Multivariate Analysis of Electron Detachment Dissociation and Infrared Multiphoton Dissociation Mass Spectra of Heparan Sulfate Tetrasaccharides Differing Only in Hexuronic acid Stereochemistry
The structural characterization of glycosaminoglycan (GAG) carbohydrates by mass spectrometry has been a long-standing analytical challenge due to the inherent heterogeneity of these biomolecules, specifically polydispersity, variability in sulfation, and hexuronic acid stereochemistry. Recent advances in tandem mass spectrometry methods employing threshold and electron-based ion activation have resulted in the ability to determine the location of the labile sulfate modification as well as assign the stereochemistry of hexuronic acid residues. To facilitate the analysis of complex electron detachment dissociation (EDD) spectra, principal component analysis (PCA) is employed to differentiate the hexuronic acid stereochemistry of four synthetic GAG epimers whose EDD spectra are nearly identical upon visual inspection. For comparison, PCA is also applied to infrared multiphoton dissociation spectra (IRMPD) of the examined epimers. To assess the applicability of multivariate methods in GAG mixture analysis, PCA is utilized to identify the relative content of two epimers in a binary mixture.
Key wordsMultivariate analysis Principal Component Analysis (PCA) Electron Detachment Dissociation (EDD) Glycosaminoglycans (GAGs) Epimers Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS)
Proteoglycans (PGs) play an important role in regulating a variety of physiologic and pathologic processes, cellular communication, and cell signaling [1, 2, 3, 4]. PGs consist of a protein core and carbohydrates known as glycosaminoglycans (GAGs). GAGs are linear biopolymers consisting of alternating hexuronic acid and hexosamine sugar residues. Variations are found in their polysaccharide type, composition, functional group substitution, stereochemistry, and distribution of polysaccharide chain lengths, resulting in great structural diversity. For example, heparin and heparan sulfate (HS), the most structurally complex GAGs, consist of a repeating disaccharide of hexuronic acid (HexA) and glucosamine (GlcN) and vary in the C5 stereochemistry with acidic residues assigned as either glucuronic acid (GlcA) or iduronic acid (IdoA). The acidic residues may also be 2-O-sulfated. Furthermore, the glucosamine may be N-sulfated or N-acetylated, 6-O-sulfated and occasionally 3-O-sulfated. These variable modifications and stereochemistry make the characterization of GAG oligosaccharides a very challenging analytical task.
Many analytical methods have been used for GAG sequencing, which include one-dimensional (1D) and two-dimensional (2D) NMR, radioactive/fluorescent labeling, polyacrylamide gel electrophoresis, or LC/CE separation [5, 6, 7, 8]. These methods are based on the analysis of the GAG oligosaccharides that are obtained from enzymatic digestion of longer GAG chains. Recently, mass spectrometry and, in particular, tandem mass spectrometry has emerged as a more sensitive and molecular specific tool for the analysis of GAG oligosaccharides [8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21]. Collision induced dissociation (CID) of GAG oligosaccharides yielded glycosidic bond and cross-ring cleavages, which provided information about the oligosaccharide sequence and position of sulfation [18, 19]. It has also been shown that the CID product ion patterns of GAG oligosaccharides are dependent upon the charge states of the precursor ion [14, 19] and that the abundances of product ions from glycosidic bond cleavage are indicative of the position of sulfation on the N-acetylgalactosamine (GalNAc) residue [10, 14, 15, 17, 19], for example, 4S/6S sulfation in chondroitin sulfate (CS) oligosaccharides . Interestingly, even hexuronic acid epimerization in CS oligosaccharides could be determined by product ion abundances generated from tandem mass spectrometry [19, 20]. Previously, Zaia and coworkers have shown that the hexuronic acid stereochemistry in CS oligomers could be determined based on the relative abundance of specific X and Y ions produced by CID . Additionally, the relative abundance of key fragment ions was used to determine the fractional abundance of IdoA versus GlcA in tetrasaccharides and hexasaccharides from an enzymatic digestion of chondroitin/dermatan sulfate GAGs [20, 22]. Tandem mass spectrometry ion abundances have also been used to quantify GAG oligosaccharide mixtures [15, 16, 20]. In these previous studies, univariate analysis, which involves a single diagnostic product ion representing each spectrum, was employed.
