Direct On-Membrane Glycoproteomic Approach Using MALDI-TOF

May 25, 2007 - Inkjet printing of proteins. Joseph T. Delaney , Patrick J. Smith , Ulrich S. Schubert. Soft Matter 2009 5 (24), 4866 ...
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Direct On-Membrane Glycoproteomic Approach Using MALDI-TOF Mass Spectrometry Coupled with Microdispensing of Multiple Enzymes Satoshi Kimura,†,‡ Akihiko Kameyama,*,† Shuuichi Nakaya,†,‡ Hiromi Ito,† and Hisashi Narimatsu*,† Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan, Shimadzu Corporation, 1, Nishinokyo-Kuwabaracho, Nakagyo-ku, Kyoto 604-8511, Japan Received February 7, 2007

We report a novel approach for direct on-membrane glycoproteomics by digestion of membrane-blotted glycoproteins with multiple enzymes using piezoelectric chemical inkjet printing technology and onmembrane direct MALDI-TOF mass spectrometry. With this approach, both N-linked glycan analyses and peptide mass fingerprinting of several standard glycoproteins were successfully performed using PNGase F and trypsin microscale digestions of the blotted spots on membrane from an SDS-PAGE gel. In addition, we performed a similar analysis for 2-DE separated serum glycoproteins as a demonstration of how the system could be used in human plasma glycoproteomics. Keywords: glycoproteins • on-membrane digestion • mass spectrometry • piezoelectric ink-jet technology • glycoproteomics

Introduction Many proteomic studies aiming at disease diagnosis and therapeutic monitoring have focused on the quantitative aspect of proteome. Differential imaging gel electrophoresis (DIGE) in gel-based proteomics and isotope-coded affinity tags (ICAT) in LC-MS/MS-based proteomics have been widely applied in the exploration of biomarkers through comparison of the quantitative profile of the proteome.1,2 Because more than half of the total proteins in human plasma are glycosylated,3 the glycoproteome has been recently recognized as a major subproteome of human plasma,4 and methodologies for the estimation of their quantitative profile have been developed.5,6 However, these methodologies mostly ignore the qualitative aspect of glycosylation which is associated with protein folding, stability, cellular localization, recognition, and immune reactions, all of which are necessary for normal biological processes.7,8 Therefore, to explore biomarkers related to functional abnormality of proteins, qualitative investigation of glycoproteins, as well as quantitative study, is necessary. In particular, fine characterization of the glycan moiety is required.9 Western blotting or lectin blotting coupled with two-dimensional gel electrophoresis (2-DE) is useful for the detection of alterations in glycosylation. However, the variety of lectins and the availability of monoclonal antibodies against glycans are far * To whom correspondence should be addressed. Akihiko Kameyama: tel, +81-29-861-3123; e-mail, [email protected]. Hisashi Narimatsu: tel, +81-29-861-3200; e-mail, [email protected]. † Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST). ‡ Shimadzu Corporation.

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more limited than the variation of glycans. Furthermore, the binding specificities of lectins are generally broad. As a result, glycoprotein characterizations in current proteomic studies tend to be significantly ambiguous. Therefore, developments of a novel, fine glycoproteomics technology which can allow rapid identification of proteins, as well as glycans attached to them, is required.10 In gel-based glycoproteomics, N-linked glycans released from a gel piece by in-gel PNGaseF digestion could be analyzed by MS.11 In-gel release of O-linked glycans by reductive β-elimination has also very recently been reported.12 However, such an in-gel digestion protocol contains multiple liquid-handling steps associated with extraction, digestion, and purification of peptides and glycans. In addition, since these in-gel procedures require at least one piece of excised gel for each reaction, duplicate 2-DE gels are normally required to perform both the PMF analysis and glycan analysis in glycoproteomics. In contrast, membranes are generally easier to handle than gels, and an on-membrane digestion method has been reported as an alternative protocol to in-gel digestion.13,14 On-membrane digestion has advantages for long-term storage of blotted membranes and for easy removal of low-molecular-weight contaminants. In fact, for O-linked glycans, reductive β-elimination has often been performed on the blotted membranes.15-17 Recently, a new method for on-membrane direct MALDITOF MS analysis was proposed.18 The technology used in the method is based on direct digestion and detection in microscale areas of blotted protein spots with a piezoelectric ink-jet device such as Chemical Inkjet Printer (CHIP-1000, Shimadzu). This 10.1021/pr070067m CCC: $37.00

 2007 American Chemical Society

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Direct On-Membrane Glycoproteomic Approach

Figure 1. Outline of an on-membrane glycoproteomic approach using microdispensing technology. A piezoelectric ink-jet device enables microscale PNGase F and trypsin digestion on a single protein spot on a blotted membrane. The digested areas can be analyzed by direct on-membrane MALDI-TOF mass spectrometry.

