Fluorinated Carbon Tag Derivatization Combined with Fluorous Solid

Apr 17, 2015 - Glycan reducing ends were derivatized with a hydrophobic fluorinated carbon tag, increasing glycan ionization efficiency during MS by m...
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Fluorinated Carbon Tag Derivatization Combined with Fluorous Solid-Phase Extraction: A New Method for the Highly Sensitive and Selective Mass Spectrometric Analysis of Glycans Lulu Li,†,§,⊥ Jing Jiao,‡,§ Yan Cai,‡ Ying Zhang,*,† and Haojie Lu*,†,‡ †

Shanghai Cancer Center and Institutes of Biomedical Sciences, and ‡Department of Chemistry and Key Laboratory of Glycoconjugates Research Ministry of Public Health, Fudan University, Shanghai 200032, China S Supporting Information *

ABSTRACT: The sensitive and specific detection of glycans via mass spectrometry (MS) remains a significant challenge due to their low abundance in complex biological mixtures, inherent lack of hydrophobicity, and suppression by other, more abundant biological molecules (proteins/peptides) or contaminants. A new strategy for the sensitive and selective MS analysis of glycans based on fluorous chemistry is reported. Glycan reducing ends were derivatized with a hydrophobic fluorinated carbon tag, increasing glycan ionization efficiency during MS by more than an order of magnitude. More importantly, the fluorinated carbon tag enabled efficient fluorous solid-phase extraction (FSPE) to specifically enrich the glycans from contaminated solutions and protein mixtures. Finally, we successfully analyzed the N-glycome in human serum using this new method.

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peptides) and impurities when applied to complex biological samples due to the relatively weak interactions between the glycans and the solid phase. Glycans are generally derivatized prior to MS analysis to increase their hydrophobicity and enhance their ionization efficiency. The most common derivatization strategies involve permethylation,14 reductive amination,15,16 and hydrazone formation.17 Permethylation converts hydroxyl and carboxyl groups to methyl groups, which increases the hydrophobicity of glycans and also stabilizes the labile sialic acid. However, the numerous wet chemical steps and relatively low reaction efficiencies limit the throughput of this method. Reductive amination and hydrazine derivatization methods are accomplished by conjugating the derivatization reagents to the glycan reducing end. The facile procedure, high yield, and availability of numerous primary amines make reductive amination the predominant derivatization method. For instance, 2-aminobenzamide (2-AB) and 2-aminopyridine (2-AP) have been widely used for glycan derivatization, enhancing the ionization response.18,19 The main disadvantage of these methods is the requirement for a cleanup/enrichment step after derivatization due to the presence of excess derivatization reagents and buffers. This cleanup/enrichment step cannot be omitted because the excess reagents often interfere with MS analysis.

lycosylation is one of the most common post-translational modifications of eukaryotic proteins.1,2 Glycans are polysaccharides that are formed during glycosylation and serve vital roles in many biological processes, such as cell growth and development, intra- and intercellular signaling, and tumor growth and metastasis.3,4 Glycans also represent key disease biomarkers and have been associated with a variety of diseases, such as cancer, inflammation, and degenerative diseases.5 Consequently, glycan profiling is critical for analyzing glycosylation and may be used to understand glycosylation function. Currently, mass spectrometry (MS) has evolved as a robust tool for glycan profiling due to its sensitivity and ability to provide glycan structural information.6−8 However, the low abundance of glycans in complex biological mixtures and suppression of glycans by other, more abundant, biological molecules or contaminants greatly limit their sensitive detection by MS. Moreover, the inherent hydrophilicity of glycans and the lack of basic sites for protonation lead to a particularly poor ionization efficiency. Low glycan abundance makes their enrichment from extremely complex biological samples necessary before MS analysis. Solid-phase extraction (SPE)-based approaches have been widely employed for glycans extraction/purification.9−11 Among these methods, porous graphitized carbon (PGC) and hydrophilic interaction chromatography (HILIC) based SPEs are commonly used.12,13 PGC- and HILIC-based SPE have both demonstrated individual glycan selectivity. However, these approaches usually exhibit low specificity for glycans and lead to the coelution of other biological molecules (proteins or © XXXX American Chemical Society

