Fully Automated Chip-Based Mass Spectrometry ... - ACS Publications

Mar 4, 2004 - Rapid and sensitive characterization of carbohydrates in biological systems by automated nanoscale liquid delivery and chip-based ...
0 downloads 0 Views 207KB Size
Download PDF
Anal. Chem. 2004, 76, 2046-2054

Fully Automated Chip-Based Mass Spectrometry for Complex Carbohydrate System Analysis Alina Zamfir,† Sergey Vakhrushev,† Alistair Sterling,‡ Hans Jo 1 rg Niebel,‡ Mark Allen,‡ and ,† Jasna Peter-Katalinic´*

Biomedical Analysis, Institute for Medical Physics and Biophysics, University of Mu¨nster, Germany, and Advion BioSciences, Ltd., Norwich, United Kingdom

Carbohydrates represent a major class of biopolymers, which occur in nature either as oligosaccharides or glycoconjugates, in which the sugar moiety is linked to proteins or lipids. The significance of mass spectrometry for highly sensitive analysis of complex carbohydrates increased after the introduction of the electrospray ionization and matrix assisted laser desorption/ionization methods and the possibility of tandem MS for sequencing of single molecular species in complex mixtures. Rapid and sensitive characterization of carbohydrates in biological systems by automated nanoscale liquid delivery and chipbased electrospray interface techniques have not been developed so far. In this contribution, the implementation and optimization of a fully automated chip-based nanoelectrospray assembly (NanoMate system), operating in the negative ion mode, in combination with QTOF-tandem MS for mapping/sequencing and computer-assisted structure assignment for carbohydrate components in complex mixtures is presented. The actual trends in analytical science toward high-throughput analysis are focused on the development of nanotechnology in automatization and miniaturization of devices.1 In biosciences, integrated, fully automated micro- and nanosystems have been demonstrated to contribute significantly toward sensitivity and speed of the analysis.2,3 Microfluidic devices and chip-based technology-driven developments have generated massive efforts for routine introduction of the “lab-on-a-chip” principle in mass spectrometry. Technological breakthrough and fair analytical perspectives of such automated micro- and nanosystems in MS have already been demonstrated for direct bioanalysis of drugs,3 peptide mapping and sequencing for protein identification and quantification,4 noncovalent protein-ligand interactions,5,6 quantitative determination of noncovalent binding interactions,7 on-chip * Corresponding author. Phone: +49-251-8352308. Fax: +49-251-8355140. E-mail: [email protected]. † University of Mu ¨ nster. ‡ Advion BioSciences, Ltd.. (1) Thorsen, T.; Maerkl, S. J.; Quake, S. R. Science 2002, 298, 580-584. (2) Groisman, A.; Enzelberger, M.; Quake, S. R. Science 2003, 300, 955-958. (3) Dethy, J. M.; Ackermann, B. L.; Delatour, C.; Henion, J. D.; Schultz, G. A. Anal. Chem. 2003, 75, 805-811. (4) Li, J.; LeRiche, T.; Tremblay, T.-L.; Wang, C.; Bonneil, E.; Harrison, D. J.; Thibault, P. Mol. Cell. Proteomics 2002, 1, 157-168. (5) Sjo ¨dahl, J.; Melin, J.; Griss, P.; Emmer, A.; Stemme, G.; Roeraade, J. Rapid Commun. Mass Spectrom. 2003, 17, 337-341.