Figure 2. An image of the blotted membrane after microdispensing of the reagents by Chemical Inkjet Printer. Protein bands were stained with Direct Blue 71, and the following microdispensed sequentially: PVP360 and PNGase F, or PVP360 and trypsin. Dispensed areas were observed as de-stained circles with approximately 0.5 mm diameter.

Figure 4. On-membrane direct MALDI-TOF MS spectra of Nlinked glycans released from the standard glycoproteins. (a) Ovalbumin, (b) transferrin, (c) R1-acid glycoprotein, (d) fetuin with neuraminidase pre-digestion, (e) fetuin without neuramidase digestion. The asterisks indicate potassium adduct ions. The signals indicated with Arabic numerals were assigned: see Table 1.

proteomics. Here, we demonstrate a novel glycoproteomic approach in which protein identification and N-linked glycan analysis of the blotted glycoprotein can be performed using a single spot on a membrane (Figure 1).

Experimental Section

Figure 3. On-membrane direct MALDI-TOF MS spectra of Nlinked glycans released from ovalbumin band by on-membrane digestion with the serially diluted PNGase F. Ovalbumin (1 µg) was loaded and run on SDS-PAGE and blotted to a membrane. PNGase F (1, 5, 10, 25, and 50 µU) was then printed on the ovalbumin band. The asterisks on the peaks correspond to N-linked glycans.

allows microdispensing of trypsin and MALDI matrix at picoliter volumes onto the membranes. This technology should enable multiple enzyme treatments on a single protein spot. For example, it would allow both trypsin and endoglycosidase digestion and thus provide a convenient approach in glyco-

Materials and Reagents. Ovalbumin, fetuin, transferrin, R1acid glycoprotein, polyvinylpyrrolidone (PVP-360), and Direct Blue 71 were purchased from Sigma Chemical (St. Louis, MO). 2,5-Dihydroxybenzoic acid (2,5-DHB) was purchased from Bruker Daltonics (Billerica, MA). PNGase F (EC 3.5.1.52) was purchased from Takara Bio (Otsu, Japan). Arthrobacter ureafaciens neuraminidase (EC 3.2.1.18) was purchased from Marukin Bio (Kyoto, Japan). Trypsin was purchased from Promega (Madison, WI), and Immobilon-PSQ PVDF membrane was purchased from Millipore (Billerica, MA). Normal human serum was from laboratory stock. Instruments. Mass spectrometry was performed using a MALDI quadrupole ion trap (QIT) time-of-flight (TOF) mass spectrometer (AXIMA-QIT; Shimadzu Corporation, Japan and Kratos Analytical, U.K.) equipped with a 337 nm nitrogen laser. For on-membrane digestion, the Chemical Inkjet Printer (CHIP1000), developed by Shimadzu Corporation with Proteome Systems Ltd. (Sidney, Australia) was used for microdispensing the reagents onto blotted protein spots. Gel Electrophoresis and Electro Blotting. Serially diluted series of standard glycoproteins (10, 5, 2, 1, and 0.5 pmol/lane) Journal of Proteome Research • Vol. 6, No. 7, 2007 2489

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Table 1. Summary of N-Linked Glycans Detected by Direct On-Membrane MALDI-TOF MS of the Standard Glycoproteinsa

a

peak no.

glycoprotein

m/z (obsd)

m/z (theor)

composition

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

ovalbumin ovalbumin ovalbumin ovalbumin ovalbumin ovalbumin ovalbumin ovalbumin ovalbumin ovalbumin ovalbumin transferrin transferrin transferrin transferrin R1-acid glycoprotein R1-acid glycoprotein R1-acid glycoprotein R1-acid glycoprotein R1-acid glycoprotein R1-acid glycoprotein R1-acid glycoprotein fetuin fetuin

1257.49 1298.51 1419.56 1460.67 1501.65 1542.66 1581.56 1663.71 1704.71 1745.77 1866.79 1663.76 1976.86 2028.87 2289.96 1663.77 1976.87 2028.94 2174.96 2276.08 2342.06 2394.14 1663.89 2029.15

