 12 L/min. The nitrogen drying gas applied at the source entrance was heated to 300 °C at a flow rate of 10 L/min. The Nebulizer gas was set to 40 psi. The source was operated in positive ESI mode with −4 kV Cap voltage and −2 kV nozzle voltage, respectively. High purity nitrogen (99.999%) was used as the buffer gas for IMS. The IMS drift tube electric field and gas pressure were maintained at 18.5 V/cm and 3.95 Torr, respectively. 2.3. Ion collision cross section (CCS) measurement The ion arrival time measured by IMS-MS was converted into the corresponding ion CCS value using Agilent's low concentration tuning 2
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mix as the calibration standards according to equation (1) [41] derived from Mason-Schamp equation [42],
tA =
z
1 mi 2 CCS mB + mi
+ t fix
(1)
where tA is the ion arrival time, z is the number of charges carried by the ion or the ion charge state, mi is the mass of the ion, mB is the molecular mass of the buffer gas, and t fix are the slope and the intercept determined experimentally. Specifically, the centroid arrival time (tA ) was first extracted by using the Agilent IMS-MS Browser (Santa Clara, CA). A calibration curve was then obtained by plotting measured tA against standardized CCS values for all the calibrant ions in the Agilent tune mix which have the lowest measurement uncertainty to date [41]. The slope ( ) and intercept (t fix ) of the calibration curve were determined by using the linear regression of the ion arrival times and the corresponding CCS values. Once the values of and t fix are determined, equation (1) can be used to calculate the CCS value of any analyte ion based on its arrival time measurement as long as the IMS operating conditions remained to be constant. Both the tuning mix and all the glycan samples were analyzed at a constant electric field at 18.5 V/cm and drift gas pressure and temperature at 3.95 Torr and 25 °C, respectively. 2.4. IMS separation resolution The IMS resolution RP equation [43],
RP
P
=
P
Fig. 2. Arrival time distribution of ISO4 and STAC incorporated with different metal ions, including (A) group I metal ions and (B) group II metal ions. The arrival times labeled in the figure were extracted from the centroid of peaks.
was calculated based on the following
2.35 tA 2 (WFWHM 1 + WFWHM 2)
(2)
[ISO4 + IA]+ ions displayed shorter arrival times than [STAC + IA]+ ions, suggesting a more compact structure of [ISO4 + IA]+ ions as compared to the corresponding [STAC + IA]+ ions. In contrast, the incorporation of group II ions with ISO4 and STAC (Fig. 2B) showed a completely opposite structure change. The IMS arrival times for [ISO4 + IIA + Cl]+ ions were all measured to be longer than the ones for the corresponding [STAC + IIA + Cl]+ ions, suggesting a more extended structure of [ISO4 + IIA + Cl]+ ions as compared to the corresponding [STAC + IIA + Cl]+ ions. In addition, the incorporation of a metal ion with the glycan can also change the conformational diversity of a specific glycan depending most likely on the number of places that a selected metal ion can be attached to the glycan [44]. This phenomenon can be well reflected by the IMS peak width changes or the appearance of multiple peaks for different glycan-metal ion complexes. For example, while the WFWHM values for all four [ISO4 + IA]+ complexes in Fig. 2A remained essentially same, the WFWHM values for [STAC + Na]+, [STAC + K]+, [STAC + Rb]+ and [STAC + Cs]+ complexes were measured at 0.59 ms, 1.11 ms, 0.67 ms and 0.57 ms, respectively. This suggests that while the incorporation of group I metal ions did not change the conformational diversity of [ISO4 + IA]+ ions, the conformational diversity of the [STAC + IA]+ complex was affected significantly by the incorporations of different group I metal ions. Specifically, the WFWHM for the [STAC + K]+ complex is almost two time broader than the WFWHM for the [STAC + Na]+ complex implying that substituting Na+ with K+ created at least two new conformations for the [STAC + K]+ complex. The leading edge shoulder for the [STAC + K]+ complex peak shown in Fig. 2A also confirmed the appearance of an additional unresolved conformation of the [STAC + K]+ complex. Even if the conformational difference was relatively large inside the peak of the [STAC + K]+ complex, these conformations were not able to be resolved in this experiment, leading to the broader peak width of this complex. These confirmations persisted for the [STAC + Rb]+ complex. Only their IMS arrival time difference decreased as indicated by a narrower peak width and a less significant shoulder on the trailing edge
where tA is the arrival time difference of any pair of selected ions, WFWHM1 and WFWHM2 are their corresponding peak widths at the half maximum. The Agilent IMS browser was used to obtain tA . The WFWHM for each ion was obtained by Gaussian fitting of the measured arrival time peak by using the Peak Analyzer of OriginPro 2017. RP P was used in this study to assess the IMS separation quality for glycan isomers. 3. Results and discussions 3.1. Conformational change of glycans by metal ion incorporation In order to optimize the separation of isomeric glycans by IMSQTOF MS, our study was initially concentrated on examining the structural changes of glycan isomers by the incorporation of different metal ions, including group I (Li, Na, K, Rb, Cs) and group II (Mg, Ca, Sr, Ba) metal ions. In general, most complexes containing an alkali cation displayed a form as [M + IA]+ except for sodium ion complexes, which presented both [M + Na]+ and [M + 2Na + Cl]+, where M is a given glycan and IA is a group I metal ion. For the incorporation of group II metal ions with glycans, more complex forms of ions, such as [M + IIA]2+; [M + IIA + Cl]+ and [2M + IIA]2+, were observed experimentally. A total of 18 different complexes for each glycan used in the current study were observed including ten singly charged ions ([M + Li]+, [M + Na]+, [M + K]+, [M + Rb]+, [M + Cs]+, [M + 2Na + Cl]+, [M + Mg + Cl]+, [M + Ca + Cl]+, [M + Sr + Cl]+ and [M + Ba + Cl]+) and eight doubly charged ions ([M + Mg]2+, [M + Ca]2+, [M + Sr]2+, [M + Ba]2+, [2M + Mg]2+, [2M + Ca]2+, [2M + Sr]2+ and [2M + Ba]2+). The protonated glycan ions were observed only for some glycans at very low abundance. The structure of each glycan was greatly changed by the incorporation of different metal ions. Fig. 2 showed an example of IMS arrival time distributions for a pair of tetrasaccharides, ISO4 and STAC, significantly affected by the incorporation of group I metal ions (Fig. 2A) and group II metal ions (Fig. 2B). For group I ion complexes,
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Fig. 3. Separation of ISO6 and CEL6 dimers incorporated with the Ba2+ metal ion by using the individual sample (colored trace) and sample mixture (inserted black trace). The arrival times labeled in the figure were extracted from the centroid of the peaks and the RP P value was calculated by using formula (2).