2046 Analytical Chemistry, Vol. 76, No. 7, April 1, 2004

proteolytic digestion,8 drug development,9 and small molecule analysis.10 Structure elucidation of N- and O-linked glycans,11-14 glycopeptides,15,16 glycoproteins17 and glycolipids18,19 by mass spectrometric methods using ESI-QTOF are well-established. However, the discrepancy between the complexity of the carbohydrate structures and variants existing in nature and the limited amount available from biological matrixes calls for a continuous improvement of mass spectrometric techniques in terms of sensitivity, accuracy, and reproducibility. Moreover, for the modern glycomics field, unlike for proteomics, novel strategies to increase the throughput by providing rapid and sensitive characterization of carbohydrates in biological systems by automated nanoscale liquid delivery and chip-based electrospray interface techniques have not been developed so far. In this context, the implementation and optimization of a high-throughput strategy comprising a fully automated chip-based nanoelectrospray robot (NanoMate system) operating in the negative ion mode in combination with QTOF-tandem MS for mapping and sequencing of carbohydrate components in complex mixtures and computerized assignment of detected species is here presented. By optimizing the NanoMate assembly for sugar ionization and (6) Benkestock, K.; Van Pelt, C. K.; Akerud, T.; Sterling, A.; Edlund, P. O.; Roeraade, J. J. Biomol. Screening 2003, 8, 247-256. (7) Zhang, S.; Van Pelt, C. K.; Wilson, D. B. Anal. Chem. 2003, 75, 30103018. (8) Lazar, I. M.; Ramsey, R. S.; Ramsey, J. M. Anal. Chem. 2001, 73, 17331739. (9) Weigl, B. H.; Bardell, R. L.; Cabrera, C. R. Adv. Drug Delivery Rev. 2003, 55, 349-377. (10) Kameoka, J.; Craighead, H. G.; Zhang, H.; Henion, J. Anal. Chem. 2001, 73, 1935-1941. (11) Zamfir, A.; Seidler, D. G.; Kresse, H.; Peter-Katalinic´, J. Rapid Commun. Mass Spectrom. 2002, 16, 2015-2024. (12) Leitao, E. A.; Bittencourt, V. C.; Haido, R. M.; Valente, A. P.; Peter-Katalinic´, J.; Letzel, M.; De Souza, L. M.; Barreto-Bergter, E. Glycobiology 2003, 10, 681-692. (13) Zamfir, A.; Seidler, D. G.; Kresse, H.; Peter-Katalinic´, J. Glycobiology 2003, 13, 733-742. (14) Sˇ agi, D.; Peter-Katalinic´, J.; Conradt, H. S.; Nimtz, M. J. Am. Soc. Mass Spectrom. 2002, 13, 1138-1148. (15) Macˇek, B.; Hofsteenge, J.; Peter-Katalinic´, J. Rapid Commun. Mass Spectrom. 2001, 15, 771-777. (16) Hofsteenge, J.; Huwiler, K. G.; Macˇek, B.; Hess, D.; Lawler, J.; Mosher, D. F.; Peter-Katalinic´, J. J. Biol. Chem. 2001, 276, 6485-6498. (17) Hanisch, F. G.; Jovanovic´, M.; Peter-Katalinic´, J. Anal. Biochem. 2001, 290, 47-59. (18) Metelmann, W.; Peter-Katalinic´, J.; Mu ¨ thing, J. J. Am. Soc. Mass Spectrom. 2001, 12, 964-973. (19) Vukelic´, Zˇ .; Metelmann, W.; Mu ¨ thing, J.; Kos, M.; Peter-Katalinic´, J. Biol. Chem. 2001, 382, 259-74. 10.1021/ac035320q CCC: $27.50

