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193 nm Ultraviolet Photodissociation of Deprotonated Sialylated Oligosaccharides Byoung Joon Ko† and Jennifer S. Brodbelt*,‡ Departments of †Chemical Engineering, and ‡Chemistry and Biochemistry, 1 University Station A5300, University of Texas at Austin, Austin, Texas, United States
bS Supporting Information ABSTRACT: The fragmentation patterns of deprotonated sialylated oligosaccharides and glycans from fetuin obtained upon collisionally induced dissociation (CID) and 193 nm ultraviolet photodissociation (UVPD) in a linear ion trap are presented. UVPD produced a more extensive series of cross-ring cleavage ions, such as A- and X-type ions, and dual-cleavage internal ions, including A/Y and X/B fragment ions. In addition, UVPD generated unique fragment ions which arise from site-specific cleavage of the triol substituent of the sialic acid residue. In contrast, CID produced more conventional glycosidic cleavages and relatively few A-type ions. UVPD of doubly deprotonated sialylated oligosaccharides produced mostly singly deprotonated fragment ions, whereas the product ions in the CID spectra were overwhelmingly doubly charged ions, an outcome attributed to the more extensive cleavages of sialic acid residues upon UVPD and products from electron photodetachment. The larger array of product ions, including those arising from extensive cross-ring cleavages and dual-cleavage ions, generated by 193 nm UVPD relative to CID gives greater confidence for identification of glycans. Several key site-specific cleavages by UVPD, such as ones involving the sialic acid moieties, provide evidence of glycan composition.
T
he oligosaccharide portions of biopolymers are known to play key roles in cellular functions including cell signaling, cellular differentiation, immune response, and inflammation.13 Glycosylation of protein is one of the most widespread posttranslational modifications, with over 50% of eukaryotic proteins being glycosylated. Establishing the functional aspects of glycosylated molecules require systematic elucidation of their structures.4 The immense diversity of oligosaccharides arises from the various nonlinear branching structures as well as their array of potential intersaccharide linkages. In addition, the possibility of two different sites of attachment to proteins, including N- and O-linked glycosylation, expands the analytical challenge. For characterization of oligosaccharides in general, the ideal tandem mass spectrometry (MS/MS) strategy would yield products both from glycosidic bond cleavages to give sequence and branching information and those from cross-ring cleavage to reveal linkages. Low-energy (eV) collisionally induced dissociation (CID), which remains the most popular method for analysis of oligosaccharides, typically affords fragment ions from both the reducing (Y-/Z-type) and nonreducing (B-/C-type) ends, but its efficiency falls off with the size of the oligosaccharide, and the lack of cross-ring cleavages generally precludes assignment of linkages.59 High-energy (keV) CID1014 typically provides more cross-ring cleavages compared to conventional low-energy CID as well as more internal ions too. The newly developed electron-based methods, including electron capture r 2011 American Chemical Society
dissociation (ECD),1517 electron detachment dissociation (EDD),1820 and electron-transfer dissociation (ETD),21,22 have been applied for characterization of oligosaccharide and glycopeptides,17 in some cases yielding abundant cross-ring cleavage ions that are useful for assignment of intersaccharide linkages. For example, electron activation results predominantly in formation of C and Z ions via glycosidic cleavage ions.17,23,24 However, due to their dependence on ample ion charging, electron-based dissociation methods have been less widely adopted for characterization of oligosaccharides compared to elucidation of other biopolymers like peptides and proteins. As an alternative strategy, photon-based dissociation methods, including infrared multiphoton dissociation (IRMPD)17,2430 and ultraviolet photodissociation (UVPD),11,14,3134 have been implemented for characterization of oligosaccharides. Lebrilla and co-workers have utilized IRMPD for characterization of oligosaccharides, as well as N- and O-linked glycans released from glycoproteins and glycopeptides, by Fourier transform ion cyclotron resonance (FTICR) mass spectrometry, finding that IRMPD afforded more numerous cross-ring cleavages and more extensive fragmentation in general compared to CID.17,2430 We have previously combined IRMPD with a boronic acid Received: July 6, 2011 Accepted: September 13, 2011 Published: September 13, 2011 8192
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Figure 1. Chemical and symbolic structures of the model oligosaccharides: (A) 60 -sialyllactose (60 -SL), (B) LS-tetrasaccharide b (LSTb), (C) disialyllacto-N-tetraose (DSLNT), (D) sialyllacto-N-fucopentaose II (SLNFP-II), and (E) monosialyllacto-N-neohexaose (MSLNnH). The nominal mass of each oligosaccharide is included in brackets.
