High-Throughput Quantitative Analysis of Total N-Glycans by Matrix

Mar 23, 2012 - Institute of Molecular Biology and Genetics and Institute of Bioengineering, Seoul National University, Shillim-dong, Seoul, 151-742,. ...
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High-Throughput Quantitative Analysis of Total N-Glycans by MatrixAssisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Hee-Jin Jeong,† Yun-Gon Kim,§ Yung-Hun Yang,# and Byung-Gee Kim*,†,‡,¶ †

School of Chemical and Biological Engineering in College of Engineering, Seoul National University, Shillim-dong, Seoul, 151-742, Korea ‡ Institute of Molecular Biology and Genetics and Institute of Bioengineering, Seoul National University, Shillim-dong, Seoul, 151-742, Korea ¶ Institute of Bioengineering, Seoul National University, Shillim-dong, Seoul, 151-742, Korea § Department of Chemical Engineering, College of Engineering, Soongsil University, 511 Sangdo-dong, Seoul 156-743, Republic of Korea # Department of Microbial Engineering, College of Engineering, Konkuk University, Seoul, Republic of Korea S Supporting Information *

ABSTRACT: Accurate and reproducible quantification of glycans from protein drugs has become an important issue for quality control of therapeutic proteins in biopharmaceutical and biotechnology industries. Mass spectrometry is a promising tool for both qualitative and quantitative analysis of glycans owing to mass accuracy, efficiency, and reproducibility, but it has been of limited success in quantitative analysis for sialylated glycans in a high-throughput manner. Here, we present a solid-phase permethylation-based total N-glycan quantitative method that includes N-glycan releasing, purification, and derivatization on a 96-well plate platform. The solid-phase neutralization enabled us to perform reliable absolute quantification of the acidic Nglycans as well as neutral N-glycans from model glycoproteins (i.e., chicken ovalbumin and porcine thyroglobulin) by only using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Furthermore, low-abundance sialylated N-glycans from human serum prostate specific antigen (PSA), an extremely valuable prostate cancer marker, were initially quantified, and their chemical compositions were proposed. Taken together, these results demonstrate that our allinclusive glycan preparation method based on a 96-well plate platform may contribute to the precise and reliable qualitative and quantitative analysis of glycans.

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preparation and little or no fragmentation of the parent molecules during desorption.6−10 There is a need for a high-throughput sample preparation method for analysis of large number of various glycan samples by using MS. Many investigators have developed 96-well platebased methods for high-throughput glycan preparation. Royle et al. have demonstrated a N-glycan preparation method that includes glycan releasing and purification,11 combined with

ukaryotic cell surfaces are highly rich in glycans which have significant roles in biological and immunological systems.1,2 The qualitative and quantitative information of glycans would be critical to correctly understand their roles in vivo. To characterize the cell surface glycans, mass spectrometry has been widely applied, owing to its ability of providing the information of chemical structures and detecting low abundant glycans with high sensitivity and resolution.3−5 Especially, the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been steadily increasing for its advantages such as convenient sample © 2012 American Chemical Society

Received: August 16, 2011 Accepted: March 12, 2012 Published: March 23, 2012 3453