of the IMS peak. Substituting Rb+ with Cs+ reduced the WFWHM for the [STAC + Cs]+ complex to about the same value as the [STAC + Na]+ complex implying one confirmation for the [STAC + Cs]+ complex. The discussion above for the tetrasaccharides pair were also observed experimentally for other glycans used in the current study. Both the conformation and the conformational diversity of glycans can be significantly altered by the incorporation of different metal ions. The detailed experimental data similar to Fig. 2 for other glycan-metal ion complexes were shown in Supplemental Figs. S2–S5. 3.2. Effect of metal cation on isomeric glycan separation The glycan structure changes provided by the incorporation of different metal ions can be potentially used to enhance the IMS separation of isomeric glycans. As shown in Supplemental Figs. S2–S5, difference of IMS arrival time for the selected isomeric glycan pair could be altered by their incorporation of different metal ions. In order to find the optimum glycan-metal ion complexes to achieve the best separation for the isomeric glycan pairs shown in Supplemental Figs. S2–S5, the resolution was further calculated by using formula (2) to assess the degree of separation for each pair of experimentally observed glycan-metal ion complexes. In the case of confirmation diversity change caused by the metal ion incorporation, as discussed above, the RP P was calculated for each corresponding structure with the separation quality determined by the smallest RP P value. One noticeable thing observed in the experiment was related to the separation of the hexasaccharide pair of ISO6 and CEL6, as shown in Fig. 3. In this case, the largest RP P for all the experimentally observed hexasaccharide-metal ion complex pairs was obtained for the dimeric glycan-Ba2+ complexes when IMS measurements were performed by using individual standards (the colored traces in Fig. 3). The separation resolution of the mixture of the two hexasaccharides (the black traces in Fig. 3) was degraded by the appearance of an additional peak at arrival time 27.58 ms between the [2ISO6 + Ba]2+ and [2CEL6 + Ba]2+ complexes at the arrival times of 26.60 ms and 28.67 ms, respectively. MS measurement further confirmed that the new peak was due to the formation of a new [ISO6 + CEL6 + Ba]2+ complex when the sample mixture was used for IMS-MS measurement. Consequently, the [2ISO6 + Ba]2+ and [2CEL6 + Ba]2+ complexes were not a good choice to achieve the optimum separation for ISO6 and CEL6 pair. Taking into account of both the structure diversity changes of the glycan-metal ion complex and the possibility of new complex formation, as discussed above, the optimum separation conditions for all four
Fig. 4. Optimal separation for selected pairs of isomeric trisaccharides, tetrasaccharides, pentasaccharides and hexasaccharides achieved by selecting the best metal ion-glycan complexes for the individual sample (colored trace) and sample mixture (black traces) IMS measurements, respectively. The arrival times labeled in the figure were extracted from centroid of peaks and the RP P values were calculated by using formula (2).
pairs of glycan-metal ion complexes under current study, corresponding to the highest resolution values, were obtained from the complete experimental measurements shown in Supplemental Figs. S2–S5. Fig. 4 showed the optimum IMS separation for these glycan-metal ion complex pairs in both pure standard and mixture sample conditions. For the isomeric trisaccharides MAL3 and ISO3, comprised of glucose units and differed in connectivity only, slightly less than baseline separation was achieved by the incorporation of two sodium ions and one chlorine ion with a calculated RP P of 0.97. The best separation for both isomeric tetrasccharide ISO4 and STAC and isomeric pentasaccharide ISO5 and CEL5 were achieved by incorporating [Ca2+ + Cl−]+ with the glycans. Baseline separation for both the [ISO4 + Ca + Cl]+ and [STAC + Ca + Cl]+complex pair and the [ISO5 + Ca + Cl]+ and [CEL5 + Ca + Cl]+ complex pair was achieved with calculated RP P of 1.31 and 2.39, respectively. For isomeric hexasaccharides ISO6 and CEL6, the best separation was achieved when complexes [CEL6 + Ba + Cl]+ and [ISO6 + Ba + Cl]+ were used with a calculated RP P value of 1.46. The optimum glycan-metal ion complexes for IMS separation obtained from Fig. 4 were further used to evaluate the effectiveness of IMS separation for all the corresponding trisaccharides, tetrasaccharides,
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Fig. 5. IMS separation of all isomeric trisaccharides, tetrasaccharides, pentasaccharides and hexasaccharides by using the optimum glycan-metal ion complexes for A) individual samples and B) sample mixtures. For comparison purpose, C) and D) showed the corresponding glycan separation for glycan-sodium ion complexes by using individual samples and sample mixtures, respectively.