In recent years, electron detachment dissociation (EDD) has been demonstrated to be a very powerful tool for the characterization of negatively charged GAG oligosaccharides [23, 24, 25, 26, 27, 28, 29] ranging from tetrasaccharides to decasaccharides [23, 24, 27]. Irradiation of multiply charged precursor anions with moderate energy (~19 eV) electrons results in extensive glycosidic and cross-ring bond cleavages, which facilitates the determination of the sites of acetylation/sulfation. Compared with threshold dissociation methods such as CID or infrared multiphoton dissociation (IRMPD), the loss of SO3 from the sites of sulfation was reduced in EDD. For the analysis and interpretation of complicated EDD spectra, it is necessary to employ more reliable, objective statistical tool that makes use of a multiple number of product ions such as multivariate analysis.
Furthermore, it was demonstrated that EDD distinguishes GlcA from IdoA in heparan sulfate (HS) tetrasaccharides based on the occurrence of diagnostic product ions . It was proposed that the diagnostic products were due to a radical-initiated mechanism, with the initial radical site generated by electron detachment from the carboxylate group on acidic residues and subsequent migration through hydrogen atom transfer. This hydrogen atom transfer is dependent upon the proximity of the initial carboxyl radical to hydrogen atoms on adjacent carbons, for example, C2, C3, or C4, thus inducing fragmentation that is sensitive to stereochemistry .
In the present study, EDD experiments were performed on four synthetic di-sulfated GAG tetrasaccharides with two epimeric sites; GlcA-GlcNAc6S-GlcA-GlcNAc6S (GG), GlcA-GlcNAc6S-IdoA-GlcNAc6S (GI), IdoA-GlcNAc6S-IdoA-GlcNAc6S (II), and IdoA-GlcNAc6S-GlcA-GlcNAc6S (IG) (shown as Scheme 1 in the Supplementary Material). Unlike tetrasaccharides prepared by enzymatic digestion of longer GAG oligosaccharides, the non-reducing end (NRE) has a saturated sugar ring, and thus the tetrasaccharides contain a stereocenter on each hexuronic acid residue. The EDD spectra of the doubly deprotonated precursor ion for each epimer are nearly identical, varying only in product ion intensity. To facilitate the differentiation of the four tetrasaccharide EDD spectra, we apply a multivariate analysis (MVA) method [30, 31, 32]. MVA has been widely applied in many scientific disciplines to extract valuable information from complicated data sets. Factor analysis has been used to help elucidate basic chemical interactions in gas chromatography and to interpret fragmentation patterns in electron impact mass spectrometry [33, 34, 35, 36]. Furthermore, it has been shown that factor analysis can separate the data from mass spectra of mixtures into the mass spectra of the pure components and give their respective concentrations . Recently, MVA has been successfully applied to discern the characteristic features of large proteomics data sets [37, 38, 39] and carbohydrate structure analysis [40, 41]. For example, comparative multivariate statistical analysis of 2D LC-MS data for proteomic samples dictated the peptide masses that were differentially expressed, and subsequently targeted during LC-MS/MS as peptides of interest [37, 38]. In the current study, we expand the use of MVA to the analysis of GAG tandem mass spectra; specifically, we use principal component analysis (PCA) to differentiate the acid stereochemistry in four synthetic HS epimers through the analysis of EDD and IRMPD spectra that contain peaks assigned to both glycosidic and cross-ring cleavages.
2.1 Tetrasaccharide Synthesis
Four HS tetrasaccharides, GlcA-GlcNAc6S-GlcA-GlcNAc6S (GG), GlcA-GlcNAc6S-IdoA-GlcNAc6S (GI), IdoA-GlcNAc6S-IdoA-GlcNAc6S (II), and IdoA-GlcNAc6S-GlcA-GlcNAc6S (IG), were synthesized as described previously and purified by silica gel column chromatography . Prepared structures were confirmed by 1H NMR and accurate mass measurement by FTICR-MS.