1257.42 1298.45 1419.48 1460.50 1501.53 1542.56 1581.53 1663.58 1704.61 1745.64 1866.66 1663.58 1976.66 2028.71 2289.74 1663.58 1976.66 2028.71 2174.77 --2341.79 2393.85 1663.58 2028.71

(Hex)2+(Man)3(GlcNAc)2 (Hex)1(HexNAc)1+ (Man)3(GlcNAc)2 (Hex)3+(Man)3(GlcNAc)2 (Hex)2(HexNAc)1+ (Man)3(GlcNAc)2 (Hex)1(HexNAc)2+ (Man)3(GlcNAc)2 (HexNAc)3+(Man)3(GlcNAc)2 (Hex)4+(Man)3(GlcNAc)2 (Hex)2(HexNAc)2+ (Man)3(GlcNAc)2 (Hex)1(HexNAc)3+ (Man)3(GlcNAc)2 (HexNAc)4+(Man)3(GlcNAc)2 (Hex)2(HexNAc)3+ (Man)3(GlcNAc)2 (Hex)2(HexNAc)2+ (Man)3(GlcNAc)2 (NeuAc)1(Hex)2(HexNAc)2+ (Man)3(GlcNAc)2 (Hex)3(HexNAc)3+ (Man)3(GlcNAc)2 (NeuAc)2(Hex)2(HexNAc)2+ (Man)3(GlcNAc)2 (Hex)2(HexNAc)2+ (Man)3(GlcNAc)2 (NeuAc)1(Hex)2(HexNAc)2+ (Man)3(GlcNAc)2 (Hex)3(HexNAc)3+ (Man)3(GlcNAc)2 (Hex)3(HexNAc)3(Deoxyhexose)1+ (Man)3(GlcNAc)2 unkown (NeuAc)1(Hex)3(HexNAc)3+ (Man)3(GlcNAc)2 (Hex)4(HexNAc)4+ (Man)3(GlcNAc)2 (Hex)2(HexNAc)2+ (Man)3(GlcNAc)2 (Hex)3(HexNAc)3+ (Man)3(GlcNAc)2

All observed ions correspond to [M + Na]+.

were subjected to SDS-PAGE. After electrophoresis, proteins were electrotransferred to Immobilon PSQ PVDF membrane and stained using Direct Blue 71. The serum was pretreated with an albumin and IgG depletion column (ProteoPrep Blue Albumin and IgG Depletion Kit, Sigma) according to the manufacturer’s procedure. The depleted serum sample was subjected to two-dimensional gel electrophoresis. Briefly, the sample containing around 200 µg of protein was mixed with sample buffer containing 8 M Urea, 2% (w/v) CHAPS, 20 mM DTT, 1% Ampholyte (pH 3.0-10, Invitrogen), and a trace of bromophenol blue (BPB). A dry IPG gelstrip (pH 3.0-10, Invitrogen) was rehydrated with the prepared sample overnight and focused on a ZOOM IPGRunner System according to the manufacturer’s procedure (Invitrogen). The focused gelstrip was subsequently equilibrated with 5 mL of buffer (50 mM TrisHCl, 10% glycerol, 2% lithium dodecyl sulfate (LDS), 0.5 mM EDTA, and 0.0025% BPB) containing 1% DTT for 15 min with gentle shaking. Another 15 min equilibration was performed with the buffer containing 2.5% iodoacetoamide, and SDSPAGE was then performed on these strips. The proteins on the 2-DE gel were electroblotted to Immobilon-PSQ membrane and stained with Direct Blue 71. On-Membrane Digestion Using Chemical Inkjet Printer. The dried blotted membrane was adhered to an Axima-series MALDI-TOF target plate (Shimadzu Corporation, Japan) using double-sided electrically conductive adhesive tape (3M, St. Paul, MN). After acquiring the image on the blotted membrane with a scanner connected to the Chemical Inkjet Printer, several printing positions were created on each protein spot. Several nanoliters of 0.25% (v/w) PVP360 in 60% methanol or 0.25% (w/v) n-octyl β-D-glucopyranoside (OGP) was printed in order to pre-wet the membrane. Enzymatic digestion was performed by printing of 50 nL of trypsin solution: 200 µg/mL in 25 mM NH4HCO3 containing 10% 2-propanol, or PNGase F solution, 1.0 Unit/mL in 25 mM NH4HCO3 onto each target protein spot. The formulation of PNGase F from the vendor (Takara Bio) was dialyzed against 25 mM NH4HCO3 prior to use. To remove sialic acids from glycans, neuraminidase solution, 5 Units/mL in 0.1% acetic acid, was printed onto the target spots. After neuraminidase digestion, the membrane was washed with deionized 2490