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Technical Note

7.0) was added and then incubated at 37 °C for 16 h. The released N-glycans were eluted from the PVDF membrane plate by adding 200 μL of water to each well and then mixed with the oligosaccharide standard mixture consisting of 10 pmol of maltotetraose, 15 pmol of maltopentaose, and 20 pmol of maltohexaose. Purification of N-Glycans. The released N-glycans and standard oligosaccharides were directly loaded onto a PGC containing 96-well plate (Waters, Milford, MA, USA) to remove the salts and other environmental contaminants. The solvent system was as follows: First, the well was prewashed with 200 μL of 30% [v/v] acetic acid/water followed 200 μL of 0.1% [v/v] trifluoroacetic acid (TFA) in 50% [v/v] acetonitrile (ACN)/water. Continually, the well was primed by 200 μL of 0.1% [v/v] TFA in 5% [v/v] ACN/water, and then, the Nglycans were loaded onto the well. Subsequently, the well was washed with 200 μL of water and 200 μL of 0.1% [v/v] TFA in 5% [v/v] ACN/water continuously. The N-glycans were eluted into the collection plate with 200 μL of 0.1% [v/v] TFA in 50% [v/v] ACN/water.23,24 The collected eluent was transferred to an eppendorf tube and then dried in a centrifugal vacuum concentrator (Hanil Research and Development, puchon, Korea). High-Throughput Solid-Phase Permethylation. Permethylation was performed using a 96-well plate-based solid-phase permethylation method, which is modified from the one-by-one solid-phase permethylation method.14 Briefly, a micro 96-well plate (Harvard, Holliston, MA, USA) was filled with NaOH beads (20−40 mesh) which were prepared in ACN, and then, ACN was discarded by suction. Next, the well was thoroughly washed with dimethyl sulfoxide (DMSO), and then, the sample which is suspended in 141.6 μL of DMSO, 52.8 μL of iodomethane (CH3I), and 5.6 μL of water was loaded and passed through the NaOH beads. This step was repeated eight times to minimize the sample loss. The salt contaminants were removed by adding 100 μL of ACN, and the samples were eluted into the collection plate by suction and then transferred to a 1.5 mL eppendorf tube. The permethylated glycans were extracted with 200 μL of chloroform and washed with water until the pH of the aqueous layer became 6−7. Finally, the chloroform was evaporated in a centrifugal vacuum concentrator. Quantitative MALDI-TOF MS Analysis. The permethylated N-glycans were dissolved with 50 μL of 50% [v/v] methanol/water. This was mixed with 2,5-dihydroxybenzoic acid (Sigma, St. Louis, MO) solution (10 mg/mL in 50% [v/v] methanol/water) and then spotted on a stainless-steel MALDI plate and dried at room temperature.23 A Bruker Daltonics Biflex IV MALDI-TOF MS equipped with a 337 nm nitrogen laser (Bruker, Bremen, Germany) was used to obtain mass spectra. The analysis parameters were as follows: positive ion and reflectron mode, detector gain = 3.9, and laser power = 70%. A total of 500 shots from 25 different spots were scanned to acquire mass spectrum data. The intensity of each ion was obtained by integrating from first to third isotopic peaks area. Data acquisition and processing were performed with Bruker X-TOF 5.1.1 (Bruker, Bremen, Germany) and flexAnalysis 2.4 software (Bruker, Bremen, Germany). Glycan cartoons representing mass peaks were built using GlycanBuilder ver. 1.2.3480.

high performance liquid chromatography (HPLC) analysis by labeling with fluorescent 2-aminobenzamide. However, the conventional quantification approach using HPLC is difficult to identify the minor glycan which can be detected in MS.12 More recently, Lee et al. introduced a high-throughput glycan profiling technique using an automated fluorescent DNA sequencer.13 The obtained N-glycans by deglycosylation of plant derived-glycoproteins were labeled using 8-amino-1,3,6pyrenetrisulfonic acid and then analyzed on a DNA sequencer. However, since the DNA sequencer is able to only read the sequence of about 1000 bp, the long sequenced glycans (e.g., animal-derived glycans) have to be divided into subsequenced glycans. Despite the various high-throughput glycan analysis methods proposed, a more facile and versatile strategy for total N-glycan analysis is still in great demand. In our previous study, we demonstrated a rapid sample preparation strategy in which N-glycans were released on a polyvinylidene fluoride (PVDF) 96-well plate and then purified using a porous graphitized carbon (PGC) 96-well plate.14 After that, purified N-glycans were derivatized with carboxymethyl trimethylammonium hydrazide (Girard’s reagent T, GT) for quantification using MALDI-TOF MS. However, the GT labeling method has a limitation for the quantitative analysis of negatively charged N-glycans such as sialic acid-containing oligosaccharides.15 Recently, Kang et al. reported a solid-phase permethylation technique using a micro spin column for a quantitative analysis of neutral and sialylated glycan.16,17 This novel permethylation technique is easier and faster for sample preparation than the traditional liquid-extract permethylation approach. It could dramatically reduce peeling reactions and oxidative degradations that are problematic in the solution-phase permethylation approach. Here, we present an improved high-throughput N-glycan preparation method that enables N-glycan liberation to permethylation using a stack of three 96-well plates (i.e., PVDF, PGC, and sodium hydroxide plates). N-glycans from chicken ovalbumin glycoprotein and porcine thyroglobulin glycoprotein have been identified by using this high-throughput manner. Moreover, the absolute quantity of total N-glycans including neutral and sialylated oligosaccharides was determined using internal standards. Finally, we applied this technique to confirm the N-glycan from human serum prostate specific antigen (PSA). To the best of our knowledge, this study presents a first attempt to develop an in situ N-glycan preparation method with a high-throughput manner.