2.2 Mass Spectrometry
Experiments were performed on a 9.4 T Bruker Apex Ultra QhFTMS (Billerica, MA, USA) equipped with an Apollo II dual source, a 25 W CO2 laser (Synrad model J48-2; Mukilteo, WA, USA) for IRMPD, and an indirectly heated hollow cathode for generating electrons for EDD. Solutions of each tetrasaccharide were introduced at a concentration of 0.1 mg/mL in 50:50:0.1 methanol:H2O:formic acid (Sigma, St. Louis, MO, USA) and ionized by nanospray using a pulled fused silica tip (model FS360-75-15-D-20; New Objective, Woburn, MA, USA). Formic acid was added to the ESI spray solvent to reduce Na/H heterogeneity . The sample solutions were infused at a rate of 10 μL/h and were examined in negative ion mode.
In EDD experiments, precursor ions were mass-selected in the external quadrupole and accumulated for 1 to 2 s in an rf-only hexapole before injection into the FTICR analyzer cell. The precursor ion selection was further refined by a coherent harmonic excitation frequency (CHEF) event . The isolated precursor ions were then irradiated with electrons for 1 s. During EDD, the cathode was biased at –19.0 V, the extraction lens was set to –18.5 V, and the cathode heater to 1.4 A. In IRMPD experiments the same experimental setup as in EDD experiments was used, except the electron irradiation period was replaced with a laser pulse. The isolated precursor ions were irradiated for 0.02 to 0.25 s with the laser beam attenuated to 60% of full laser power. In both experiments, 24 acquisitions were signal averaged per mass spectrum. For each mass spectrum, 512 K points were acquired, padded with one zero fill, and apodized using a sinebell window.
2.3 Data Analysis
Product ion assignments were made based on accurate mass measurement and aided by Glycoworkbench . Monoisotopic peaks corresponding to assigned glycosidic and cross-ring cleavages and their respective abundances were compiled for each mass spectrum in Bruker Data Analysis ver. 3.4. External calibration of mass spectra produced mass accuracy of 5 ppm. Internal calibration was also performed using confidently assigned glycosidic bond cleavage products as internal calibrants, providing mass accuracy of <1 ppm. Due to the larger number of low intensity products formed by EDD, only peaks with S/N > 10 are reported. All products are reported using the Domon and Costello nomenclature . The annotation of glycosidic fragmentation products that occur with the loss of additional hydrogen (compared with products found in conventional ion activation experiments) are modified by appending single or double quotes to the B, C, Y, or Z designation, to indicate the loss of 1 or 2 hydrogen atoms, respectively, consistent with the EDD literature [23, 24, 25, 26, 27, 28].
Principal component analysis (PCA) was performed using PLS Toolbox (Eigenvector Research, Inc., Wenatchee, WA). The abundances of 59 assigned fragment ions were normalized with respect to total ion abundance in each EDD spectrum. An input data matrix was constructed with each row containing the mass spectrum of a single tetrasaccharide epimer (samples) and each column, the normalized abundance of an assigned fragment ion (variables). For each tetrasaccharide, five EDD spectra were obtained in the same day. Prior to PCA, each data set was mean-centered and cross-validated. In brief, PCA analysis explains the variance in the examined datasets by generation of a system of linear equations, i.e., principal components. The plot of principal component scores shows the relationship between the samples, e.g., similar samples will be grouped. Examination of the PCA loading plots reveals the variables that contribute to the sample distinction, e.g., minimally contributing variables will be located near the origin whereas variables with high contribution will have relatively large values.
3 Results and Discussion
3.1 EDD of HS Tetrasaccharide Epimers
The doubly deprotonated anion, [M – 2H]2–, of each tetrasaccharide shown in Scheme 1 (Supplementary Material) was subjected to a 1.0 s, 19 eV electron irradiation to obtain their EDD mass spectra. As previously stated, these compounds differ only in their hexuronic acid stereochemistry; therefore, the four potential doubly deprotonated precursor ions are isobaric and cannot be distinguished at the MS level.