complex was well separated from the complexes [ISO3 + 2Na + Cl]+ and [RAFF + 2Na + Cl]+ (Fig. 5A). This was confirmed further from the sample mixture data shown in Fig. 5B and D by three IMS peaks for complexes [M + 2Na + Cl]+ as compared to two IMS peaks for complexes [M + Na]+. In the case of tetrasaccharides, the features of sodiated complexes [STAC + Na]+, [MAN4 + Na]+ and [ISO4 + Na]+ were almost overlapped and centered at tA 29.48 ms, 29.49 ms and 29.51 ms, respectively. The resolution values between any two of these complexes were measured at 0.01–0.03. The complex [MAL4 + Na]+ was only slightly separated from the remaining three tetrasaccharide complexes. In contrast, the IMS separation of tetrasaccharide complexes [M + Ca + Cl]+ were significantly improved, as
pentasaccharides and hexasaccharides by using both pure standards and sample mixtures. Fig. 5 showed the IMS separation of four trisaccharides, tetrasaccharides, pentasaccharides and three hexasaccharides by using both pure standards (Fig. 5A and C) and sample mixtures (Fig. 5B and D). In Fig. 5A and B, the best glycan-metal ion complexes were used for IMS separation. For comparison purpose, Fig. 5C and D showed the IMS separation of all the sodiated glycans. It is clearly indicated that the separation of isomeric glycans were greatly improved by using the optimum glycan-metal ion complexes. Specifically, for the IMS separation of isomeric trisaccharides, while sodiated complexes [MAL3 + Na]+, [ISO3 + Na]+ and [RAFF + Na]+ were hardly separated from each other (Fig. 5C), the [MAL3 + 2Na + Cl]+
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glycans would be even higher if a better resolving power IMS is used. 3.3. IMS trendlines for glycan-metal ion complexes It is well known that the IMS-MS measurements of a specific class of compound such as peptides, lipids, glycans and polymers display their distinctive charge state based trendlines in the 2-dimensional display of IMS arrival time and MS m/z, a very useful feature of IMS-MS measurements for the identification of different classes of chemical compounds [40]. It is interesting to see if the IMS-MS measurement trendline for glycan is still preserved after the incorporation of different metal ions. Fig. 7A displayed IMS-MS measurements of different glycans and glycan clusters incorporated with sodium and calcium metal ions for singly and doubly charged complex ions. Two well separated trendlines were clearly observed corresponding to singly charged glycan-sodium ion complexes [M + Na]+ and doubly charged glycancalcium ion complexes [M + Ca]2+. The results in Fig. 7A was also consistent with a previous report indicating that doubly charged species generally presented higher mobility than singly charged species with the same m/z [45]. In an attempt to further generalize the trendline discussion, a total of 280 complexes observed in this study, which include 270 glycanmetal ion complexes and 10 protonated glycan ions, were included to see if the IMS-MS measurement trendlines for glycan would still hold. To make the experimental data IMS instrument independent, the IMS arrival times of these complexes were converted into their corresponding collision cross section (CCS) values by using equation (1) as listed in Supplemental Tables S6–S9. To ensure the CCS calculation accuracy, a subset of the CCS values was compared with the literature values [46]. The difference of our calculated CCS values (Supplemental Table S5) were all less than 0.75% to the literature CCS values. The projection of complexes [M + IIA + Cl]+ in Fig. 7B occupied relatively more extended area than other single charged species. Similar trends were also held for complexes [2M + IIA]2+. These two examples were consistent with the above results that the combination of multiple ions may lead to larger structure change. In addition, Fig. 7B showed a CCS vs m/z plot for all the experimentally observed glycan-metal ion complexes which clearly indicated that the trendlines for singly and doubly charged glycan-metal ion complexes were still preserved and appeared to be glycan dependent only. The incorporation of any metal ions with glycans has little effect on the IMS-MS measurement trendline. From the linear fits of m/z and CCS for 1 + and 2 + charge state trendlines, it was estimated that the singly charged complexes [M + H]+, [M + IA]+, [M + IIA + Cl]+ and [M + 2Na + Cl]+ distributed around the singly charged trendline with ± 16% deviation and the doubly charged complexes [M + IIA]2+ and [2M + IIA]2+ distributed around the doubly charged trendline with ± 17% deviation. This firmly suggests that although the gas-phase structure of glycans were significantly influenced by metal ion incorporation, the conformational space of these complexes still mainly depended on the structure and charge state of glycans.
Fig. 6. Distribution of IMS separation resolution for all glycan-metal ion complex pairs in three different categories.
shown in Fig. 5A and B. Specifically, the RP P was measured at 0.65 for the separation of complexes [STAC + Ca + Cl]+ and [MAN4 + Ca + Cl]+, 0.64 for the complexes [MAN4 + Ca + Cl]+ and [ISO4 + Ca + Cl]+, which is more than six fold improvement in IMS separation. Similar conclusion to trisaccharide IMS separation can also be reached for pentasaccharides by using the optimum [M + Ca + Cl]+ complexes as compared to their corresponding sodiated complexes [M + Na]+. For hexasaccharide IMS separation, the features of all hexasaccharides were almost baseline separated for the optimum complexes [M + Ba + Cl]+ as compared to almost no separation for sodiated hexasaccharide ions. To draw a broader conclusion on the effect of metal ion incorporation on the glycan IMS separation from the current systematic study, all the glycan-metal ion complex pairs used in the experiments were grouped in Fig. 6, according to their measured IMS resolutions listed in Supplemental Tables S1–S4, into three different categories. Each resolution category in Fig. 6 represents a distinctive IMS separation characteristic. RP P in the range of 0–0.5 represents overlapping IMS peaks for the corresponding glycan-metal ion complex pair. RP P in the range of 0.5–1 represents partially separated peaks for the glycanmetal ion complex pair. RP P > 1 represents baseline separated peaks for the glycan-metal ion complex pair [43]. With a total 21 glycan pairs used in this study, a total of 378 glycan-metal ion complex pairs was observed experimentally by incorporating each glycan pair with different group I and group II metal ions (i.e. 18 different glycan-metal ion complex pairs were observed for each glycan pair). To simplify the discussion, each glycan pair in the form of any glycan-metal ion complex pair can only appear in each resolution category once at most to eliminate counting redundancy. Based on this definition, 100% for a specific resolution group in Fig. 6 implies that one can always find at least one glycan-metal ion complex pair having the IMS separation resolution indicated in the group for all the glycan pairs in the current study. The results in Fig. 6 confirmed the striking effect of metal ion incorporation on the IMS separation of isomeric glycans. For any unresolved isomeric glycan pair, one can always find a good glycan-metal ion complex pair to partially separate them in IMS. In addition, there are 76% of the chance that the IMS separation of the glycan pair can be baseline resolved if an optimum glycan-metal ion complex is used even with the modest IMS resolving power of 50–80 used in the current study. It is expected that the chance to baseline separate isomeric
4. Conclusions The incorporation of different types of metal ions with glycans can change both the structure and the conformational diversity of glycans which can be effectively explored to enhance IMS separation of isomeric glycans. The systematic IMS-MS measurements demonstrated that partial IMS separation of a given isomeric pair can always be realized by carefully selecting a glycan-metal ion complex. Under the optimum glycan-metal ion complex conditions, even baseline IMS separation can be realized for many unresolvable isomeric glycans in their common sodiated form even with the modest IMS resolving power of 50–80. In addition, even after the gas-phase structure of the glycans
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Fig. 7. A) An experimental 2D plot of arrival time versus m/z for the incorporation of calcium ion or sodium ion with each glycan presented in Fig. 1. The features of each ion in plot were numerically labeled, and the corresponding species were (1) [TRI + Ca]2+, (2) [TETRA + Ca]2+, (3) [PENTA + Ca]2+, (4) [HEX + Ca]2+, (5) [TRI + TETRA + Ca]2+, (6) [2TETRA + Ca]2+ (7) [TETRA + PENTA + Ca]2+ (8) [2PENTA + Ca]2+, (9) [TRI + Na]+, (10) [TETRA + Na]+, (11) [PENTA + Na]+ and (12) [HEX + Na]+, where TRI, TETRA, PENTA and HEX represented trisaccharide, tetrasccharide, pentasaccharide and hexasaccharide, respectively. B) A 2D plot of CCS versus m/z for all complexes obtained in this study. The insert lines contained the linear fits of 1 + and 2 + charged state glycans (colored dotted traces) and these complexes' data included area (colored solid traces).
were significantly altered by the metal ion incorporation, the wellknown IMS-MS measurement trendlines were still preserved for all the glycan-metal ion complexes which were glycan and charge state dependent only.