© 2004 American Chemical Society Published on Web 03/04/2004

sequence requirements, we have developed a solid methodology that is advantageous in comparison with the capillary-based nanoESI. Analytical potential of this advanced system was explored on complex carbohydrate mixtures from urine of two patients. To test the feasibility of the NanoMate device for carbohydrate ionization and sequencing, we first investigated a mixture of O-glycosylated sialylated amino acids and peptides which was identified by mass spectrometry and described by us before.20-22 The sample was obtained from urine of a patient suffering from a hereditary N-acetyllactosamine deficiency (Schindler’s disease). The potential of the NanoMate approach for discovery of novel carbohydrate variants in complex biological mixtures has been demonstrated on a mixture of oligosaccharides and glycosylated amino acids from urine of a patient suffering from congenital disorder of glycosylation (CDG)23,24 which was not previously investigated. For structural assignment of single components in this unknown complex carbohydrate mixture, which can belong to the N- or O-glycan type, either with a free reducing end or with the reducing end linked to an amino acid or a peptide, a program for computer-assisted assignment was developed. The advantage of the fully automated chip-ESI-QTOF for high performance screening, sequencing and identification of components is demonstrated here for the first time in glycomics. This integrative high-throughput approach, including the chip and robot-based mass spectrometry followed by computer-assisted assignment, provides a novel basis for carbohydrate system analysis and automatization in glycomics. EXPERIMENTAL SECTION Reagents and Samples. Methanol was obtained from Merck (Darmstadt, Germany) and used without further purification. Distilled and deionized water (Milli-Q Water Systems Millipore, Bedford, MA) was used for the preparation of the sample solutions. Biological samples investigated in this work were a mixture of O-glycosylated sialylated peptides, denoted BPy, which was obtained and previously purified from urine of patient B.P. suffering from a hereditary N-acetylhexosaminidase deficiency known as Schindler’s disease, as we described before,20-22 and a native mixture of glycans and O- and N-glycosylated sialylated peptides, denoted M5, from the urine of patient K.L. suffering from congenital disorder of glycosylation (CDG),23 which was not previously investigated. For isolation of the components, the patients’ urine was first filtered and submitted to gel filtration chromatography on Biogel P2. The glycans were separated by gel filtration chromatography performed on a Fractogel TSK HW 50 in 0.01 M pyridinium acetate, pH 5.4, as eluting buffer.24 The collected fractions were desalted by overnight incubation with H+ beads in water at 22 °C followed by 10-min centrifugation. Aqueous solutions of the mixtures were dried in a Speed Vac SPD 111V system (Savant, Du¨sseldorf, Germany). For ESI-MS analysis, stock solutions of (20) Linden, H. U.; Klein, R. A.; Egge, H.; Peter-Katalinic´, J.; Dabrowski, J.; Schindler, D. Biol. Chem. Hoppe-Seyler 1989, 370, 661-672. (21) van Diggelen, O. P.; Schindler, D.; Kleijer, W. J.; Huijmans, J. M.; Galjaard, H.; Linden, H. U.; Peter-Katalinic´, J.; Egge, H.; Dabrowski, U.; Cantz, M. Lancet 1987, 2, 804. (22) Peter-Katalinic´, J.; Williger, K.; Egge, H.; Green, B.; Hanisch, F.-G.; Schindler, D. J. Carbohydr. Chem. 1994, 13, 447-456. (23) Jaeken, J.; Carchon, H. J. Inherited Metab. Dis. 1993, 16, 813-817. (24) Williger, K. Ph.D. Thesis, 1993, University of Bonn, Bonn, Germany.