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MATERIALS AND METHODS Release of N-Glycans from Glycoproteins. Different amounts (50, 125, 250, and 500 μg) of glycoproteins (i.e., chicken ovalbumin and porcine thyroglobulin, Sigma, St. Louis, MO, USA) and 25 μg of human serum PSA (Sigma, St. Louis, MO, USA) were dissolved with 50 μL of distilled water and then denatured on a PCR 96-well plate (Axygen, California, CA, USA) by heating at 95 °C for 3 min at thermocycler (BioRad, California, CA, USA). After cooling down at room temperature, the samples were loaded onto a 96-well PVDF membrane filter plate (Millipore, Billerica, MA, USA) which was prewashed with 200 μL of 70% [v/v] ethanol/water followed 200 μL of water. After equilibrating the well with 200 μL of 50 mM sodium phosphate buffer (pH 7.0), 5 units of peptide N-glycosidase F (PNGase F, Roche, Mannheim, Germany) in 50 μL of 50 mM sodium phosphate buffer (pH 3454

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Figure 1. Overall strategy of total N-glycan analysis based on 96-well plate platform.

Figure 2. MALDI-TOF MS spectra of (A) standard mixture and (B) permethylated standard mixture; M, oligosaccharide of standard mixture mixture (10 pmol of permethylated maltotetraose, 15 pmol of permethylated maltopentaose, and 20 pmol of permethylated maltohexaose); M′, oligosaccharide of permethylated standard mixture; lines between the spectra indicate changes in mass after permethylation.

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RESULTS AND DISCUSSION

dithiothreitol and iodoacetic acid, heat denaturation has been applied, because the remained chemicals can affect the enzyme activity for deglycosylation and interfere with the subsequent MS analysis. After the denatured samples were transferred to a PVDF membrane filter 96-well plate, PNGase F was added to liberate N-glycans from glycoprotein. The released N-glycans

Strategy for High-Throughput N-Glycan Analysis. The comprehensive workflow of total N-glycan analysis is shown in Figure 1. The glycoproteins were applied onto a PCR 96-well plate and then denatured by heating with a thermocycler. Instead of chemical denaturation of the proteins using 3455

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Figure 3. MALDI-TOF MS spectra of permethylated N-glycans from (A) 500 ng, (B) 1 μg, (C) 2.5 μg, and (D) 5 μg of chicken ovalbumin glycoprotein with the addition of internal standard mixture; (E) calibration curve of internal standard mixture; (F) linear relationship between amount of chicken ovalbumin glycoprotein and peak intensity which was majorly observed in MALDI-TOF MS spectra; asterisk denotes internal standard mixture (∗, 10 pmol of permethylated maltotetraose; ∗∗, 15 pmol of permethylated maltopentaose; ∗∗∗, 20 pmol of permethylated maltohexaose).

mixed with internal standard oligosaccharides were purified using a PGC packed 96-well plate and then subsequently permethylated in the 96-well plate packed with solid NaOH. These two steps, purification and solid-permethylation, were carried out in less than 1 h, and up to 96 samples were treated at the same time. Finally, the permethylated N-glycans were followed by MALDI-TOF MS analysis. All the procedures from glycan release to MALDI-TOF MS analysis were carried out within 2 days, demonstrating its capability of rapid preparation of a large number of samples. Mass Profiling of N-Glycans from Glycoproteins. The MALDI spectra of N-glycans derived from chicken ovalbumin glycoprotein and porcine thyroglobulin glycoprotein are shown

in Supplementary Figure 1 (Supporting Information), and the identified peaks are summarized in Supplementary Tables 1 and 2 (Supporting Information), respectively. As shown in Supplementary Figure 1A (Supporting Information), 24 of the N-glycans were clearly observed from 100 μg (100 pmol) of chicken ovalbumin glycoprotein, which correspond to the previous reports.18−20 Moreover, 23 of the N-glycans from 5 μg (7 pmol) of porcine thyroglobulin glycoprotein were identified (Supplementary Figure 1C, Supporting Information) and matched with the previously reported N-glycan structures of porcine thyroglobulin.21,22 In the case of the sensitivity, Nglycan profiles of chicken ovalbumin and porcine thyroglobulin could be obtained with up to a 500 ng amount of glycoprotein 3456

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Figure 4. MALDI-TOF MS spectra of permethylated N-glycans from (A) 500 ng, (B) 1 μg, (C) 2.5 μg, and (D) 5 μg of porcine thyroglobulin glycoprotein with the addition of internal standard mixture; (E) calibration curve of internal standard mixture; (F) the linear relationship between amount of porcine thyroglobulin glycoprotein and peak intensity that was majorly observed in MALDI-TOF MS spectra; asterisk denotes internal standard mixture (∗, 10 pmol of permethylated maltotetraose; ∗∗, 15 pmol of permethylated maltopentaose; ∗∗∗, 20 pmol of permethylated maltohexaose).