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Acknowledgements This work was supported by National Natural Science Foundation of China (Grant Nos. 61971248), National Key Research and Development Program of China (Grant No. 2017YFC1001700), Key Research and Development Program of Zhejiang Province (Grant No. 2020C03064), Science and Technology Major Project of Ningbo (Grant No. 2018B10075), Scientific Research Foundation of Graduate School of Ningbo University and sponsored by K.C. Wong Magna Fund in Ningbo University. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.talanta.2020.120719. References [1] A. Varki, Biological roles of glycans, Glycobiology 27 (2017) 3–49. [2] R.A. Dwek, Glycobiology: toward understanding the function of sugars, Chem. Rev.
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Improving glycan isomeric separation via metal ion incorporation for drift tube ion mobility-mass spectrometry
T
Chengyi Xiea,b, Qidi Wua,b, Shulei Zhanga,b, Chenlu Wangb,c, Wenqing Gaob,c, Jiancheng Yua,b,∗∗, Keqi Tangb,c,∗ a
Faculty of Electrical Engineering and Computer Science, Ningbo University, Ningbo, 315211, PR China Institute of Mass Spectrometry, Ningbo University, Ningbo, 315211, PR China c School of Material Science and Chemical Engineering, Ningbo University, Ningbo, 315211, PR China b
ARTICLE INFO
ABSTRACT
Keywords: Ion mobility spectrometry Isomeric glycan separation Glycan-metal ion complex Collision cross section Trendline
Glycosylated proteins are an essential class of molecules playing critical roles in complex biological systems. Understanding their biological functions remains extremely difficult due to the extremely broad compositions and structure variations of glycans. Although the combination of ion mobility spectrometry and mass spectrometry (IMS-MS) has become a promising technique in glycan structure characterization and composition identification, the insufficient resolving power of most IMS-MS instruments has limited its utility in performing the comprehensive structure characterization of glycans. To mitigate the low IMS resolving power, metal ion incorporation has been employed to enhance the separation of isomeric glycans. Here, we present a systematic investigation of many different glycan-metal ion complexes in an attempt to optimize the IMS separation of different isomeric glycans. By selecting optimum glycan-metal ion complexes, partial IMS separation was realized for all the 21 isomeric glycan pairs used in the experimental study. Baseline IMS separation was achieved for 76% of these isomeric glycan pairs. The best IMS separation of isomeric glycans was achieved in some cases by incorporating multiple ions with a glycan, such as the complex [glycan + Ca + Cl]+. In addition, the wellknown IMS-MS measurement trendlines, often used to identify specific compound classes, were preserved for glycans even for all the 270 glycan-metal ion complexes observed in IMS-MS spectra.
1. Introduction Glycosylation plays critical roles in many biological processes, including the control and regulation of cell signaling pathways, intrinsic and extrinsic recognition, and disease pathogenesis [1–5]. Due to their extreme diversities of chemical compositions and structure variations, it is rather difficult to understand the basic biological functions of glycans [3,6,7]. The combination of changing compositions, different connectivity and diverse structural configurations has also made the identification and quantification of glycans in biological specimen extremely challenging. Nuclear magnetic resonance (NMR) has often been used to obtain glycan structure information [8]. The method typically requires a large amount of sample and the measurement is also time-consuming. In parallel, liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has also been developed to perform more sensitive and higher throughput glycomics analysis in which hundreds to
∗
thousands of glycans could be identified in a single analysis [9]. Different dissociation techniques (CID, ExD and UVPD, etc.) [10–16] have been used for the sequence identification and structure characterization of glycans. However, the tandem MS step cannot easily discriminate many isomers, which has limited its utility in accurate structural identification of glycans. The measurements by NMR and LC-MS/MS techniques can also be used together to increase the confidence of glycan identification and structure determination. Recently, ion mobility spectrometry (IMS) has emerged as a promising alternative technique [17–24] for effective glycan analysis. IMS is a post-ionization separation method in gas phase, where ions are driven by a constant electric field [25,26] or the combination of a changing electric field and a constant gas flow field [27] and separated/ fractionated based on their mobilities. The combination of IMS and mass spectrometry (MS) has been demonstrated for discerning glycan isomers efficiently [28,29]. A recent development of the Waters cyclic IMS system has increased the resolving power of as much as 750
Corresponding author. Institute of Mass Spectrometry, Ningbo University, Ningbo, 315211, PR China Corresponding author. Faculty of Electrical Engineering and Computer Science, Ningbo University, Ningbo, 315211, PR China E-mail addresses: [email protected] (J. Yu), [email protected] (K. Tang).
∗∗
https://doi.org/10.1016/j.talanta.2020.120719 Received 22 November 2019; Received in revised form 29 December 2019; Accepted 3 January 2020 Available online 07 January 2020 0039-9140/ © 2020 Elsevier B.V. All rights reserved.