derivatization protocol to increase the photoabsorption cross sections of oligosaccharides and simplify the fragmentation patterns by selective cleavages from the nonreducing ends.35 More recently, UVPD has generated substantial interest for characterization of oligosaccharides due to the ability to promote a greater array of cross-ring cleavages. For example, Reilly and coworkers have employed an 157 nm F2 excimer laser to dissociate glycans11,14,31 and glycopeptides,36 demonstrating comprehensive elucidation of glycan linkages through extensive cross-ring cleavage (X) products as well as glycosidic (Y, Z) fragments. Lemoine and co-workers have applied 220 and 240 nm UVPD for characterization of oligosaccharide anions.33,34 UVPD produced a richer pattern of cross-ring cleavages (i.e., A- and X-type ion series). We have reported 355 nm UVPD for oligosaccharides labeled at their reducing ends with fluorophores, a strategy that selectively enhanced the formation of A- and C-type ions containing the nonreducing end.37 In addition, we have demonstrated that different cross-ring cleavages, yielding A-type ions, occurred upon UVPD at 355 nm of chromophore-labeled oligosaccharides produced by hydrazide conjugation versus reductive amination.38 Since UVPD results in the richest array of fragment ions, including those arising from both glycosidic bond cleavages and cross-ring cleavages, this activation method provides the most promising opportunity to obtain sequence, branching, and linkage information for oligosaccharides. Our two previous UVPD studies at 355 nm required derivatization of the oligosaccharides to endow them with appropriate UV chromophores, and a streamlined strategy that does not depend on derivatization steps offers a more compelling strategy for characterization of glycans from biological molecules. This motivated our interest in implementing UVPD at 193 nm specifically for analysis of acidic sialylated oligosaccharides and glycans without derivatization in the present study. The characterization of sialylated oligosaccharides proves to be particularly challenging by most MS/MS methods because of the lability
of the sialic acid groups and/or lack of sufficient product ions to confirm linkage information. Sialic acid is a hydrophilic nine-carbon monosaccharide typically located at the nonreducing termini of glycans, presented both on cell surfaces and in secreted proteins. Sialic acids are overexpressed in some cancer cells39 and serve as binding sites for a number of pathogens.40 In addition, sialylated glycans displayed at cell surfaces play a critical role in cellcell communication and antigen recognition in the immune system.41 Sialylated oligosaccharides and glycans have been characterized extensively by tandem mass spectrometry. Despite the natural acidities of sialic acids, many previous mass spectrometric studies have been performed in the positive mode, typically after esterification, amination, or permethylation of the sialic acid residues to stabilize the sialic acid groups and/or improve their ionization efficiencies and subsequent detection as metalcationized species.10,11,30,4246 Positively charged nonderivatized sialylated glycans typically undergo facile neutral loss of sialic acid, a factor which can impede localization of the sialic acid groups.47 Mechref and co-workers10,43 characterized sialylated glycans after permethylation and observed extensive A- and X-type ions. Recently, Wu et al.42 performed structural analysis of sialylated human milk oligosaccharides and developed a library of 30 human milk oligosaccharides based on retention times, accurate masses, and MS/MS data in positive mode. Since sialylated glycans produce numerous metal adducts in the positive mode, MS/MS characterization of deprotonated sialylated glycans has also been explored.18,20,44,4751 Sagi et al. sequenced tri- and tetra-antennary N-glycans to identify the positions of the sialic acids.48 Deguchi et al.47 performed structural determination of deprotonated monosialylated biantennary N-linked glycans using an MSn strategy. Although these studies successfully characterized sialylated glycans in the negative mode, both studies relied on CID which resulted primarily in glycosidic bond cleavages without sufficient cross-ring cleavages to confirm linkages. 8193
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Figure 2. MS/MS data of deprotonated [60 -SL H] (m/z 632) (A) by CID and (B) by 193 nm UVPD and the product ions assigned from (C) CID and (D) UVPD. A superscript o represents the loss of H2O. The precursor ion is labeled with an asterisk.