Received: November 27, 2014 Accepted: April 17, 2015

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Fluorous Derivatization of Glycans. Standard glycans (DP7 or dextran 1000) as well as the glycans from glycoproteins were dissolved in 50% ACN containing 5% HAc (v/v) and were allowed to react with HFUA (the molar ratio of derivatization reagent to glycan was 10:1), and then the reducing reagent NaBH3CN was added. The derivatization reaction was carried out at 65 °C for 2 h. Fluorous Solid-Phase Extraction. Fluorinated carbon derivatized glycans were captured by FluoroFlashNuTips packed with fluorinated carbon-linked silica gel. NuTips were prewashed and equilibrated by aspirating with 20 μL of 100% MeOH three times, then washed with 20 μL of 30% MeOH containing 10 mM ammonium formate five times. The labeled samples were dissolved in 30% MeOH with 10 mM ammonium formate and loaded onto NuTips. Nonspecifically bound proteins, peptides, and salts were removed by washing NuTips with 30% MeOH in 10 mM ammonium formate five times and with 60% MeOH in 10 mM ammonium formate five times. Finally, fluorous tagged glycans were eluted using 100% MeOH as the elution buffer, and then the eluate was dried for further analysis. Matrix-Assisted Laser Desorption Ionization Time-ofFlight Mass Spectrometry Analysis. The dried eluate was dissolved in 50% ACN and 1 μL of sample was spotted on the MALDI plate and dried in the air. Then, 1 μL of matrix (5 mg/ mL CHCA dissolved in 50% ACN with 0.1% TFA) was also spotted on the MALDI plate and dried in the air for MS analysis. Matrix-assisted laser desorption ionization time-offlight (MALDI-TOF) mass spectrometry analysis was performed in positive reflectron mode on a 5800 Proteomic Analyzer (Applied Biosystems, Framingham, MA, U.S.A.) with a Nd:YAG laser at 355 nm, a repetition rate of 400 Hz, and an acceleration voltage of 20 kV. The range of laser energy was optimized, and the laser energy was set as 5500 for further analysis. External mass calibration was performed by using standard peptides from myoglobin digests. GlycoWorkbench software was applied for mass spectrometric data interpretation and glycoform analysis. We also confirmed our results by comparing the assigned glycans from glycoproteins or human serum to those in previous studies.25−27

Therefore, we attempted to develop a one-pipeline strategy for the specific and sensitive glycan analyses, simultaneously improving the ionization efficiency and enriching the glycans selectively. To achieve this goal, we designed an enrichmentoriented tag derivatization method for glycans, accompanied by tag-specific SPE. Perfluoroalkyl compounds are ideal tags due to their unique properties, such as low polarizability, strong hydrophobicity, and good chemical stability. Thus, these unique characteristics make perfluoroalkyl tags ideal derivatization reagents for increasing glycan hydrophobicity. Furthermore, the strong selectivity of perfluoroalkyl groups through their dipole−dipole interactions allows fluorous solid-phase extraction (FSPE) to be used to isolate highly fluorinated targets from nonfluorinated compounds. Taking advantages of these distinct properties, fluorous-based methods have been previously introduced for biochemical applications, such as proteomics and metabolomics analyses. 20−23 Herein, a fluorous-based method was introduced to N-glycomics for the first time, proving to be effective for the highly specific and sensitive analysis of the N-glycome from complex samples.