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making the IMS significantly more effective in separating glycan isomers [30–32]. However, the resolving power for most commercial IMS instruments has been limited to a couple of hundreds, insufficient to separate glycan isomers in most cases. Alternatively, glycan isomer separation can be effectively improved by the incorporation of metal ions [33–35] and ligands [36,37]. When group I metal ions were used to form glycan-metal ion complexes, the arrival times (tA) of the complexes was shown to increase with the increase of metal ion radius in general [35]. It has also been demonstrated that metal ion incorporation can change the conformation of glycans [34], providing promising opportunities to further optimize the separation of glycan isomers via metal ion incorporation technique. Here, a systematic experimental investigation was performed on the incorporation of different types of metal ions with glycans and its effect on the structural changes of different isomeric glycans in an attempt to optimize the ion mobility separation of isomeric glycans in an IMSQTOF mass spectrometer. While most previous studies mainly focused on the incorporation of single metal ion with a glycan [7,33–35], our experimental study also included the multiple ion incorporation with glycans. The optimum separation of glycan isomers was achieved in some cases by incorporating multiple ions with a glycan, such as [glycan + Ca + Cl]+ ions. As a result, the separation of glycan isomers was further improved by selecting different types of glycan-metal ion complexes. In addition, we also explored the structure similarity of different glycan-metal ion complexes by demonstrating the well-known measurement trendlines based on the collision cross section (CCS) and the mass to charge ratio (m/z) of each glycan-metal ion complex.
Fig. 1. Structures of the glycans used in all experiments. The symbols of constituent monosaccharides and glycosidic bonds were provided at bottom.
2. Materials and method
each tri-, tetra- and pentasaccharide, 16.7 μM of each hexasaccharide. To explore the structure similarity of different glycan-metal ion complexes, the mixture of fifteen glycans (Fig. 1) was prepared with the final concentration at 3.3 μM of each glycan. The salt concentration in all samples was maintained at 50 μM to ensure the complete glycanmetal ion complexation.
2.1. Sample preparation Mannotetraose (MAN4), mannopentaose (MAN5), isomaltohexaose (ISO6) were purchased from Zzstandard Shanghai Zzbio Co., Ltd. (Shanghai, China). Isomaltotetraose (ISO4), isomaltopentaose (ISO5) were purchased from TCI Development Co., Ltd. (Shanghai, China). Raffinose (RAFF), isomaltotriose (ISO3), melezitose (MELE)·H2O, stachyose (STAC)·4H2O, maltopentaose (MAL5), cellopentaose (CEL5), maltohexaose (MAL6), RbCl were purchased from Shanghai Yuanye Biological Technology Co., Ltd. (Shanghai, China). Maltotriose (MAL3), Maltotetraose (MAL4), CsCl were purchased from Solarbio Science and Technology Co. Ltd. (Beijing, China). Cellohexaose (CEL6) was purchased from Toronto Research Chemicals Inc. (North York, ON, Canada). LiCl, NaCl, KCl, CaCl2, SrCl2, BaCl2 were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). MgCl2 was purchased from Aladdin Co. Ltd. (Shanghai, China). Acetonitrile (ACN) was purchased from Merck, Inc. (Darmstadt, Germany). Formic acid (FA) was purchased from Fisher Scientific Inc. (Pittsburgh, PA, USA). An ESI low concentration tune mix was obtained from Agilent Technologies (Santa Clara, CA, USA). The structures of the glycans used in the current experimental study, including four trisaccharide isomers, four tetrasaccharide isomers, four pentasaccharide isomers and three hexasaccharide isomers, was illustrated in Fig. 1 by using the component symbols proposed by Varki et al. [38] and Harvey et al. [39] All the sample solutions were prepared by using deionization water from a Milli-Q water purification system (Millipore Corp., Bedford, MA). Each glycan and salt were individually dissolved into an acetonitrile/water/formic acid solution (49.95:49.95:0.1, v/v/v). For individual glycan analysis, 30 μM or 100 μM of each glycan solution was first prepared and then mixed with 100 μM salt solution at a 1:1 vol ratio. For IMS separation studies, a set of sample solutions containing a mixture of two different glycans at 25 μM each and different salts at 50 μM concentrations were prepared. Four series of glycans mixtures including tri-, tetra-, penta- and hexasaccharides were prepared with the final concentration at 12.5 μM of
2.2. Ion mobility spectrometry and mass spectrometry All glycan analysis experiments were performed on an Agilent 6560 IMS-QTOF MS (Santa Clara, CA) [40], equipped with a dual Agilent Jet Stream electrospray ionization (AJS ESI) source. An Agilent 1290 Infinity liquid chromatography (LC) system was used for flow injection analysis (i.e. direct sample injection without the use of a LC column). Mobile phase A of 0.1% formic acid in 99.9% water (v/v) and Mobile phase B of 0.1% formic acid in 99.9% ACN (v/v) were used in all sample analyses. 5 μL prepared sample was first loaded into the sample loop of the LC system and subsequently injected into the ESI source using 50% B at a flow rate of 0.4 mL/min. A typical extracted ion chromatograms (XIC) of the [MAL3 + Ca + Cl]+ complex (m/z 579.1009, at 0.189 min after the sample injection) was shown in Supplemental Fig. S1. The dual AJS ESI source was operated with a nitrogen sheath gas heated between 300 °C and 350 °C with a flow rate of 12 L/min. The nitrogen drying gas applied at the source entrance was heated to 300 °C at a flow rate of 10 L/min. The Nebulizer gas was set to 40 psi. The source was operated in positive ESI mode with −4 kV Cap voltage and −2 kV nozzle voltage, respectively. High purity nitrogen (99.999%) was used as the buffer gas for IMS. The IMS drift tube electric field and gas pressure were maintained at 18.5 V/cm and 3.95 Torr, respectively. 2.3. Ion collision cross section (CCS) measurement The ion arrival time measured by IMS-MS was converted into the corresponding ion CCS value using Agilent's low concentration tuning 2
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mix as the calibration standards according to equation (1) [41] derived from Mason-Schamp equation [42],
tA =
z
1 mi 2 CCS mB + mi
+ t fix
(1)
where tA is the ion arrival time, z is the number of charges carried by the ion or the ion charge state, mi is the mass of the ion, mB is the molecular mass of the buffer gas, and t fix are the slope and the intercept determined experimentally. Specifically, the centroid arrival time (tA ) was first extracted by using the Agilent IMS-MS Browser (Santa Clara, CA). A calibration curve was then obtained by plotting measured tA against standardized CCS values for all the calibrant ions in the Agilent tune mix which have the lowest measurement uncertainty to date [41]. The slope ( ) and intercept (t fix ) of the calibration curve were determined by using the linear regression of the ion arrival times and the corresponding CCS values. Once the values of and t fix are determined, equation (1) can be used to calculate the CCS value of any analyte ion based on its arrival time measurement as long as the IMS operating conditions remained to be constant. Both the tuning mix and all the glycan samples were analyzed at a constant electric field at 18.5 V/cm and drift gas pressure and temperature at 3.95 Torr and 25 °C, respectively. 2.4. IMS separation resolution The IMS resolution RP equation [43],
RP
P
=
P
Fig. 2. Arrival time distribution of ISO4 and STAC incorporated with different metal ions, including (A) group I metal ions and (B) group II metal ions. The arrival times labeled in the figure were extracted from the centroid of peaks.