Previously, EDD of HS tetrasaccharide epimers has produced diagnostic product ions that can be used to distinguish IdoA from GlcA . From a mechanistic aspect, the proximity of the initially generated carboxy radical to a neighboring hydroxyl group on the sugar ring directs different hydrogen transfer rates and, therefore, distinct cross-ring cleavage patterns. With this in mind, the EDD mass spectra in Figure 1 are compared. A striking distinction cannot be found between the four spectra in terms of the overall fragment ion distributions. (This result is based on the examination of EDD spectra generated from the activation of a doubly deprotonated ion generated from a tetrasaccharide with two sulfate groups. The [M – 2H]2– precursor ion is the most easily obtained species and presents two ionized sulfates based on pKa. To obtain EDD spectra containing products consistent with prior results, it is necessary to ionize and detach from a carboxyl group. This mechanistic aspect is the subject of a manuscript in preparation.) However, a close inspection of relative abundances of fragments shows a number of diagnostic product ion peaks that may enable the differentiation of one epimer from another. Notable fragment peaks are the followings: (B2 + Y3)–, 0,2A2–, Y32–, B2–, C2–, 3,5A3–, (B3-SO3)– at, C3″–, (Z3″– H2SO4)–, Z3–, Y3–, and 1,3 X3 or 2,4X3–. (Masses are provided as Supplementary Data.) More specifically, 0,2A2– fragment appears in high abundance for the tetrasaccharides containing GlcA at the NRE. In the case of the C2– and 3,5A3– fragments, their abundances stand out for II. As another example, the abundance of the C3″- peak is relatively higher when IdoA is located at the third saccharide residue from the NRE, which is consistent with prior EDD results when IdoA is located at this position . Although visual inspection of these spectra can provide some insight into the assignment of hexuronic acid stereochemistry, it is necessary to consider as many diagnostic peaks as possible in order to characterize an unknown EDD mass spectrum in a reliable manner.
3.2 Principal Component Analysis (PCA) of EDD Mass Spectra
In the present study, a statistical method is employed as a reliable, objective tool for distinguishing the EDD spectra obtained from four HS tetrasaccharide epimers. As briefly described above, it is difficult to gain discriminant information using a univariate analysis. Multivariate analysis (MVA) is therefore used for the differentiation of the obtained mass spectra. Specifically, principal component analysis (PCA), one of the most common multivariate analysis tools, is used in the present study. This method is known to be very useful for the extraction and visualization of the most important features from complicated data sets [30, 32]. PCA was carried out for 20 EDD mass spectra obtained from quintuplicate experiments for each of the four tetrasaccharides. The relative abundances of 59 fragment ions shown in Figure 1 were normalized with respect to the total abundance and used to construct an input data matrix for PCA.
As shown in Figure 2a, when the four tetrasaccharides are projected onto the PC1 axis (solid lines), they are all well separated from each other, as the high value of variance explained by PC1 (51%) indicates. Specifically, the GG replicates have the highest positive scores along the PC1 axis, while IG replicates have the most negative scores. These two epimers are most readily distinguished by PC1 alone. Closer examination of the PCA score plot reveals differentiation of the stereochemistry of the non-reducing end hexuronic acid by PC1. Tetrasaccharides containing IdoA at the NRE have negative PCA scores whereas GlcA results in positive scores for PC1. Further differentiation can be made in PC2 for the stereochemistry of the third saccharide residue. The epimers containing GlcA at the third position have positive scores in PC2 while the tetrasaccharides with IdoA have negative scores. In this plot, however, II and GI have the lowest degree of separation in PC1, but the separation between II and GI is more distinct in the plot of PC3 versus PC1 visualized in Figure 3a. The separation between the clustered groups can be more clearly demonstrated in a 3D plot displayed in Figure 3b.
In general, principal components are constructed by a weighted combination of fragment ion intensities in a manner that best explains the variance in the data, revealing the internal structure of the data. The relative contribution of each fragment in constructing principal components can be determined from the loading plots. For example, Figure 2b shows that the following peaks have high positive loading values on PC1: 1,3X3 or 2,4X3–, Y1–, B2–, and 0,2A2–. High abundances of these peaks can be collectively used as characteristics of epimers containing GlcA at the NRE. In contrast, high negative loading values on PC1 were observed for (Z3″-H2SO4)–, (B3-SO3)–, B3–, and Z3–, are diagnostic for epimers containing IdoA at the NRE.