UVPD at 193 nm has been widely applied to biopolymer analysis due the ability to promote more diverse fragmentation pathways likely arising from the higher energy deposition of UV photoabsorption.52,53 In the present study, we compare the CID and 193 nm UVPD fragmentation patterns obtained for deprotonated sialylated oligosaccharides. Whereas CID predominantly produces B/C- and Y/Z-type ions from cleavages at glycosidic linkages, 193 nm UVPD yields a more diverse array of complementary fragment ions, as well as unique fragment ions from cleavages at sialic acid. We also apply 193 nm UVPD for the analysis of glycans enzymatically released from fetuin, a protein which contains a heterogeneous mixture of acidic glycans.
’ EXPERIMENTAL SECTION Reagents. The five model sialylated oligosaccharides were purchased from V-Laboratories (Covington, LA). 60 -Sialyllactose (60 -SL), LS-tetrasaccharide b (LSTb), silalyllacto-N-fucopentaose II (SLNFP-II), monosialyllacto-N-neohexaose (MSLNnH), and disialyl-lacto-N-tetraose (DSLNT) are illustrated in Figure 1. Glycerol-free peptide N-glycosidase F (PNGase F) and deglycosylation buffers were purchased from New England Biolabs (Ipswich, MA). Fetuin from fetal calf serum was obtained from Sigma (St. Louis, MO). All other chemicals were purchased from
Fisher Scientific (Pittsburgh, PA) and used without any further purification. Deglycosylation. The glycans were prepared from fetuin with enzymatic deglycosylation using PNGase F. The procedure followed the manufacturer’s protocol. An amount of 20 μg of fetuin was dissolved in 10 μL of denaturing buffer and heated to 100 °C for 10 min. Then, 2 μL of 500 mM Na2PO4, 2 μL of 10% Nonidet P-40, and 5 units of PNGase F were added. The solution was incubated at 37 °C for 2 h prior to purification of sialylated glycans a porous graphitic carbon column. Desalting. After the deglycosylation with PNGase F, the released glycans were cleaned up using porous graphitic carbon solid-phase extraction (SPE) columns (Alltech Associate, Deerfield, IL). The cleanup was based on a procedure previously described by Kim et al.46 Briefly, the column was washed with 30% acetic acid, followed by 0.1% trifluoroacetic acid (TFA) in 50% acetonitrile/water. The washed columns were primed with 3 mL of 0.1% TFA in 5% acetonitrile/water. The oligosaccharide samples were loaded, then washed with water and 0.1% TFA in 5% acetonitrile/water. The oligosaccharides were eluted with 0.1% TFA in 50% acetonitrile/water, then dried with a SpeedVac prior to MS analysis. Mass Spectrometry. All experiments were performed with a Thermo Fisher LTQ XL linear ion trap mass spectrometer (San Jose, CA) equipped with a Coherent ExciStar XS excimer laser 8194
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Figure 3. MS/MS data of deprotonated [LSTb H] (m/z 997) by (A) CID and (B) 193 nm UVPD with 30 pulses and the product ions assigned from (C) CID and (D) UVPD. A superscript o represents the loss of H2O. The precursor ion is labeled with an asterisk.
(Santa Clara, CA) producing 193 nm photons at 8 mJ/pulse. The laser setup was the same as previously described.53 For CID, a 30 ms activation time and 0.25 qz value were used. For UVPD, 10 laser pulses per spectrum were used in a total activation period of 18 ms unless specified otherwise. The qz value for UVPD was fixed at 0.1. The concentration of the oligosaccharide solution was ∼10 μM in 50/50 acetonitrile/water. The oligosaccharides were injected via direct infusion into an electrospray ionization (ESI) source at a flow rate of 3 μL/min with a Harvard PHD 2000 syringe pump (Holliston, MA). Typically up to 20100 scans were averaged with each scan composed of five microscans. The photodissociation efficiency was calculated based on the equation below: PD efficiency ð%Þ ¼
∑ðarea of product ionsÞ
∑ðarea of product ions þ area of surviving precursorÞ
100
’ RESULTS AND DISCUSSION As reported herein, CID and 193 nm UVPD are used to characterize both model sialylated oligosaccharides and sialylated glycans from fetuin in a linear ion trap mass spectrometer. Since the oligosaccharides and glycans all have at least one acidic monosaccharide at the nonreducing ends, they are naturally
conducive for analysis in the negative ESI mode. In the positive ESI mode, adduction of metals is rather extensive, either requiring sample cleanup or causing spectral congestion. In some cases, the variation in the number of types of metal adducts complicates the ESI mass spectra and subdivides the ion current. Our specific interest focused on evaluation of the performance metrics of UVPD for oligosaccharide anions in terms of the type and diversity of diagnostic ions relative to CID. Five model oligosaccharides, 60 -SL, LSTb, DSLNT, SLNFP-II, and MSLNnH, were used to establish benchmark CID and UVPD data prior to analysis of glycans released from fetuin. Fragment ions in the MS/MS data described in the following sections were assigned manually based on the nomenclature of Domon and Costello54 as illustrated in Supporting Information Figure 1. In addition, the deprotonation sites were fixed at the sialic acid residues in the schemes because of the high acidity of the carboxylic acid moieties relative to the hydroxyl groups of the other saccharides. In some cases there are several isobaric assignments possible for a particular product ion in the MS/MS spectra (whether CID or UVPD), and in these cases one reasonable assignment is labeled to prevent excessive labeling congestion of the spectra. MS/MS of the Deprotonated Model Oligosaccharides. The CID and UVPD mass spectra obtained for [60 -SL H] are shown in Figure 2. The types of fragments observed upon CID of 8195
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Figure 4. MS/MS data of doubly deprotonated [DSLNT 2H]2 (m/z 643) by (A) CID and (B) 193 nm UVPD with 10 pulses and the product ions assigned from (C) CID and (D) UVPD. The precursor ion is labeled with an asterisk.