was calculated based on the following
2.35 tA 2 (WFWHM 1 + WFWHM 2)
(2)
[ISO4 + IA]+ ions displayed shorter arrival times than [STAC + IA]+ ions, suggesting a more compact structure of [ISO4 + IA]+ ions as compared to the corresponding [STAC + IA]+ ions. In contrast, the incorporation of group II ions with ISO4 and STAC (Fig. 2B) showed a completely opposite structure change. The IMS arrival times for [ISO4 + IIA + Cl]+ ions were all measured to be longer than the ones for the corresponding [STAC + IIA + Cl]+ ions, suggesting a more extended structure of [ISO4 + IIA + Cl]+ ions as compared to the corresponding [STAC + IIA + Cl]+ ions. In addition, the incorporation of a metal ion with the glycan can also change the conformational diversity of a specific glycan depending most likely on the number of places that a selected metal ion can be attached to the glycan [44]. This phenomenon can be well reflected by the IMS peak width changes or the appearance of multiple peaks for different glycan-metal ion complexes. For example, while the WFWHM values for all four [ISO4 + IA]+ complexes in Fig. 2A remained essentially same, the WFWHM values for [STAC + Na]+, [STAC + K]+, [STAC + Rb]+ and [STAC + Cs]+ complexes were measured at 0.59 ms, 1.11 ms, 0.67 ms and 0.57 ms, respectively. This suggests that while the incorporation of group I metal ions did not change the conformational diversity of [ISO4 + IA]+ ions, the conformational diversity of the [STAC + IA]+ complex was affected significantly by the incorporations of different group I metal ions. Specifically, the WFWHM for the [STAC + K]+ complex is almost two time broader than the WFWHM for the [STAC + Na]+ complex implying that substituting Na+ with K+ created at least two new conformations for the [STAC + K]+ complex. The leading edge shoulder for the [STAC + K]+ complex peak shown in Fig. 2A also confirmed the appearance of an additional unresolved conformation of the [STAC + K]+ complex. Even if the conformational difference was relatively large inside the peak of the [STAC + K]+ complex, these conformations were not able to be resolved in this experiment, leading to the broader peak width of this complex. These confirmations persisted for the [STAC + Rb]+ complex. Only their IMS arrival time difference decreased as indicated by a narrower peak width and a less significant shoulder on the trailing edge
where tA is the arrival time difference of any pair of selected ions, WFWHM1 and WFWHM2 are their corresponding peak widths at the half maximum. The Agilent IMS browser was used to obtain tA . The WFWHM for each ion was obtained by Gaussian fitting of the measured arrival time peak by using the Peak Analyzer of OriginPro 2017. RP P was used in this study to assess the IMS separation quality for glycan isomers. 3. Results and discussions 3.1. Conformational change of glycans by metal ion incorporation In order to optimize the separation of isomeric glycans by IMSQTOF MS, our study was initially concentrated on examining the structural changes of glycan isomers by the incorporation of different metal ions, including group I (Li, Na, K, Rb, Cs) and group II (Mg, Ca, Sr, Ba) metal ions. In general, most complexes containing an alkali cation displayed a form as [M + IA]+ except for sodium ion complexes, which presented both [M + Na]+ and [M + 2Na + Cl]+, where M is a given glycan and IA is a group I metal ion. For the incorporation of group II metal ions with glycans, more complex forms of ions, such as [M + IIA]2+; [M + IIA + Cl]+ and [2M + IIA]2+, were observed experimentally. A total of 18 different complexes for each glycan used in the current study were observed including ten singly charged ions ([M + Li]+, [M + Na]+, [M + K]+, [M + Rb]+, [M + Cs]+, [M + 2Na + Cl]+, [M + Mg + Cl]+, [M + Ca + Cl]+, [M + Sr + Cl]+ and [M + Ba + Cl]+) and eight doubly charged ions ([M + Mg]2+, [M + Ca]2+, [M + Sr]2+, [M + Ba]2+, [2M + Mg]2+, [2M + Ca]2+, [2M + Sr]2+ and [2M + Ba]2+). The protonated glycan ions were observed only for some glycans at very low abundance. The structure of each glycan was greatly changed by the incorporation of different metal ions. Fig. 2 showed an example of IMS arrival time distributions for a pair of tetrasaccharides, ISO4 and STAC, significantly affected by the incorporation of group I metal ions (Fig. 2A) and group II metal ions (Fig. 2B). For group I ion complexes,
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Fig. 3. Separation of ISO6 and CEL6 dimers incorporated with the Ba2+ metal ion by using the individual sample (colored trace) and sample mixture (inserted black trace). The arrival times labeled in the figure were extracted from the centroid of the peaks and the RP P value was calculated by using formula (2).