the samples at 0.1 mg/mL were prepared by dissolving the dried material in 1:1 v/v methanol/water and frozen at -20 °C. Dilution of the stock solution in 1:1 v/v methanol/water yielded the working aliquots at a concentration of ∼3 (BPy) and 5 pmol/µL (M5). Method and Instrumentation. Mass Spectrometry. Mass spectrometry was performed on an orthogonal hybrid quadrupole time-of-flight mass spectrometer (QTOF Micromass, Manchester, U.K.) in the Micromass Z-spray geometry. The QTOF mass spectrometer was interfaced to a PC computer running the MassLynx software to control the instrument and acquire and process MS data. Nitrogen was used as a dissolvation gas, and the source block temperature was kept at 80 °C. All mass spectra were acquired in the negative ion mode, which was shown to be advantageous for carbohydrate MS analysis.25 Tandem mass spectrometry was performed by CID at low energies using Ar as a collision gas. To obtain a maximum coverage of sequence ions, the collision energy was adjusted during the experiment within a 25-40-eV range, as described before.11 Automated Chip-Based Nanoelectrospray. Fully automated chipbased nanoelectrospray was performed on a NanoMate 100 incorporating ESI chip technology (Advion Biosciences, Ithaca, NY) which was mounted to the QTOF mass spectrometer. The electrospray chip was positioned a few millimeters from the sampling cone potential. Aliquots (6 µL) of the working sample solutions were transferred into the 96-well plate. The robot was programmed to aspirate 4 µL of sample, followed by 2 µL of air, into the pipet tip and then deliver the sample to the inlet side of the microchip. The microchip consists of a 10 × 10 array of nozzles etched into the planar surface of a silicon wafer and was fabricated using a variety of techniques, including deep reactive ion etching. The preparation of the microchip is described in detail elsewhere.26 A channel extended from the nozzle through the microchip to an inlet on the opposite surface, and during analysis the conductive pipet tip containing the sample was engaged against this inlet, forming a pressure seal. Each nozzle had an internal diameter of 10 µm. Electrospray was initiated from the nozzle by applying a voltage of -1.55 kV and a head pressure of 0.5-0.8 psi, depending on the sample in the pipet tip. Following sample infusion and MS analysis, the pipet tip was ejected. A fresh tip and nozzle were used for each sample, thus preventing any cross-contamination or carryover. RESULTS Automated Chip-Based NanoESI-QTOF-MS and Tandem MS on BPy Mixture of O-Glycosylated Amino Acids and Peptides. In Figure 1, the total ion current (TIC) of the BPy mixture of O-glycosylated sialylated amino acids and peptides acquired for about 1.3 min by automated chip-based nanoESIQTOF in the negative ion mode at 30 V sampling cone potential is presented. The TIC profile indicates a sustained and constant spray as well as an efficient ionization proved by the high intensity of ion current. By combining across the entire recorded TIC, a spectrum of high signal/noise was obtained (Figure 2), although a fair signal/noise ratio could be obtained also within only 30 s of acquisition. In Table 1, the assignment of major singly and doubly charged ions derived from tri-, tetra-, penta-, hexa-, hepta-, and (25) Peter-Katalinic´, J. Mass Spectrom. Rev. 1994, 13, 77-90. (26) Schultz, G. A.; Corso, T. N.; Prosser, S. J.; Zhang, S. Anal. Chem. 2000, 72, 4058-4063.

Analytical Chemistry, Vol. 76, No. 7, April 1, 2004

2047

Figure 1. Stability of the total ion current (TIC) in the automated chip-based (-) nanoESI-QTOF-MS experiment on BPy O-glycopeptide mixture. Substrate concentration, 3 pmol/µL. Sampling cone potential, 30 V.

Figure 2. Automated chip-based (-) nanoESI-QTOF-mass spectrum of BPy O-glycopeptide mixture, derived by combining across the TIC depicted in Figure 1. The single (1-) and double (2-) ion charge states are assigned.

octasaccharide amino acids and peptides detected by the automated chip-based nanoESI/MS is listed in terms of building blocks, expressing patterns typical for GalNAc-type O-glycosylation.20 The most abundant ions are assigned to singly or doubly charged pseudomolecular species originating from tri-, tetra-, penta- hexa-, hepta, octa-, and decasaccharide glycans O-linked 2048 Analytical Chemistry, Vol. 76, No. 7, April 1, 2004

either to serine (Ser), threonine (Thr), or the Thr-Pro dipeptide (Table 1). Singly charged ions assigned to free oligosaccharides, such as NeuAc at m/z 308.11, NeuAcHex at m/z 470.13, and NeuAcHexHexNAc at m/z 673.24, were present in the mixture as well. We assume that these sugars are the fragments from O-glycans of the type of NeuAcGalGalNAc.