linear 60 -SL are consistent with those reported previously in which glycosidic bond cleavages are dominant, yielding mainly B and C ions, along with formation of lower abundances of A-type ions such as 0,2A.4749 This dominant 0,2A3 ion conceivably could also be assigned as 0,4A3 or 1,3A3, all which are isobaric and not differentiable based on m/z alone. UVPD produced a more diverse array of fragment ions including a new cross-ring cleavage product, 4,5X3, as well as new product ions arising from the loss of 62 Da which may occur in conjunction with the loss of the reducing end sugar (labeled as 62/C3). These three new UVPD products all evolve from unique cleavages involving the sialic acid group at the nonreducing end, and they are consistently observed upon UVPD for the other sialylated oligosaccharides in the present study, as described later. The loss of 62 Da is uniquely promoted upon UVPD via cleavage of the triol substituent of the sialic acid group, thus affording a very specific reporter pathway for sialylated oligosaccharides. In both the CID and UVPD spectra of deprotonated 60 -SL, complete series of B- and C-type ions (B1, B2, C1, and C2; m/z 290, 452, 308, and 470, respectively) were detected, whereas the complementary Y- and Z-type ions are not seen in the spectra. The production of abundant B- and C-type ions and absence of Y- and Z-type ions correspond to the localization of the single charge site on the
sialic acid moiety, thus meaning that all reducing end product ions are uncharged. Another notable difference was the twohydrogen loss from the C2 ion which has been observed upon EDD of sialylated oligosaccharides18 or glycosaminoglycans.20 These two-hydrogen loss products are presumed to form upon rearrangement of the oligosaccharides.18 The MS/MS data of three larger and more highly branched sialylated oligosaccharides, including LSTb, DSLNT, and MSLNnH, are displayed in Figure 3 and Supporting Information Figures 2 and 3, respectively. These three model oligosaccharides have the same core consisting of glucose, galactose, N-acetylglucosamine, and galactose in series from the reducing end. The CID fragmentation pattern of deprotonated LSTb is dominated by glycosidic bond cleavages with low abundances of cross-ring cleavages (Figure 3), similar to that noted above for 60 -SL. This type of CID pattern is consistent for the other sialylated oligosaccharides. Although these CID patterns are useful for sequencing the oligosaccharides, there are few cross-ring cleavages that reveal the branching patterns. An even more diverse array of fragments, including those arising from cross-ring cleavages, are produced upon 193 nm UVPD. Moreover, the unique fragment ions stemming from the two unusual cleavages of the triol substituent of sialic acid (loss of 62 Da and formation of the 4,5X4α ion) were 8196
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Figure 5. MS/MS data of deprotonated [2Ant1SiA H] (m/z 1930) by (A) CID and (B) 193 nm UVPD with 20 pulses and the product ions assigned from (C) CID and (D) UVPD. The precursor ion is labeled with an asterisk.