of the IMS peak. Substituting Rb+ with Cs+ reduced the WFWHM for the [STAC + Cs]+ complex to about the same value as the [STAC + Na]+ complex implying one confirmation for the [STAC + Cs]+ complex. The discussion above for the tetrasaccharides pair were also observed experimentally for other glycans used in the current study. Both the conformation and the conformational diversity of glycans can be significantly altered by the incorporation of different metal ions. The detailed experimental data similar to Fig. 2 for other glycan-metal ion complexes were shown in Supplemental Figs. S2–S5. 3.2. Effect of metal cation on isomeric glycan separation The glycan structure changes provided by the incorporation of different metal ions can be potentially used to enhance the IMS separation of isomeric glycans. As shown in Supplemental Figs. S2–S5, difference of IMS arrival time for the selected isomeric glycan pair could be altered by their incorporation of different metal ions. In order to find the optimum glycan-metal ion complexes to achieve the best separation for the isomeric glycan pairs shown in Supplemental Figs. S2–S5, the resolution was further calculated by using formula (2) to assess the degree of separation for each pair of experimentally observed glycan-metal ion complexes. In the case of confirmation diversity change caused by the metal ion incorporation, as discussed above, the RP P was calculated for each corresponding structure with the separation quality determined by the smallest RP P value. One noticeable thing observed in the experiment was related to the separation of the hexasaccharide pair of ISO6 and CEL6, as shown in Fig. 3. In this case, the largest RP P for all the experimentally observed hexasaccharide-metal ion complex pairs was obtained for the dimeric glycan-Ba2+ complexes when IMS measurements were performed by using individual standards (the colored traces in Fig. 3). The separation resolution of the mixture of the two hexasaccharides (the black traces in Fig. 3) was degraded by the appearance of an additional peak at arrival time 27.58 ms between the [2ISO6 + Ba]2+ and [2CEL6 + Ba]2+ complexes at the arrival times of 26.60 ms and 28.67 ms, respectively. MS measurement further confirmed that the new peak was due to the formation of a new [ISO6 + CEL6 + Ba]2+ complex when the sample mixture was used for IMS-MS measurement. Consequently, the [2ISO6 + Ba]2+ and [2CEL6 + Ba]2+ complexes were not a good choice to achieve the optimum separation for ISO6 and CEL6 pair. Taking into account of both the structure diversity changes of the glycan-metal ion complex and the possibility of new complex formation, as discussed above, the optimum separation conditions for all four
Fig. 4. Optimal separation for selected pairs of isomeric trisaccharides, tetrasaccharides, pentasaccharides and hexasaccharides achieved by selecting the best metal ion-glycan complexes for the individual sample (colored trace) and sample mixture (black traces) IMS measurements, respectively. The arrival times labeled in the figure were extracted from centroid of peaks and the RP P values were calculated by using formula (2).
pairs of glycan-metal ion complexes under current study, corresponding to the highest resolution values, were obtained from the complete experimental measurements shown in Supplemental Figs. S2–S5. Fig. 4 showed the optimum IMS separation for these glycan-metal ion complex pairs in both pure standard and mixture sample conditions. For the isomeric trisaccharides MAL3 and ISO3, comprised of glucose units and differed in connectivity only, slightly less than baseline separation was achieved by the incorporation of two sodium ions and one chlorine ion with a calculated RP P of 0.97. The best separation for both isomeric tetrasccharide ISO4 and STAC and isomeric pentasaccharide ISO5 and CEL5 were achieved by incorporating [Ca2+ + Cl−]+ with the glycans. Baseline separation for both the [ISO4 + Ca + Cl]+ and [STAC + Ca + Cl]+complex pair and the [ISO5 + Ca + Cl]+ and [CEL5 + Ca + Cl]+ complex pair was achieved with calculated RP P of 1.31 and 2.39, respectively. For isomeric hexasaccharides ISO6 and CEL6, the best separation was achieved when complexes [CEL6 + Ba + Cl]+ and [ISO6 + Ba + Cl]+ were used with a calculated RP P value of 1.46. The optimum glycan-metal ion complexes for IMS separation obtained from Fig. 4 were further used to evaluate the effectiveness of IMS separation for all the corresponding trisaccharides, tetrasaccharides,
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Fig. 5. IMS separation of all isomeric trisaccharides, tetrasaccharides, pentasaccharides and hexasaccharides by using the optimum glycan-metal ion complexes for A) individual samples and B) sample mixtures. For comparison purpose, C) and D) showed the corresponding glycan separation for glycan-sodium ion complexes by using individual samples and sample mixtures, respectively.
complex was well separated from the complexes [ISO3 + 2Na + Cl]+ and [RAFF + 2Na + Cl]+ (Fig. 5A). This was confirmed further from the sample mixture data shown in Fig. 5B and D by three IMS peaks for complexes [M + 2Na + Cl]+ as compared to two IMS peaks for complexes [M + Na]+. In the case of tetrasaccharides, the features of sodiated complexes [STAC + Na]+, [MAN4 + Na]+ and [ISO4 + Na]+ were almost overlapped and centered at tA 29.48 ms, 29.49 ms and 29.51 ms, respectively. The resolution values between any two of these complexes were measured at 0.01–0.03. The complex [MAL4 + Na]+ was only slightly separated from the remaining three tetrasaccharide complexes. In contrast, the IMS separation of tetrasaccharide complexes [M + Ca + Cl]+ were significantly improved, as
pentasaccharides and hexasaccharides by using both pure standards and sample mixtures. Fig. 5 showed the IMS separation of four trisaccharides, tetrasaccharides, pentasaccharides and three hexasaccharides by using both pure standards (Fig. 5A and C) and sample mixtures (Fig. 5B and D). In Fig. 5A and B, the best glycan-metal ion complexes were used for IMS separation. For comparison purpose, Fig. 5C and D showed the IMS separation of all the sodiated glycans. It is clearly indicated that the separation of isomeric glycans were greatly improved by using the optimum glycan-metal ion complexes. Specifically, for the IMS separation of isomeric trisaccharides, while sodiated complexes [MAL3 + Na]+, [ISO3 + Na]+ and [RAFF + Na]+ were hardly separated from each other (Fig. 5C), the [MAL3 + 2Na + Cl]+
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glycans would be even higher if a better resolving power IMS is used. 3.3. IMS trendlines for glycan-metal ion complexes It is well known that the IMS-MS measurements of a specific class of compound such as peptides, lipids, glycans and polymers display their distinctive charge state based trendlines in the 2-dimensional display of IMS arrival time and MS m/z, a very useful feature of IMS-MS measurements for the identification of different classes of chemical compounds [40]. It is interesting to see if the IMS-MS measurement trendline for glycan is still preserved after the incorporation of different metal ions. Fig. 7A displayed IMS-MS measurements of different glycans and glycan clusters incorporated with sodium and calcium metal ions for singly and doubly charged complex ions. Two well separated trendlines were clearly observed corresponding to singly charged glycan-sodium ion complexes [M + Na]+ and doubly charged glycancalcium ion complexes [M + Ca]2+. The results in Fig. 7A was also consistent with a previous report indicating that doubly charged species generally presented higher mobility than singly charged species with the same m/z [45]. In an attempt to further generalize the trendline discussion, a total of 280 complexes observed in this study, which include 270 glycanmetal ion complexes and 10 protonated glycan ions, were included to see if the IMS-MS measurement trendlines for glycan would still hold. To make the experimental data IMS instrument independent, the IMS arrival times of these complexes were converted into their corresponding collision cross section (CCS) values by using equation (1) as listed in Supplemental Tables S6–S9. To ensure the CCS calculation accuracy, a subset of the CCS values was compared with the literature values [46]. The difference of our calculated CCS values (Supplemental Table S5) were all less than 0.75% to the literature CCS values. The projection of complexes [M + IIA + Cl]+ in Fig. 7B occupied relatively more extended area than other single charged species. Similar trends were also held for complexes [2M + IIA]2+. These two examples were consistent with the above results that the combination of multiple ions may lead to larger structure change. In addition, Fig. 7B showed a CCS vs m/z plot for all the experimentally observed glycan-metal ion complexes which clearly indicated that the trendlines for singly and doubly charged glycan-metal ion complexes were still preserved and appeared to be glycan dependent only. The incorporation of any metal ions with glycans has little effect on the IMS-MS measurement trendline. From the linear fits of m/z and CCS for 1 + and 2 + charge state trendlines, it was estimated that the singly charged complexes [M + H]+, [M + IA]+, [M + IIA + Cl]+ and [M + 2Na + Cl]+ distributed around the singly charged trendline with ± 16% deviation and the doubly charged complexes [M + IIA]2+ and [2M + IIA]2+ distributed around the doubly charged trendline with ± 17% deviation. This firmly suggests that although the gas-phase structure of glycans were significantly influenced by metal ion incorporation, the conformational space of these complexes still mainly depended on the structure and charge state of glycans.