Figure 3. Fragmentation spectrum obtained by automated chip-based (-) nanoESI-QTOF-MS/MS using NeuAc2GalGalNAc-Thr detected as a doubly charged ion at m/z 532.19 as a precursor ion. Collision energy, 20-40 eV. Assignment of fragment ions is according to Domon and Costello.29 Table 1. Assignment of Major Ionic Species Detected in the BPy Mixture by Automated Chip-Based (-) NanoESI-QTOF-MS [M - 2H]2m/z

[M - H]m/z 470.13

525.19 532.19 673.24 707.79 714.77 760.26 774.28 853.31 871.29 890.31 897.32 912.88 919.88 1051.35 1065.35

composition

theoretical m/z

NeuAcHex NeuAc2HexHexNAc-Ser NeuAc2HexHexNAc-Thr NeuAcHexHexNAc NeuAc2Hex2HexNAc2-Ser NeuAc2Hex2HexNAc2-Thr NeuAcHexHexNAc-Ser NeuAcHexHexNAc-Thr NeuAc3Hex2 HexNAc2-Ser NeuAcHexHexNAc-Thr-Pro NeuAc2Hex3HexNAc3-Ser NeuAc2Hex3HexNAc3-Thr dHex4Hex2HexNAc4-Ser dHex4Hex2HexNAc4-Thr NeuAc2HexHexNAc-Ser NeuAc2HexHexNAc-Thr

470.15 525.17 532.18 673.23 707.73 714.74 760.26 774.27 853.32 871.33 890.30 897.31 912.83 919.83 1051.35 1065.37

The tetrasaccharide-threonine NeuAc2GalGalNAc-Thr detected in the MS1 as an abundant doubly charged [M - 2H]2- ion at m/z 532.19 was manually selected and subjected to the chip nanoESI-tandem MS by CID at variable low energies (VE-CID), as described previously.13 The MS/MS spectrum, resulting after 3 min of signal acquisition within the collision energy range of 20-40 eV (Figure 3), is dominated by the Y ions at m/z 774.28 (singly charged) and m/z 386.13 (doubly charged) generated by the cleavage of the NeuAc moiety attached to the core GalNAc. In addition, the Y2R/B1β singly charged ion at m/z 483.16 indicates further stripping of the NeuAc moiety attached to Gal. The structure of the molecule can be assigned according to the C2R

Figure 4. Fragmentation scheme of NeuAc2GalGalNAc-Thr detected as a doubly charged ion at m/z 532.17.

singly charged ion at m/z 470.11, the B3/B1R at 673.22 and the C3 doubly charged ion at m/z 481.63, the latest illustrating the Thr detachment from the saccharide moiety and the formation of the [NeuAc2GalGalNAc]2- ion (Figure 4). The reduced signal-to-noise ratio of low-abundance molecular precursor ions in complex mixtures obtained by classical nanoESIMS/MS can generally be overcome by long signal accumulation in an off-line approach,27 but it is frequently associated with the spray instability or signal interruptions. The performance of the NanoMate system to provide long-lasting electrospray signal rendered reliable conditions for high sensitivity, particularly useful (27) Alving, K.; Paulsen, H.; Peter-Katalinic´, J. J. Mass Spectrom. 1999, 34, 395407.

Analytical Chemistry, Vol. 76, No. 7, April 1, 2004

2049

Figure 5. Total ion current (TIC) profile for ion isolation (0 eV) and fragmentation (25 and 40 eV) applied in the automated chip-based (-) nanoESI-QTOF-MS/MS of NeuAc2Gal3GlcNAc2GalNAc-Ser detected as a doubly charged ion at m/z 890.32 in the course of the sampling cone potential at 30 V.

Figure 6. Fragmentation spectrum obtained by automated chip-based (-) nanoESI-QTOF-MS/MS derived by using NeuAc2Gal3GlcNAc2GalNAc-Ser detected as a doubly charged ion at m/z 890.32 across the collision energy range depicted in Figure 5.