consistently generated. Neither of these types of pathways occurred upon CID of any of the sialylated oligosaccharides nor glycans from fetuin. An interesting characteristic of the spectra shown in Figure 3B and Supporting Information Figures 2B and 3B is the highly abundant internal ions (dual cleavages such as 2,4A4/Y3β and 2,4 X4/C3). For the LSTb oligosaccharide, CID generated a few internal ions such as B3/Y3β and C3/Y3β which are dual cleavages at only a single glycosidic linkage. UVPD produced the same internal ions, as well as additional internal ions such as 2,4A4/Y3β and 2,4X4/C3 which arise from both cleavages of glycosidic linkages and cross-ring cleavages. These dual-cleavage products are a hallmark of high-energy activation methods. The mass spectra obtained for doubly deprotonated [DSLNT 2H]2 upon CID or UVPD are shown in Figure 4. In general, UVPD of the doubly deprotonated oligosaccharides upon 193 nm irradiation generates a significantly different series of fragment ions than that observed upon CID. Far more A/Y and A/Z dual-cleavage ions were observed in the m/z range from 700 to 1000 upon UV irradiation. Another notable feature of the UVPD spectra for the doubly deprotonated oligosaccharides relates to the charge states of the product ions. Whereas CID of doubly deprotonated oligosaccharides generated predominantly doubly deprotonated fragment ions, UVPD instead produced
mainly singly charged fragment ions except for the lone doubly charged 4,5X5α2 product ion observed in the DSLNT spectrum in Figure 4B. This striking difference in the charge states of the UVPD product ions relative to the CID product ions may be rationalized by two factors: the number of the sialic acid moieties retained in the product ions and the potential for electron photodetachment upon UVPD. With respect to the former, CID of doubly deprotonated DSLNT produced A/B/C-type ions containing both sialic acids or Y/Z-type ions containing just one sialic acid. On the other hand, UVPD of doubly deprotonated oligosaccharides produced extensive internal ions which contain only one sialic acid. The consistent loss of at least one sialic acid residue for the dual-cleavage pathways upon UVPD results in formation of singly charged product ions. Electron photodetachment, as previously observed for negatively charged peptides, nucleic acids, and lipids,5557 and in many ways analogous to electron detachment dissociation,1820,58 is another process that may generate singly charged product ions. Upon UV irradiation, photoabsorption may lead to loss of an electron from the selected precursor, resulting in an intact chargedreduced species that may undergo subsequent dissociation. For the doubly charged sialylated oligosaccharides in the present study, the resulting products would be singly charged, thus affording another source of singly charged product ions in the 8197
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Figure 6. MS/MS data of doubly deprotonated [2Ant2SiA 2H]2 (m/z 1110) by (A) CID and (B) 193 nm UVPD with 10 pulses and the product ions assigned from (C) CID and (D) UVPD. The precursor ion is labeled with an asterisk.
UVPD spectra. Inspection of the higher m/z region of the spectrum in Figure 4B indicates a very low abundance of an apparent charge-reduced species that would correspond to the intact electron photodetachment ion. MS/MS of the Deprotonated Glycans from Fetuin. The 193 nm UVPD method was also applied to glycans from a glycoprotein, fetuin from fetal calf serum. Fetuin contains a number of branched sialylated glycans. The glycan containing two antenna and one sialic acid (2Ant1SiA) was subjected to CID and UVPD, and the resulting spectra are displayed in Figure 5. Similar comparisons are shown for doubly deprotonated [2Ant2SiA 2H]2 and [3Ant2SiA 2H]2 in Figure 6 and Supporting Information Figure 4. For 2Ant1SiA, several characteristic cross-ring cleavage ions A- as well as B- and C-type ions evolving from glycosidic bond cleavages are observed upon CID, but the spectrum is dominated by the uninformative loss of water. In addition, just one X-type ion, 0,3X6β, was detected. In contrast, the UVPD spectrum of deprotonated 2Ant1SiA produced a far more extensive array of fragment ions such as numerous dual-cleavage internal ions and cross-ring cleavages similar to the model oligosaccharides described above. For example, UVPD of deprotonated 2Ant1SiA produced a complete series of B- and C-type ions as well as Y- and Z-type ions (Figure 5D), whereas CID generated only a few B- and C-type ions close to the reducing end (Figure 5C). For all of the glycans