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EXPERIMENTAL SECTION Materials and Chemicals. Maltoheptaose (DP7, 95%) was purchased from Hayashibara Biochemical Laboratories (Okayama, Japan). Human serum was offered by Shanghai Zhongshan Hospital from a healthy volunteer. Dextran 1000 analytical standard (dextran 1000, 98%), A1 glycan was purchased from Ludger (Oxford, U.K.). α-Cyano-4-hydroxycinnamic acid (CHCA), trifluoroacetic acid (TFA), ammonium bicarbonate (ABC), ammonium formate, trypsin (proteomics grade), ovalbumin (OVA), bovine serum albumin (BSA), sodium cyanoborohydride (NaBH3CN), and Supel-Tips carbon pipette tips were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.). FluoroFlashNuTips and heptadecafluoroundecylamine (HFUA) were purchased from Fluorous Technologies, Inc. (Pittsburgh, PA). Peptide N-glycosidase (PNGase F) was obtained from New England Biolabs (Ipswich, MA, U.S.A.). Sodium chloride (NaCl) was purchased from Shanghai Chemical Reagent Company (Shanghai, China). HPLC-grade methanol (MeOH) and acetonitrile (ACN) were purchased from Merck (Darmstadt, Germany). Analytical-grade acetic acid (HAc) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Distilled water was purified by a Milli-Q system (Milford, MA, U.S.A.). Preparation of Glycans from Glycoproteins. OVA glycoproteins were dissolved in 50 mM ABC buffer (pH 8.0) at a concentration of 2 mg/mL, protein solutions were then treated with PNGase F solution to release the glycans, and the enzymatic deglycosylation reaction was carried out at 37 °C for 18 h. For serum sample preparation, the sample was mixed with an equal volume of 20 mM dithiothreitol solution was denatured under mild conditions as previously described.24 Glycans were released using the procedure described above. Finally, the obtained sample mixture including glycans, proteins, and other contaminates was stored at −20 °C until further use. Glycan permethylation treatment was conducted as follows. Briefly, 10 μg of DP7 (in 0.5 μL water) was suspended in 300 μL of DMSO, to which 30 mg of NaOH powder and 5 μL of methyl iodide were added; this reaction was conducted at room temperature for 10 min. Permethylated glycans were then extracted with chloroform and washed repeatedly with water. Then permethylated sample was dried and resuspended in 50:50 ACN/water solution.

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RESULTS AND DISCUSSION Ionization Efficiency Enhancement by Fluorinated Carbon Tag Derivatization. As shown in Scheme 1, C8F17functionalized amine (heptadecafluoroundecylamine, HFUA) was used as a representative fluorinated carbon tag for glycan derivatization via reductive amination. The perfluoroalkyl group (C8F17) also functioned as an affinity tag, and thus, the fluorinated glycans were retained in fluorous-phase NuTips packed with fluorinated, carbon-linked silica gel, whereas the underivatized substances (i.e., proteins, peptides, and other contaminants) were removed. The fluorinated glycans were then eluted for ultimate MS analysis. A simple glycan maltoheptaose (DP7) was used as a model to first investigate the derivatization reaction. Before derivatization, the DP7 signals were observed as alkali metal adducts, [DP7 + Na]+ (m/ z 1175.2) and [DP7 + K]+ (m/z 1191.2) (Supporting Information Figure S1a). After derivatizing with HFUA, the fluorinated DP7 appeared at m/z 1636.3 ([DP7 + HFUA + Na]+), m/z 1652.3 ([DP7 + HFUA + K]+), and m/z 1614.3 ([DP7 + HFUA + H]+), while the native DP7 signal disappeared, suggesting complete derivatization (Supporting Information Figure S1b). Because perfluoroalkyl-containing B