for detection and sequencing of minor glycopeptide components, previously accessible for fragmentation from such complex mixtures only by on-line capillary electrophoresis/ESI-QTOF-MS using the automatic MS-to-MS/MS mode switching in a datadependent analysis.28 In the case of the chip nanoESI-MS/MS, even the very low abundant ions, such as the one detected as a doubly charged species at m/z 890.32, could be successfully 2050 Analytical Chemistry, Vol. 76, No. 7, April 1, 2004

fragmented. The doubly charged ion at m/z 890.32 was manually selected and submitted to CID-VE-MS/MS to obtain a possibly high coverage of fragment ions, as described before.13 The TIC profile of the isolation/sequencing events (Figure 5) demonstrates the stability during the MS/MS experiment for each of the (28) Zamfir, A.; Peter-Katalinic´, J. Electrophoresis 2001, 22, 2448-2457. (29) Domon, B.; Costello, C. E. Glycoconjugate J. 1988, 5, 397-409.

Table 2. Computer-Assisted Assignment of the Major Ions Detected in the M5 Mixture by Automated Chip-Based (-) NanoESI-QTOF-MS [M - 2H]2- [M - H]m/z m/z 461.14 470.15 481.65 493.15 525.16 532.15 583.18 656.18 664.19 673.19 707.72 718.68 826.21 846.73 899.24 Figure 7. Fragmentation scheme of NeuAc2Gal3GlcNAc2GalNAcSer detected as a doubly charged ion at m/z 890.31.

collision energy values applied during the ongoing experiment and the corresponding ion current dependence on this parameter. The MS/MS spectrum combined over the 25- and 40-eV range of the TIC is presented in Figure 6. The most abundant fragment ions were Y5β (or Y2R) assigned to the ion arising after the cleavage of one NeuAc unit and Y5β/B1R (or Y2R/B1β) attributed to the loss of two NeuAc units. The structure of the molecule deduced from the typical fragmentation pattern of the disialooctasaccharide O-linked to Ser is depicted in Figure 7. Automated Chip-Based NanoESI-QTOF-MS and Tandem MS on M5 Mixture Containing Glycans and Glycosylated

964.26 986.23 1008.74 1037.72 1110.30 1139.26 1175.28 1183.30 1212.26

composition determined by computer algorithm

theoretical m/z

NeuAc2Hex2 NeuAcHex NeuAc2HexHexNAc NeuAcHexNAc (-H2O) NeuAc2HexHexNAc-Ser NeuAc2HexHexNAc-Thr NeuAc2HexHexNAc2 NeuAc2dHexHexHexNAc2 NeuAc2Hex2HexNAc2 NeuAcHexHexNAc NeuAc2Hex2HexNAc2-Ser NeuAc2Hex2HexNAc2-Ser (Na) NeuAc2Hex4HexNAc2 NeuAc2Hex3HexNAc3 NeuAc2dHexHex4HexNAc2 NeuAc2HexHexNAc NeuAc2HexHexNAc (Na) NeuAc2Hex5HexNAc3 NeuAcdHexHex5HexNAc4 NeuAc2Hex5HexNAc4 NeuAcdHexHex5HexNAc5 NeuAc2HexHexNac-Ser (2Na + P) NeuAc2dHexHex5HexNAc4 NeuAcdHex2Hex5HexNAc5

461.15 470.15 481.66 493.17 525.18 532.18 583.20 656.23 664.23 673.23 707.74 718.74 826.28 846.80 899.31 964.33 986.33 1008.84 1037.87 1110.38 1139.41 1175.32 1183.41 1212.43

Peptides. A urine fraction containing sialylated species from patient K.L. suffering from an unknown type of motor and neurodegenerative disease showing clinical picture of that of CDG was submitted to glycoscreening by negative ion automated chipbased nanoESI-QTOF-MS. The mass spectrum was acquired for 3 min by automated chip-based nanoESI-QTOF in the negative

Figure 8. Automated chip-based (-) nanoESI-QTOF mass spectrum of the M5 mixture containing glycans and glycopeptides. The average concentration was 5 pmol/µL. Sampling cone potential, 50 V.