from fetuin, the increased number of A- and X-type ions upon UVPD resulted in more diagnostic fragmentation patterns that afford both sequence and branching information (Figure 6 and Supporting Information Figure 4). We speculated that the greater array of dual-cleavage internal ions such as A/Y and X/B ions upon UVPD might be generated upon consecutive dissociation of primary product ions because 10 consecutive laser pulses were used. Thus, the influence of the number of UV pulses on the distributions of product ions and dissociation efficiencies was evaluated as summarized in Supporting Information Figure 5 for [LSTb H] and in Supporting Information Figure 6 for [DSLNT 2H]2. As the number of laser pulses increased, there was actually little change in the relative portions of products arising from glycosidic cleavages, cross-ring cleavages, or internal ions (see Supporting Information Figures 5A and 6A). This result indicates that the dualcleavage internal ions are not predominantly due to consecutive dissociation pathways after cross-ring or glycosidic cleavages but instead are formed directly from the precursor ions upon the high-energy activation from absorption of the 193 nm photons. There is a minor reduction in the portion of electron detachment product and small variations in the percentages of internal ions and glycosidic cleavages produced from the doubly charged DSLNT precursor (Supporting Information Figure 6A), but the variations are not sufficiently substantial to confirm a direct 8198
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Analytical Chemistry genealogical relationship from the electron photodetachment product to any particular secondary fragment ions. As illustrated in Supporting Information Figures 5B and 6B, the overall dissociation efficiencies increase with the number of UV pulses (while maintaining the pulse energy at ∼8 mJ per pulse). Thus, using a larger number of laser pulses results in conversion of more precursor ions into diagnostic product ions but does not significantly change the types or distributions of product ions. To further explore the contribution of electron photodetachment products to the UVPD spectra, a number of MS/MS/MS experiments were undertaken in which two separate stages of UVPD were used to produce the presumed charge-reduced electron photodetachment products, followed by isolation and subsequent UVPD. Comparative examples are shown in Supporting Information Figure 7 for UVPD of [DSLNT 2H]2 and UVPD/UVPD of [DSLNT 2H]2• for the process [DSLNT 2H]2 f [DSLNT 2H]• f . The resulting product ions in the latter spectrum have little overlap with the primary fragment ions formed directly from UVPD, with many shifted by 2 Da. This MS/MS/MS data does not provide convincing support that electron photodetachment is the dominant origin of the singly charged fragment ions upon UVPD of the doubly charged precursors.
’ CONCLUSIONS UVPD (193 nm) of deprotonated sialylated oligosaccharides and glycans results in more elaborate fragmentation patterns than those obtained upon CID with far more cross-ring cleavages and internal fragment ions, thus yielding more extensive branching information. UVPD also resulted in several unique fragmentation pathways, such as the loss of 62 Da and the formation of 4,5X-type ions, not observed upon CID. These latter products result from UVPD-specific cleavages of the triol substituent appended to the sialic acid residue. The differences in the fragmentation patterns between CID and UVPD were even more dramatic for doubly deprotonated sialylated oligosaccharides, with UVPD generating singly deprotonated fragment ions, whereas CID produced mainly doubly deprotonated fragment ions. These differences in the CID and UVPD fragmentation patterns are rationalized in part by the fact that energy deposition of UVPD at 193 nm is significantly higher than the average internal energy deposition of CID, as well as the ability to trap the lower m/z fragment ions upon UVPD. The ability to generate diagnostic fragmentation patterns in the negative mode by UVPD offers a streamlined strategy for characterization of acidic oligosaccharides and glycans and alleviates the need for derivatization or metal complexation. The more extensive fragmentation offered by UVPD, as well as the greater array of product ions in the resulting spectra, has the potential to offer great scoring confidence affiliated with database search algorithms in future glycomics applications. ’ ASSOCIATED CONTENT
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Supporting Information. Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected].