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1932.7) was derivatized using our method. After derivatization, the MW of fluorous derivatized-A1 is 2392.8 Da in theory. After fluorous derivatization, the loss of sialic acid was also sometimes inevitable in the positive mode of MALDI-MS analysis (Supporting Information Figure S3), corresponding to the observed peaks at m/z 2102.6 [A1 + HFUA − SA + H]+ and at m/z 2124.6 [A1 + HFUA − SA + Na]+. However, this issue could be addressed by analysis of sialylated glycans in the negative mode, with the observed peak at m/z 2391.7 [A1 + HFUA − H]− (Supporting Information Figure S3d). Similar to DP7, the signal intensity of A1 was enhanced dramatically after fluorous derivatization, meaning that our method was applicable to sialylated glycans. Permethylation is one of the most common derivatization methods for MS-based glycan analysis. Compared to the native glycans, permethylated glycans have much higher ionization efficiency. Ionization efficiency of fluorous derivatized glycans and permethylated glycans were compared using model glycan DP7. DP7 glycans were permethylated according to the procedure of Ciucanu and Costello.29 The permethylated DP7 appeared at m/z 1497.7 ([PerDP7 + Na]+) in mass spectrum. We mixed equal amounts of permethylated DP7 and fluorinated DP7 (10 pmol) for MALDI-MS analyses. As shown in Supporting Information Figure S4, the relative intensity for the permethylated glycan [PerMeDP7 + Na]+ was 11.8, while fluorinated DP7 [DP7 + HFUA + H]+, [DP7 + HFUA + Na]+, and [DP7 + HFUA + K]+ were detected with relative intensities of 18.1, 97.8, and 5.1, respectively. The total fluorinated DP7 signal intensity increased over 10-fold relative to permethylated DP7, demonstrating that fluorinated DP7 boasted higher ionization efficiency than the permethylated one. Besides, permethylation has its own inherent limitations. Before permethylation, glycans should be purified from the protein and glycan mixtures while glycans can be directly derivatized in the presence of proteins in our strategy. Therefore, permethylation treatment requires time-consuming and labor-intensive process which certainly will cause sample loss. Our method solved the problem by recovering the glycans using fluorous solid-phase extraction but not liquid−liquid extraction in permethylation. Additionally, as mentioned before, the fluorous derivatization can be applied to sialylated glycans and detection under negative mode to avoid the loss sialic acid (Supporting Information Figure S3). For glycan identification, tandem mass spectrometry (MS/ MS) is demonstrated to be a valuable tool in the structural characterization. Fluorous derivatization will influence the fragmentation patterns of analytes.30 The fragmentation behaviors of native and derivatized glycans were evaluated separately. The MALDI-TOF MS/MS spectra of native DP7 and fluorinated DP7 are shown in Figure 2. For native DP7 without derivatization (Figure 2a), only five fragment ions (m/z 671.6 [B4], 689.6 [Y4], 833.8 [B5], 851.8 [Y5], and 1013.8 [Y6]) are detected (S/N > 3) from the precursor ion [DP7 + Na]+ with low intensity, which gives limited structural information. After derivatization, obviously improved MS/MS signal intensity and spectrum quality are obtained from fluorinated DP7 (precursor ion [DP7 + HFUA + H]+ and [DP7 + HFUA + Na]+). In Figure 2, parts b and c, a series of consecutive fragmentation peaks could be observed due to the successive loss of hexose unit formed through glycosidic bond breakage. MS/MS fragmentation of [DP7 + HFUA + Na]+ provides consecutive B, Y, and A series of product ions which provide more information for spectral interpretation, while

Scheme 1. (a) Illustration of the Fluorous Derivatization of Glycans with Heptadecafluoroundecylamine through Reductive Amination Reaction; (b) Schematic Diagram of the Fluorous Amine-Based Glycan Derivatization, FSPE Enrichment, and MS Analysis

molecules have unique hydrophobic properties, the DP7 signals before and after fluorous derivatization were compared to determine the influence that the fluorous tag had on the glycan ionization efficiency. As shown in Figure 1, equal amounts of

Figure 1. MALDI-TOF mass spectrum of the mixture of equimolar (10 pmol) native DP7 and fluorinated DP7. “∗” denotes [M + H]+ signals, “●” denotes [M + Na]+ signals, and “⧫” denotes [M + K]+ signals.

DP7 and fluorinated DP7 were mixed for MALDI-MS analyses. The relative intensity for the native glycan [DP7 + Na]+ was 2.8, while derivatized [DP7 + HFUA + H]+, [DP7 + HFUA + Na]+, and [DP7 + HFUA + K]+ were detected with relative intensities of 13.5, 96.3, and 11.4, respectively. The total fluorinated DP7 signal intensity increased over 40-fold relative to the native DP7. Additionally, dextran 1000, a branched polysaccharide composed of glucose units with differing degrees of polymerization, was used to further evaluate fluorous tag performance. A degree of polymerization from 4 to 15 was easily detected with enhanced intensities and higher signal-tonoise ratios (S/N) after fluorous tagging (Supporting Information Figure S2b) relative to underivatized samples (Supporting Information Figure S2a). Therefore, the fluorous tag can greatly increase glycan hydrophobicity, and obvious ionization efficiency enhancements were achieved. Due to the lability of sialic acid, the loss of sialic acid under positive mode of MALDI-MS was sometimes observed in the previous report.28 To address the concern in analysis of sialylated glycans, a standard sialylated glycan A1 (MW: C

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Figure 3. MALDI-TOF/TOF tandem mass spectra of a pair of isomeric glycans after HFUA derivatization (m/z 2124.6): (a) the glycan is from OVA with a hybrid structure; (b) the glycan is from ASF with a complex structure.