Analytical Chemistry, Vol. 76, No. 7, April 1, 2004

2051

Figure 9. Automated chip-based (-) nanoESI-QTOF-MS/MS of the doubly charged ion at m/z 1008.74 in the M5 mixture assigned to the truncated N-glycan NeuAc2Hex5HexNAc3. A) Mass range m/z 150-850. B) Mass range m/z 850-1800. Sampling cone potential, 70 V; collision energy, 45 eV.

ion mode at 50-V sampling cone potential (Figure 8). A high level of heterogeneity concerning the type of glycans and glycopeptides as well as the degree of sialylation was observed. The high complexity of this sample could arise from the presence of Oand N-linked glycans appearing either as free oligosaccharides or linked to one or more amino acids. A computer algorithm was developed in our laboratory for the preliminary analysis and 2052 Analytical Chemistry, Vol. 76, No. 7, April 1, 2004

assignment of glycoforms in such a heterogeneous mixture never previously investigated. The experimental data for the input are the m/z values and the respective charge states determined from MS and MS/MS experiments. All possible structures containing Hex, HexNAc, dHex and NeuAc as monosaccharide building-block units were calculated by this algorithm. In the second step, the modeling of the theoretical fragmentation pattern from different

Table 3. Computer-Assisted Assignment of Fragment Ions Obtained by Automated Chip-Based (-) nanoESI-QTOF-MS/MS of the Doubly Charged Ion at m/z 1008.74, Assigned to the Truncated N-Glycan NeuAc2Hex5HexNAc3a m/z singly charged exptl

calcdb

202.09 220.10

202.08 220.08 220.09

263.10 290.10 306.13 308.13 350.15 364.13 372.09 382.14 424.15 452.14 470.15 645.14 655.21 673.22 715.25

doubly charged exptl

calcdb

290.09 308.10 350.12 364.14 424.16 452.15 470.15 655.23 673.23 715.25 817.78

835.26

835.28 847.75 898.29 907.26 928.29 949.27 958.28 999.80

1095.38 1113.32 1275.36 1524.47 1566.46 1626.48 1709.50 1727.55

1709.59 1727.59

898.26 907.31 928.32 958.25

fragment ion assignment Z1 Y1 0,2A , 0,2A 1R 1β nac B1R, B1β 2,4C 2,4A - CO 2R - CO2, 2β 2 C1R, C1β 2,4A , 2,4A 2R 2β 3,5A , 3,5A 2R 2β c na c na 1,5A , 1,5A 2R 2β B2R, B2β C2R, C2β nac B3R, B3β C3R, C3β 1,3A , 1,3A 4R 4β nac C4R, C4β nac 0,2X , 0,2X , B 6R 6β 5 C5 2,4A 6 nac 0,2A 6 [M - H]- - H2O 2,4A /Z /Y ′ 6 4 5 2,4A /Y /Y ′ 6 4 5 2,4A /Y /Y ′ 6 5 5 Y5/C5 2,4A /Y 6 5 0,2A /Y 6 5 Z5R, Z5β Y5R, Y5β

a Theoretical values, calculated upon the assignment of experimental m/z mass values of 34 ions by the computer program are in good correlation. b Computerized assignment. c Not assigned.