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water and dried, after which PNGase F was printed. All enzymatic reactions were performed for 16 h at 30 °C (for trypsin) or 37 °C (for PNGase F and neuraminidase) in a humidified chamber. After the enzyme reactions, 100 nL of 10 mg/mL 2,5-DHB in 25% (v/v) acetonitrile was printed to each position on the membrane after drying. Mass Spectrometry. For sample preparation, 100 nL of 2,5DHB solution (10 mg/mL in 25% (v/v) acetonitrile) was printed to each position digested by enzyme as described above. Direct on-membrane MALDI MS analysis was performed in positiveion mode using external calibration with a mixture of angiotensin II and the peptide fragment of the adrenocorticotropic hormone corresponding to amino acid residues 18-39. Target positions were inputted to the AXIMA-QIT by loading the X,Ycoordinates created by CHIP-1000. Searching of the database was performed using Mascot (Matrix Science Ltd., London , U.K.) for peptide mass fingerprinting (PMF), and GlycoMod (http://www.expasy.ch/tools/glycomod/) for matches of the glycan compositions.

Results and Discussion Optimization of On-Membrane Release of N-Linked Glycans. We attempted to optimize the on-membrane reaction with PNGase F for direct MALDI-TOF MS analysis of N-linked glycans. This process is affected by several factors, including efficiency of enzymatic reaction and electroblotting of protein and ionization of glycans in the presence of other kinds of molecules on nonconductive membrane. The commercially available formulation of PNGase F contains significant amounts of nonvolatile chemicals such as phosphate, Tris, and EDTA. These chemical additives cause ion suppression and/or result in strong background signals in the mass spectra. In particular, the signals from EDTA interfere with glycan signals due to overlap with the mass range of glycans. To obtain clear mass signals of glycans, it is necessary to remove these chemical additives before dispensing on the membrane. Dialysis of the commercially available formulation of PNGase F against a volatile buffer such as 25 mM NH4HCO3 solution successfully removed these chemical additives giving clear signals from the

Direct On-Membrane Glycoproteomic Approach

glycans in the mass spectrum (data not shown). Thus dialyzed PNGase F was used for further optimization of the onmembrane digestion of glycoproteins. As a model glycoprotein, we employed ovalbumin whose N-linked glycans have been well-characterized.19,20 Ovalbumin (1 µg) was loaded, run on SDS-PAGE, and transferred to PVDF membrane by electroblotting. The membrane was stained with Direct Blue 71 which does not give any signals derived from the dye during MALDI MS analysis. To test the efficiency of on-membrane digestion, a serially diluted series of dialyzed PNGase F (1-50 µUnits/ printing area) was microdispensed onto the stained ovalbumin band with the Chemical Inkjet Printer. Temperature and duration of digestion were 37 °C and 16 h, respectively, which are the parameters generally used for release of N-linked glycans from glycoproteins in solution. After incubation in a humidified chamber, 2,5-DHB solution was microdispensed onto each digested position. Figure 2 shows an image of PVDF membrane after microdispensing of reagents. The microdispensed area looks like a circular de-stained spot which is 0.5 mm in diameter. Consequently, it was estimated that the area corresponded to approximately 20% of the protein band being used for the direct MS analysis on the membrane. This figure correlates to around 4 pmol of ovalbumin. Although sensitivity of glycans in MALDI MS is relatively low compared with that of peptides, more than 1 pmole of glycans should be enough to acquire mass spectra with good S/N ratio when using normal sample preparation. Figure 3 shows the MS spectra acquired by direct MALDI MS of N-linked glycans released by onmembrane digestion with the serially diluted PNGase F. The intensity and S/N ratios of peaks derived from glycans increased as the amount of dispensed enzyme increased (asterisks in Figure 3). Peaks with sufficient intensity were observed when more than 25 µUnits of PNGase F were dispensed onto one spot. In addition, PNGase F itself did not affect MS spectra. Because of the expense of high concentration enzyme, it was cost-inhibitive to add it to the large reservoir (500 µL) of the Chemical Inkjet Printer. Therefore, 50 µUnits of PNGase F was used for each spot in further experiments. On-Membrane Analysis of N-Linked Glycans. Using the optimized conditions, N-linked glycans of four standard glycoproteins, ovalbumin, transferrin, and R1-acid glycoprotein together with those of fetuin were analyzed using the direct on-membrane MALDI MS analysis. The glycoproteins were run on SDS-PAGE (1 µg/lane), transferred to PVDF mambrane, and stained as described above. Areas of the stained bands 0.5 mm in diameter on the membrane were digested with 50 µU of PNGase F, and direct MALDI-TOF MS analyses were performed. Figure 4 shows the MS spectra of the glycans released from the tested glycoproteins by the on-membrane digestion. Major glycans of ovalbumin, transferrin, and R1-acid glycoprotein were directly detected from the membrane after microdispensing of PNGase F. The observed signals are summarized and assigned in Table 1. The results of the on-membrane analyses of N-linked glycans of these glycoproteins are in agreement with previous reports.19-22 However, no distinct signals were observed from the fetuin band (Figure 4e). This might be due to difficulty of detection of sialylated glycans using MALDITOF MS in positive ion mode as it is known that the N-linked glycans of fetuin are highly sialylated.23,24 Predigestion of the band with neuraminidase using microdispensing before PNGase F digestion allowed detection of the glycans of fetuin in an asialo-form (Figure 4d). The optimal pH for the neuraminidase is 4-6, which is not compatible with the conditions for