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’ ACKNOWLEDGMENT Support from the NSF (CHE-1012622) and the Welch Foundation (F-1155) is acknowledged. ’ REFERENCES (1) Bertozzi, C. R.; Kiessling, L. L. Science 2001, 291, 2357–2364. (2) Dwek, R. A. Chem. Rev. 1996, 96, 683–720. (3) Raman, R.; Raguram, S.; Venkataraman, G.; Paulson, J. C.; Sasisekharan, R. Nat. Methods 2005, 2, 817–824. (4) Bielik, A. M.; Zaia, J. In Functional Glycomics; Li, J., Ed.; Humana Press, New York, NY, 2010; Vol. 600, pp 930. (5) Harvey, D. J. Mass Spectrom. Rev. 2011, 30, 1–100. (6) Asam, M. R.; Glish, G. L. J. Am. Soc. Mass Spectrom. 1997, 8, 987–995. (7) Park, Y. M.; Lebrilla, C. B. Mass Spectrom. Rev. 2005, 24, 232–264. (8) Weiskopf, A. S.; Vouros, P.; Harvey, D. J. Anal. Chem. 1998, 70, 4441–4447. (9) Zhang, Y.; Go, E. P.; Desaire, H. Anal. Chem. 2008, 80, 3144– 3158. (10) Mechref, Y.; Baker, A. G.; Novotny, M. V. Carbohydr. Res. 1998, 313, 145–155. (11) Devakumar, A.; Mechref, Y.; Kang, P.; Novotny, M. V.; Reilly, J. P. Rapid Commun. Mass Spectrom. 2007, 21, 1452–1460. (12) Mechref, Y.; Novotny, M. V.; Krishnan, C. Anal. Chem. 2003, 75, 4895–4903. (13) Harvey, D. J.; Bateman, R. H.; Green, M. R. J. Mass Spectrom. 1997, 32, 167–187. (14) Devakumar, A.; Thompson, M. S.; Reilly, J. P. Rapid Commun. Mass Spectrom. 2005, 19, 2313–2320. (15) Adamson, J. T.; Hakansson, K. Anal. Chem. 2007, 79, 2901–2910. (16) Deguchi, K.; Ito, H.; Baba, T.; Hirabayashi, A.; Nakagawa, H.; Fumoto, M.; Hinou, H.; Nishimura, S. I. Rapid Commun. Mass Spectrom. 2007, 21, 691–698. (17) Hakansson, K.; Chalmers, M. J.; Quinn, J. P.; McFarland, M. A.; Hendrickson, C. L.; Marshall, A. G. Anal. Chem. 2003, 75, 3256–3262. (18) Adamson, J. T.; Hakansson, K. J. Am. Soc. Mass Spectrom. 2007, 18, 2162–2172. (19) Wolff, J. J.; Amster, I. J.; Chi, L. L.; Linhardt, R. J. J. Am. Soc. Mass Spectrom. 2007, 18, 234–244. (20) Wolff, J. J.; Chi, L. L.; Linhardt, R. J.; Amster, I. J. Anal. Chem. 2007, 79, 2015–2022. (21) Alley, W. R.; Mechref, Y.; Novotny, M. V. Rapid Commun. Mass Spectrom. 2009, 23, 161–170. (22) Zhang, Q. B.; Frolov, A.; Tang, N.; Hoffmann, R.; van de Goor, T.; Metz, T. O.; Smith, R. D. Rapid Commun. Mass Spectrom. 2007, 21, 661–666. (23) Zhao, C.; Xie, B.; Chan, S. Y.; Costello, C. E.; O’Connor, P. B. J. Am. Soc. Mass Spectrom. 2008, 19, 138–150. (24) Wolff, J. J.; Laremore, T. N.; Busch, A. M.; Linhardt, R. J.; Amster, I. J. J. Am. Soc. Mass Spectrom. 2008, 19, 790–798. (25) Goldberg, D.; Bern, M.; Li, B. S.; Lebrilla, C. B. J. Proteome Res. 2006, 5, 1429–1434. (26) Li, B. S.; An, H. J.; Hedrick, J. L.; Lebrilla, C. B. In Methods in Molecular Biology, Glycomics: Methods and Protocols; Packer, N. H., Kalsson, N. G., Eds.; Humana Press, New York, NY, 2009; Vol. 534, pp 2335. (27) Seipert, R. R.; Dodds, E. D.; Clowers, B. H.; Beecroft, S. M.; German, J. B.; Lebrilla, C. B. Anal. Chem. 2008, 80, 3684–3692. (28) Seipert, R. R.; Dodds, E. D.; Lebrilla, C. B. J. Proteome Res. 2009, 8, 493–501. (29) Xie, Y. M.; Lebrilla, C. B. Anal. Chem. 2003, 75, 1590–1598. (30) Lancaster, K. S.; An, H. J.; Li, B. S.; Lebrilla, C. B. Anal. Chem. 2006, 78, 4990–4997. (31) Devakumar, A.; Mechref, Y.; Kang, P.; Novotny, M. V.; Reilly, J. P. J. Am. Soc. Mass Spectrom. 2008, 19, 1027–1040. 8199
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dx.doi.org/10.1021/ac201751u |Anal. Chem. 2011, 83, 8192–8200