structures with the same m/z ratio was processed. Such data are presented as a combination of B, C, Y, and Z ions, related to the cleavage of the glycosidic bonds, as well as the ring cleavage ions A and X aligned to the proposed structure of the chosen molecular ion. In Table 2, the first-step MS analysis along with the proposed composition of single components detected by automated (-) chip-based nanoESI-QTOF spectrum presented in Figure 8 is given. Singly charged ions were assigned to NeuAcHex at m/z 470.15, NeuAcHexNAc at m/z 493.15, NeuAcHexHexNAc at m/z 673.19, and NeuAc2HexHexNAc (Na) at m/z 986.23. As doubly charged ions, NeuAc2HexHexNAc at m/z 481.65, NeuAc2Hex2HexNAc2 at m/z 664.19, NeuAc2Hex4HexNAc2 at m/z 826.21, NeuAc2Hex5HexNAc3 at m/z 1008.74, and NeuAc2Hex5HexNAc4 at m/z 1110.30 were assigned. The ratio of Hex and HexNAc moieties indicated that the structures detected may originate from both N- and O-glycans. The doubly charged molecular ion at m/z 1008.74 was manually selected and submitted to the automated chip-based nanoESI-QTOF-MS/MS by CID at low energies (Figure 9), and the experimental data were correlated with the computed fragment ions (Table 3). In the first step of computer analysis, performed for the range of the 0.3-Da mass window, two possible compositions were proposed for the m/z value of 1008.74: NeuAc2Hex3HexNAc1dHex5 and NeuAc2Hex5HexNAc3. According to biosynthetic pathways for assembly of carbohydrate oligomers, the NeuAc2Hex3HexNAc1dHex5 is a less probable structure because of the insufficient number of Hex and HexNAc moieties as attachment sites for the dHex, whereas the second proposed structure, NeuAc2Hex5HexNAc3, is one of a higher probability. The presence of NeuAcHex, NeuAcHexHexNAc, NeuAcHex2HexNAc, and NeuAc2Hex5HexNAc2 as C2, C3, C4, and C5 fragment ions, respectively, in the MS/MS spectrum of the doubly charged precursor ion at m/z 1008.74 (Figure 9) allows consideration to assign this ion as a truncated N-linked biantennary glycan missing the terminal HexNAc at the reducing end. The fragmentation scheme is presented in Figure 10. This structure proposal was imported into the computer program, which generated the

Figure 10. Fragmentation scheme of NeuAc2Gal2Man3GlcNAc3 detected as a doubly charged ion at m/z ) 1008.74.

Analytical Chemistry, Vol. 76, No. 7, April 1, 2004

2053

possible A, B, C, X, Y, and Z negatively charged fragment ions. According to these data, ∼80% of abundant fragment ions from MS/MS spectra could be automatically assigned. CONCLUSIONS An advanced system consisting of a robot and chip-based mass determination, followed by computer-assisted data analysis, was implemented for glycomics. Two complex carbohydrate mixtures containing glycoforms from different structural systems, from patients suffering from two different hereditary diseases, Schindler’s disease and congenital disorder of glycosylation (CDG), were explored by this approach. The feasibility of the fully automated chip-ESI-QTOF-MS for high-performance glycoscreening and sequencing, followed by computer-based identification of components in an unknown carbohydrate mixture, could be demonstrated. The potential of here-developed automated chip-based ESIQTOF-MS methodology for glycomics will be beneficial for discovery of novel carbohydrate variants in complex biological mixtures, due to increased sensitivity, reproducibility, ionization efficiency, and ability to generate a sustained and constant electrospray. For structural assignment of single components in unknown complex carbohydrate mixtures by mapping and sequencing, computer-assisted assignment provided the rapid and accurate basic structure identification essential for glycomics automatization.

2054

Analytical Chemistry, Vol. 76, No. 7, April 1, 2004

Considering the ESI chip as the interface between the mass spectrometer and the NanoMate 100, future developments for online experimental procedures by coupling to the ESI chip are viable, such as on-line sample cleanup and monolithic support material packed into either the ESI chip or the conductive pipet tips. Work in these and related areas is being actively followed in our laboratory. ACKNOWLEDGMENT We thank Prof. Dr. Detlev Schindler, Institute for Human Genetics, University of Wu¨rzburg, Germany, for fruitful long-term cooperation and Dr. Kerstin Williger and Mrs. Claudia Nahrings, Institute for Physiological Chemistry, University of Bonn, for sample preparation. Financial support of this work was provided by Deutsche Forschungsgemeinschaft within Sonderforschungsbereich 492, project Z2 to J.P.-K. The ESI-QTOF mass spectrometer was obtained with a HbfG Grant (Land Nordrhein Westfalen) to J.P.-K.

Received for review November 8, 2003. Accepted January 21, 2004. AC035320Q