rather than the complex glycan from ASF. The fragment ion Y3 at m/z 1575.6 appeared in Figure 3b, indicating that the corresponding precursor was the complex glycan from ASF. The above results demonstrated fluorous derivatization effectively improves MS/MS analysis of glycans with enhanced signal intensity and better spectrum quality and could help discriminate isomeric glycans. Selective Glycan Purification and Enrichment via FSPE. In addition to the inherently low ionization efficiency of glycans, their low abundance in complex biological mixtures and their suppression by more abundant biological molecules or contaminants also lead to poor detection sensitivity via MS. Therefore, selective glycan enrichment is also significant for increasing the detection sensitivity. Here, the perfluoroalkyl glycan tags provided an innovative way for glycan enrichment via FSPE. Thus, we investigated the enrichment of poorly abundant glycans from diluted solutions using FSPE. Fluorinated DP7 with concentrations of 0.5 and 0.1 pmol μL−1 were subjected to FSPE. Before enrichment, the S/N for fluorinated DP7 was extremely low (Supporting Information Figure S5, parts a and b). After enrichment with FluoroFlashNuTips, the S/N for fluorinated DP7 increased from 20.9 (Supporting Information Figure S5b) to 556.6 (Supporting Information Figure S5d), and the glycan signals were clearly distinguishable in the mass spectrum. These results indicate that FSPE had good enrichment capabilities for low-abundance fluorinated glycans. We next evaluated whether FSPE could purify glycans from salt buffers. The purification of glycans from complex mixtures of salts and detergents prior to MS detection is critical because the salts and detergents used during sample preparation can interfere with glycans ionization. Supporting Information

Figure 2. MALDI-TOF/TOF tandem mass spectra of native DP7 and fluorinated DP7: (a) [DP7+ Na]+ (precursor ion m/z 1175.2), (b) [DP7 + HFUA + Na]+ (precursor ion m/z 1636.3), and (c) [DP7 + HFUA + H]+ (precursor ion m/z 1614.3).

MS/MS fragmentation of [DP7 + HFUA + H]+ predominantly provides Y series of product ions which simplifies the spectrum. To further verify the ability of the fluorous derivatization to structural interpretation of glycans, a pair of glycan isomers at m/z 1663.6 from two different glycoproteins was analyzed. One from OVA is a hybrid-type glycan, and the other from ASF is a complex-type glycan. After derivatization, the fragmentation patterns of the glycan isomers were compared. Figure 3 displays the MS/MS spectra of the corresponding precursor at m/z 2124.6. The fragment ion Y3α at m/z 1616.8 and Y3γ at m/z 1900.1 only appeared in Figure 3a, indicating that the corresponding precursor was the hybrid glycan from OVA D