As illustrated in Figure 3a, II and GI isomers are well separated on PC3, on which 1,3 X3 or 2,4X3–, 3,5A3–, and C2– have high positive loadings, while B2–, (Z3″-H2SO4)–, and 0,2A2– have high negative loadings (Figure 3c). To make the above-mentioned important loadings easily noticeable, these loadings are denoted with dotted lines and assignments are circled in Figure 1.
3.3 Binary Mixture Analysis
The determination of mixture composition is one of the primary aims during the analysis of real world samples. Here it is examined whether PCA can be employed to determine the composition of a binary mixture based on EDD mass spectra. As a demonstrative example, four binary mixtures of IG and GG, 7:3, 5:5, 3:7, 1:9 (IG:GG), were separately subjected to EDD mass spectrometry and the resulting mass spectra analyzed using PCA.
3.4 PCA Analysis of IRMPD Spectra
We have shown above that PCA analysis is a very useful tool for the analysis of complex EDD mass spectra of GAGs, but the question may arise whether PCA analysis is also beneficial for the analysis of tandem mass spectra of GAGs due to threshold activation methods such as CID or IRMPD, which generally produce fewer product ions. Included in the Supplemental Material are the IRMPD mass spectra for the four synthetic HS epimeric isomers. Compared with the EDD mass spectra in Figure 1, IRMPD mass spectra exhibit fewer fragments. The dominant fragment ions are Y1–, Y3–2, B2–, [(M – 2H) – SO3]2–, and B3–. When the four IRMPD mass spectra are compared, it is clearly visible that the relative abundances of B3– vary significantly from one spectrum to another; the relative abundance was the highest for II isomer, while the lowest for GG isomer. In contrast, B2 has a low abundance for the II isomer, but high abundance for GG isomer.
A MVA approach to the analysis of EDD mass spectra of GAG tetrasaccharide anions is described. In general, EDD spectra of GAG oligomer anions generally exhibit a large number of fragment ions that arose from cleavages of glycosidic and/or cross-ring bonds. Indeed, in the present study for synthetic HS tetrasaccharide anions that contain two epimeric sites, i.e., GlcA-GlcNAc6S-GlcA-GlcNAc6S (GG), GlcA-GlcNAc6S-IdoA-GlcNAc6S (GI), IdoA-GlcNAc6S-GlcA-GlcNAc6S (IG), and IdoA-GlcNAc6S-IdoA-GlcNAc6S (II), EDD produced highly complex product ion spectra. Due to the high spectral complexity, it was neither simple nor straightforward to distinguish one spectrum from another by visual inspection. To aid the differentiation of the obtained EDD spectra, PCA was performed to extract the information necessary to characterize each spectrum. In the PCA score plots, EDD spectra of the four tetrasaccharides were well grouped on the basis of their epimeric states. Furthermore, the loading plots indicated the characteristic fragments that contributed significantly to differentiating each of EDD spectra. It was also demonstrated that the compositions of binary tetrasaccharide mixtures could be broadly analyzed by projecting their EDD spectra results onto the score plot of ‘pure’ tetrasaccharides. PCA was also shown to be very useful for analyzing the IRMPD spectra of the four synthetic HS tetrasaccharide anions. This study clearly demonstrates that PCA is a promising tool for characterizing GAG oligosaccharides, particularly, isobaric or epimeric GAG mixtures, in combination with EDD and IRMPD tandem mass spectrometry.
H.B.O. thanks the National Research Foundation of Korea (NRF-2009-0075245) for the financial support. F.E.L. III and I.J.A. gratefully acknowledge financial support from the National Institutes of Health grant no. 2R01-GM038060-20. F.E.L. III, I.J.A., S.A., K.A.-M, A.V., and G.-J.B. gratefully acknowledge financial support from the Center for Research Resource of the National Institutes of Health grant no P41RR005351.
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