Figure 3A−D shows the MALDI spectra of permethylated Nglycans from chicken ovalbumin glycoproteins (500 ng, 1 μg, 2.5 μg, and 5 μg) spiked with the equivalent internal standards. As shown in the Figure 3E, the calibration curve of the internal standard mixture showed a good linearity according to the concentration of the three oligosaccharides (maltotetraose/ maltopentaose/maltohexaose = 2:3:4), and as shown in the Figure 3F, the ratio of peak areas of each glycan obtained from Figure 3A−D was close to 1:2:5:10, corresponding to the proportion of protein concentrations. The linearity of the Nglycans is shown in Figure 3F (R2 values are greater than 0.99). To determine the absolute amount of N-glycans, we calculated

(Supplementary Figure 1B,D, Supporting Information), suggesting that this approach enables one to identify the Nglycans from a picomole scale of glycoprotein. Absolute Quantitative Analysis of Glycans Using Internal Standard. When we analyzed three different concentrations of the standard oligosaccharide mixture (i.e., 10 pmol of maltotetraose, 15 pmol of maltopentaose, and 20 pmol of maltohexaose), the MALDI spectra showed only [M + Na]+ ions not [M + H]+ or [M + K]+ ions (Figure 2). Consequently, it simplified the overall MALDI profile and also facilitated the quantitative analysis from the peak area in MALDI-TOF MS. 3457

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Figure 5. MALDI-TOF MS spectra of permethylated N-glycans from 25 μg of human serum PSA. (A) Solid-phase permethylated N-glycans; (B) liquid-extract permethylated N-glycans.

weight N-glycans,25 is an important biomarker for prostate cancer,26,27 since the concentrations of PSA increase in prostate cancer.28 In particular, as the change in glycosylation is reflected cancer progression,29 a characterization of the glycan of PSA is required to discriminate prostate cancer. Several studies have been performed to identify the glycan constituents of PSA by 1 H-nuclear magnetic resonance spectroscopy,30 oligosaccharide sequencing,31 and HPLC.32−34 However, because these techniques require large amounts of sample for glycan analysis, only the major components of glycans could be resolved. Moreover, since there are low concentrations of PSA in human serum (approximately 0.1−2.6 ng/mL35), a more sensitive approach is required to characterize PSA. Recently, a more detailed structural characterization of the N-glycan of the PSA was carried out with MS.36,37 However, this approach did not clearly show the quantitative glycan components. Therefore, we applied the high-throughput N-glycan profiling method for the qualitative and quantitative analysis of total N-glycans from human serum PSA. As a result, 49 putative compositions of Nglycans were identified from 1 nmol of human serum PSA and the relative quantity of the component glycans were estimated from peak areas of each N-glycan among the areas of the total N-glycans (Figure 5A, Table 1). In this study, human serum PSA were characterized as having some high-mannose type glycans (16.1%) and mostly complex type glycans, including sialylated complex type (6.4%), sialylated fucosyl complex type (12.4%), and desialylated

the mole concentration of each N-glycan using the internal standard calibration curve (Supplementary Table 1, Supporting Information). Overall, 1.18 mol of N-glycans is included in 1 mol of chicken ovalbumin, which is slightly higher than the previously published result.12 As shown in Figure 4 and Supplementary Table 2 (Supporting Information), N-glycans released from porcine thyroglobulin were also quantified absolutely with the internal standard mixture, and the peak areas from major four peaks (peak numbers 5, 6, 9, 13) were plotted in Figure 4F, which demonstrates the linearity between the amount of glycoprotein and peak area of each glycan. The summed N-glycans per 1 mol of porcine thyroglobulin was 36.2 mol. Moreover, it is confirmed that the amount of sialylated N-glycan per 1 mol of porcine thyroglobulin glycoprotein is 14.2 mol (39.2%) in similar proportions to those seen previously. Although the 96well format method has been validated with a three point calibration in here, the number of glycosylation sites was reliably identified with this quantitative analysis. Moreover, according to the results, we could theoretically estimate limit of detection (LOD) and limit of quantitation (LOQ) from “leastsquares analysis” (Supplementary Result, Supporting Information). Therefore, the reproducibility and reliability of our highthroughput method was successfully demonstrated by using well-known glycoproteins. Relative Quantification of N-Glycans from Human Serum PSA. PSA, a glycoprotein with 8.3% of its molecular 3458