Fig. 6. Distribution of IMS separation resolution for all glycan-metal ion complex pairs in three different categories.
shown in Fig. 5A and B. Specifically, the RP P was measured at 0.65 for the separation of complexes [STAC + Ca + Cl]+ and [MAN4 + Ca + Cl]+, 0.64 for the complexes [MAN4 + Ca + Cl]+ and [ISO4 + Ca + Cl]+, which is more than six fold improvement in IMS separation. Similar conclusion to trisaccharide IMS separation can also be reached for pentasaccharides by using the optimum [M + Ca + Cl]+ complexes as compared to their corresponding sodiated complexes [M + Na]+. For hexasaccharide IMS separation, the features of all hexasaccharides were almost baseline separated for the optimum complexes [M + Ba + Cl]+ as compared to almost no separation for sodiated hexasaccharide ions. To draw a broader conclusion on the effect of metal ion incorporation on the glycan IMS separation from the current systematic study, all the glycan-metal ion complex pairs used in the experiments were grouped in Fig. 6, according to their measured IMS resolutions listed in Supplemental Tables S1–S4, into three different categories. Each resolution category in Fig. 6 represents a distinctive IMS separation characteristic. RP P in the range of 0–0.5 represents overlapping IMS peaks for the corresponding glycan-metal ion complex pair. RP P in the range of 0.5–1 represents partially separated peaks for the glycanmetal ion complex pair. RP P > 1 represents baseline separated peaks for the glycan-metal ion complex pair [43]. With a total 21 glycan pairs used in this study, a total of 378 glycan-metal ion complex pairs was observed experimentally by incorporating each glycan pair with different group I and group II metal ions (i.e. 18 different glycan-metal ion complex pairs were observed for each glycan pair). To simplify the discussion, each glycan pair in the form of any glycan-metal ion complex pair can only appear in each resolution category once at most to eliminate counting redundancy. Based on this definition, 100% for a specific resolution group in Fig. 6 implies that one can always find at least one glycan-metal ion complex pair having the IMS separation resolution indicated in the group for all the glycan pairs in the current study. The results in Fig. 6 confirmed the striking effect of metal ion incorporation on the IMS separation of isomeric glycans. For any unresolved isomeric glycan pair, one can always find a good glycan-metal ion complex pair to partially separate them in IMS. In addition, there are 76% of the chance that the IMS separation of the glycan pair can be baseline resolved if an optimum glycan-metal ion complex is used even with the modest IMS resolving power of 50–80 used in the current study. It is expected that the chance to baseline separate isomeric
4. Conclusions The incorporation of different types of metal ions with glycans can change both the structure and the conformational diversity of glycans which can be effectively explored to enhance IMS separation of isomeric glycans. The systematic IMS-MS measurements demonstrated that partial IMS separation of a given isomeric pair can always be realized by carefully selecting a glycan-metal ion complex. Under the optimum glycan-metal ion complex conditions, even baseline IMS separation can be realized for many unresolvable isomeric glycans in their common sodiated form even with the modest IMS resolving power of 50–80. In addition, even after the gas-phase structure of the glycans
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Fig. 7. A) An experimental 2D plot of arrival time versus m/z for the incorporation of calcium ion or sodium ion with each glycan presented in Fig. 1. The features of each ion in plot were numerically labeled, and the corresponding species were (1) [TRI + Ca]2+, (2) [TETRA + Ca]2+, (3) [PENTA + Ca]2+, (4) [HEX + Ca]2+, (5) [TRI + TETRA + Ca]2+, (6) [2TETRA + Ca]2+ (7) [TETRA + PENTA + Ca]2+ (8) [2PENTA + Ca]2+, (9) [TRI + Na]+, (10) [TETRA + Na]+, (11) [PENTA + Na]+ and (12) [HEX + Na]+, where TRI, TETRA, PENTA and HEX represented trisaccharide, tetrasccharide, pentasaccharide and hexasaccharide, respectively. B) A 2D plot of CCS versus m/z for all complexes obtained in this study. The insert lines contained the linear fits of 1 + and 2 + charged state glycans (colored dotted traces) and these complexes' data included area (colored solid traces).
were significantly altered by the metal ion incorporation, the wellknown IMS-MS measurement trendlines were still preserved for all the glycan-metal ion complexes which were glycan and charge state dependent only.
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Acknowledgements This work was supported by National Natural Science Foundation of China (Grant Nos. 61971248), National Key Research and Development Program of China (Grant No. 2017YFC1001700), Key Research and Development Program of Zhejiang Province (Grant No. 2020C03064), Science and Technology Major Project of Ningbo (Grant No. 2018B10075), Scientific Research Foundation of Graduate School of Ningbo University and sponsored by K.C. Wong Magna Fund in Ningbo University. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.talanta.2020.120719. References [1] A. Varki, Biological roles of glycans, Glycobiology 27 (2017) 3–49. [2] R.A. Dwek, Glycobiology: toward understanding the function of sugars, Chem. Rev.
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