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Analytical Chemistry Figure S6 displays the MALDI-TOF mass spectra for the fluorinated DP7 enriched from solutions containing different types of salts. Although the fluorinated DP7 could be directly analyzed, the signal intensities were severely suppressed by the salts (Supporting Information Figure S6, parts a, c, and e). However, after FSPE treatment, high-quality mass spectra could be obtained, even in the presence of high inorganic salt (2.6 M NH4HCO3, Supporting Information Figure S6b, and 6.2 M NaCl, Supporting Information Figure S6d) and chaotrope (2 M urea, Supporting Information Figure S6f) concentrations. Therefore, FSPE could desalt the glycans for effective sample preparation. Furthermore, the feasibility of using FSPE for glycan purification from protein mixtures was evaluated. For real sample analyses, the first step in the downstream glycan analyses of glycoproteins is glycan release. However, the residual proteins often prevent glycan detection. Therefore, extracting glycans from the mixture of residual proteins is essential. A mixture of fluorinated DP7 and BSA (1:10 molar ratio) was used as a model sample. As shown in Figure 4a, while the fluorinated DP7 could still be detected, the S/N was poor (S/N 44.3) due to BSA suppression fraction (Figure 4b, inset). These results indicate that the FSPE had good selectivity for the fluorinated glycans. For comparison, we purified the sample mixture using a traditional graphitized carbon-based enrichment method. Carbon pipet tips packed with a graphitized carbon adsorbent were used to enrich the DP7 glycans from proteins. Before enrichment, no native DP7 signals could be detected (Figure 4c). After enrichment, DP7 signals were observed; however, their signal-to-noise ratios were relatively low (S/N 73.8) (Figure 4d), and the BSA signal was detected in the eluate fraction (Figure 4d, inset). The BSA residue may compete with glycans for ionization. Our comparison between FSPE and carbon-based SPE indicated that fluorous derivatization and FSPE are more advantageous for purifying glycans from proteins. The above results demonstrate that fluorous derivatization can be used to enhance glycan ionization efficiency and that FSPE can be employed to effectively purify fluorinated glycans. With these results, glycans released from glycoproteins underwent direct fluorous derivatization and subsequent purification via FSPE for MS analysis using our one-pipeline strategy. Chicken ovalbumin (OVA), a well-characterized glycoprotein that is glycosylated primarily with high mannose and hybrid glycan structures,32 was used as the model sample. OVA was dissolved in 50 mM ammonium bicarbonate buffer and treated with PNGase F to release the glycans from the glycoprotein core. This mixture, which contained the released glycans, residual protein, and buffer solvent, was dried, redissolved, and derivatized with HFUA. After derivatization, the fluorinated glycans were selectively separated via FSPE. As shown in Figure 5a, direct analyses of the glycans released from OVA without treatment only yielded four glycan peaks with low signal intensities. After fluorous derivatization and FSPE treatment, the derivatized glycans from OVA were effectively enriched and could be sensitively detected (Figure 5b). The number of detected glycans increased from 4 to 24, with enhanced signal intensities. Supporting Information Table S1 overviews the detected fluorinated glycan peaks and their proposed structures. Therefore, the one-pipeline strategy described herein provides a simple and effective means of sensitively detecting glycans from glycoproteins. Glycan Analysis in Serum via Fluorous Derivatization and FSPE Strategy. To further investigate the capabilities of

Figure 4. MALDI-TOF mass spectra of a mixture of fluorinated DP7 and BSA (1:10, molar ratio) (a) directly analyzed without any enrichment, (b) enriched by FSPE, inset shows the detected BSA signal in the eluate fraction and MALDI-TOF mass spectra of the mixture of native DP7 and BSA (1:10, molar ratio), (c) directly analyzed without any enrichment, and (d) enriched by carbon material based SPE, inset shows the detected BSA signal in the eluate fraction. “∗” denotes [M + H]+ signals, “●” denotes [M + Na]+ signals, and “⧫” denotes [M + K]+ signals.

our fluorous derivatization and FSPE strategy for analyzing glycans in real samples, human serum from a healthy volunteer was tested. After treating the serum with PNGase F, the released N-glycans were derivatized with HFUA. The E

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effective glycan derivatization and efficient removal of contaminants, including proteins, lipids, salts etc. This result further indicates that the presented fluorous derivatization and FSPE strategy is successful for highly complex sample analyses. Fluorous derivatization and FSPE, which enhances ionization efficiency of glycans, effectively purifies and enriches glycans. For the first time, fluorous derivatization and purification are introduced for the analyses of glycans using MS. Glycan intensities were greatly enhanced by hydrophobic fluorous derivatization. Additionally, enrichment-oriented fluorous derivatization provides an innovative means for glycan

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CONCLUSIONS

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ASSOCIATED CONTENT

In conclusion, we have developed a novel approach to analyze glycans from complex mixtures and real samples using purification, as FSPE can be used to enrich and desalt lowabundant glycans. Notably, FSPE was used to purify and isolate fluorinated glycans from protein mixtures; therefore, glycans released from glycoproteins can be fluorous derivatized directly and purified by FSPE using the one-pipeline strategy. In recent years, some novel and effective methods have been developed to improve the MS detection of glycans from glycoproteins.9−11,27,31 For example, taking advantage of the size exclusion effect of the mesoporous silicon to proteins and the specific interaction between carbon and oligosaccharides, mesoporous silica−carbon composite nanoparticles were prepared and utilized to enrich N-linked glycans from complex biological samples with high selectivity and efficiency.10,11,27 Also, based on the conjugation of glycans to hydrazide beads through the formation of reversible bond (e.g., hydrazine), captured glycans can be specifically released after washing out unbound nonglycans.9 Compared with the enrichment method for native glycans, our method realized a combination of enrichment-oriented fluorous derivatization and FSPE, which showed particular merits. First, signals of glycans were significantly enhanced after fluorous derivatization; second, improved MS/MS signal intensity could be obtained after derivatization thereby facilitating the structural interpretation; third, the tolerance of contaminants such as salt (2.6 M NH4HCO3 and 6.2 M NaCl) and chaotrope (2 M urea) were investigated by our method which were not included in previous reports. High-quality mass spectra could be obtained after FSPE treatment, even in the presence of high inorganic salt or chaotrope. Moreover, this one-pipeline workflow combined with the commercial availability of fluorinated carbon tag and FluoroFlashNuTips avoids the tedious and time-consuming preparation of enrichment materials in above methods, making our method has great potential to be accessible to other laboratories. In a word, a new method for the highly sensitive and selective mass spectrometric analysis of glycans by fluorinated carbon tag derivatization and FSPE was established and evaluated, showing great application prospect in MS-based glycomics analysis.