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Table 1. Identification of N-Glycan of Human Serum PSA

results demonstrated more detailed qualitative and quantitative information of N-glycans from PSA that has not been previously reported.37 Moreover, when sialylated glycan components of solid-phase permethylated N-glycan spectra from 25 μg of human serum PSA (Figure 5A) were compared with those of liquid-extract permethylated N-glycan spectra from 25 μg of human serum PSA (Figure 5B), sialylated glycans (permethylated m/z = 1951.6, 1981.5, 2155.6, 2185.5, 2313.6, 2360.6, 2400.6, 2430.6, 2604.5, 2645.8, 2909.5, 2966.5, and 3007.6) were more detected at solid-phase permethylated Nglycan spectra (total component of sialylated glycans was 18.8%) than liquid-extract permethylated N-glycan spectra (total component of sialylated glycans was 1.2%). It indicates that the solid-phase permethylation is able to more stably preserve sialic acid from peeling reactions and oxidative degradations that occur in a traditional permethylation approach. Thus, these results show that our MALDI-TOF MS-based N-glycan analysis method is highly sensitive and accurate for N-glycan profiling.

compositionsb peak no.

[M + Na] m/za

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49

1171.6 1345.5 1375.5 1416.6 1519.5 1549.5 1579.5 1590.5 1620.5 1661.6 1753.5 1764.5 1783.5 1794.5 1824.5 1835.5 1865.5 1927.5 1951.6 1968.5 1981.5 1998.6 2028.6 2039.6 2069.6 2080.6 2155.6 2172.6 2185.5 2213.6 2226.6 2243.6 2284.6 2313.6 2346.6 2360.6 2387.6 2400.6 2417.6 2430.6 2518.6 2591.6 2604.5 2621.5 2645.8 2766.5 2909.5 2966.5 3007.6

+

H

HN

3 3 4 3 3 4 5 3 4 3 5 3 6 4 5 3 4 5 3 4 4 5 6 4 5 3 4 5 5 4 4 5 4 3 5 5 4 4 5 5 6 5 5 6 4 5 6 5 4

2 2 2 3 2 2 2 3 3 4 2 3 2 3 3 4 4 2 3 3 3 3 3 4 4 5 3 3 3 4 4 4 5 3 3 3 4 4 4 4 5 4 4 4 5 4 5 4 5

dH NA 0 1 0 0 2 1 0 1 0 0 1 2 0 1 0 1 0 2 1 2 0 1 0 1 0 1 1 2 0 2 1 1 1 1 3 1 3 1 2 0 0 3 1 2 1 4 0 1 1

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 1 0 1 0 0 0 0 2 0 1 0 1 0 1 0 0 1 0 1 0 0 2 2

NG

percentage of total [%]

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 total

4.7 1.8 6.4 1.0 0.5 0.6 7.6 3.2 1.6 0.9 0.6 1.4 2.1 2.3 2.2 0.7 1.0 1.6 1.0 1.3 1.3 1.5 0.4 1.8 1.6 0.4 1.3 4.2 2.8 1.0 0.7 3.9 0.6 1.1 1.2 0.5 1.3 0.7 4.8 1.6 0.9 12.4 4.4 0.7 0.4 2.2 0.7 2.1 0.7 100.0

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CONCLUSIONS This paper has focused on the development of a highthroughput N-glycan preparation method for reproducible qualitative and quantitative analysis. The multistack 96-well plates were able to release total N-glycans from picomoles of glycoproteins and subsequently neutralize them for absolute and relative quantitative analysis by MALDI-TOF MS. This work showed the potential application of glycosylation analysis for disease diagnosis with a high-throughput manner. As a supplementary tool, exoglycosidase digestion coupled MALDITOF MS would be applied to this system to provide more detailed structural information such as specific glycosyl-linkages (alpha- or beta-) and sequences. In the case of glycosylated recombinant proteins used for therapeutic applications, this unbiased and systematic glycomics technology will afford a valuable approach to verify biosimilarity in protein drugs.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +82-2-880-6774. Fax: +822-883-6020. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS H.-J.J. and Y.-G.K. contributed equally to this work as first authors. This research was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (No. 20090083035) and World Class University program (R322009000102130). This research was supported by a grant (M10417060004-04N1706-00410) from Korea Biotech R&D Group of Next-generation growth engine project of the Ministry of Education, Science and Technology, Republic of Korea. This work was also supported by Basic Science Research Program (2010-0009942) through the National Research Foundation grant funded by the Korean Government (MEST).

a Permethylated mass value. bH, hexose; HN, N-acetylhexosamine; dH, Deoxyhexose; NA, N-acetylneuraminic acid; NG, N-glycolylneuraminic acid.

fucosyl complex type (50.7%). Interestingly, these glycans compositions are in good agreement with those obtained previously37 and showed excellent reliability and reproducibility with better resolution and higher sensitivity. Furthermore, our 3459

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Technical Note

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dx.doi.org/10.1021/ac203440c | Anal. Chem. 2012, 84, 3453−3460