Figure 5. MALDI-TOF mass spectra of N-glycans released from 40 pmol OVA (a) directly analyzed without any enrichment and (b) derivatized with HFUA and enriched by FSPE using the one-pipeline strategy. “∗” denotes [M + H]+ signals, “●” denotes [M + Na]+ signals, and “☆” denotes [M + H − H2O]+ signals.

unconjugated molecules present in the complex sample were then removed via FSPE, and ultimately, the fluorinated glycans (corresponding to 0.25 μL of serum) were eluted for MALDITOF MS analyses. As shown in Figure 6, the fluorous derivatization and FSPE strategy detected 34 derivatized Nglycans spanning all three N-glycosylation forms (high mannose, complex, and hybrid structures). The observed derivatized glycan signals and their proposed structures are presented in Supporting Information Table S2. The successful detection of N-glycans from serum was attributed to the

S Supporting Information *

Figure 6. MALDI-TOF mass spectrum of N-glycans identified from 0.25 μL of human serum after HFUA derivatization and FSPE treatment in one-pipeline strategy. “●” denotes [M + Na]+ signals, “★” denotes [M + Na − H2O]+ signals, and “☆” denotes [M + H − H2O]+ signals of fluorinated glycans.

Additional information as noted in text. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ac504437h. F

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(25) Aldredge, D. H.; An, J.; Tang, N.; Waddell, K.; Lebrilla, C. B. J. Proteome Res. 2012, 11, 1958−1968. (26) An, H. J.; de Leoz, M. L.; Lebrilla, C. B.; Miyamoto, S.; Leiserowitz, G. S. Proteomics 2009, 9, 2986−2994. (27) Sun, N. R.; Deng, C. H.; Li, Y.; Zhang, X. M. Anal. Chem. 2014, 86, 2246−2250. (28) Nie, H.; Li, Y.; Sun, X. L. J. Proteomics 2012, 75, 3098−3112. (29) Ciucanu, I.; Costello, C. E. J. Am. Chem. Soc. 2003, 125, 16213− 16219. (30) Li, Y.; Arigi, E.; Eichert, H.; Levery, S. B. J. Mass Spectrom. 2010, 45, 504−519. (31) Zhang, W.; Han, H.; Bai, H.; Tong, W.; Zhang, Y.; Ying, W.; Qin, W.; Qian, X. Anal. Chem. 2013, 85, 2703−2709. (32) Harvey, D.; Wing, D.; Küster, B.; Wilson, I. J. Am. Soc. Mass Spectrom. 2000, 11, 564−571.

AUTHOR INFORMATION

Corresponding Authors

*Phone: +86 21 54237618. Fax: +86 21 54237961. E-mail: [email protected]. *Phone: +86 21 54237618. Fax: +86 21 54237961. E-mail: [email protected]. Present Address ⊥

L.L.: Shanghai Applied Protein Technology Co. Ltd., Shanghai 200233, China.

Author Contributions §

L.L. and J.J. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work was supported by NST (2012CB910602 and 2012AA020203), NSF (21335002 and 21375026), MOE (20130071110034), and Shanghai Projects (Eastern Scholar, Rising star 15QA1400600 and B109).

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REFERENCES

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DOI: 10.1021/ac504437h Anal. Chem. XXXX, XXX, XXX−XXX