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Figure 5. On-membrane glycoproteomic approach with human serum. (a) An image of the blotted membrane from a 2-DE of human serum. The membrane was stained with Direct Blue 71. The indicated spots with Roman numerals could be identified by on-membrane PMF analysis using Chemical Inkjet Printer. Identified proteins: see Table 2. (b) On-membrane direct MALDITOF MS spectra of the N-linked glycans from spots I, III, and IV. The asterisks indicate potassium adduct ions. The signals indicated with Arabic numerals were assigned: see Table 3.

PNGase F (pH 7-9). Therefore, the membrane was washed with ultrapure water after the neuraminidase treatment, and then PNGase F printing was performed at the same position that the neuraminidase was printed. In this case, dialysis of neuraminidase solution was not required because enzymes and other extra chemicals were washed away from the membrane before MS analysis. The MALDI-TOF mass spectrum of N-linked glycans of ovalbumin afforded the clearest signals of the four tested glycoproteins because the N-linked glycans of ovalbumin have relatively low molecular weights and are not sialylated glycans (Figure 4). The other three glycoproteins contain sialylated N-linked glycans. While the glycans of fetuin could not be detected without predigestion with neuraminidase as described above, sialylated glycans of transferrin and R1-acid glycoprotein were detected without neuraminidase treatment (Figure 4b,c). The reason for this observation is unclear. However, the presence of sialylated O-linked glycans in fetuin may be one Journal of Proteome Research • Vol. 6, No. 7, 2007 2491

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Table 2. Proteins Identified by Direct On-Membrane Glycoproteomic Approach with Human Seruma accession MW peptide coverage no. (KDa) pI countc (%)

spot no.b

protein

I II III IV V VI VII

R-2-macroglobulin ceruloplasmin serum transferrin serum albumin IgA1d vitamin D-binding protein Apo A1

CAA01532 163.2 6.0 BAA08084 115.4 5.4 AAA61141 77.0 6.8 CAA01217 66.4 5.7 1IGAA 51.0 5.3 AAA61704 52.9 5.4 CAA00975 28.1 5.3

15 13 11 12 1 7 18

14 12 17 18 4 12 58

a Theoretical molecular mass and pI are derived from the amino acid sequences in NCBI. b Spot no. corresponds to Figure 5a. c Peptide count means the number of peptides matched in MS analysis. d Spot V could be identified by sequence query of MS/MS spectrum of m/z 1835.90.

possible reason. No O-linked glycans have been reported for transferrin and R1-acid glycoprotein. In the microcircumstance of on-membrane reaction, high concentrations of acidic sialylated O-linked glycans in the band on the PVDF membrane might perturb the PNGaseF digestion for which the optimal pH is 7-9. Structural analyses of glycans on the PVDF membrane by using a multistage tandem mass spectrometry could not be performed due to low-sensitivity of on-membrane direct MALDI MS of glycans. Although this technique provides a convenient method to give qualitative information in glycoproteomics, the sensitivity needs to be improved to allow further analysis such as accurate assignment of glycans on the membrane using tandem or multistage tandem MS. Compared to peptides, glycans give relatively low-response in MALDI. In direct, on-membrane MALDI-TOF MS analysis, the sensitivities are further reduced compared to that of normal stainless MALDI. To overcome this problem, we are studying onmembrane labeling with sensitizer for MALDI MS detection of glycans. On-Membrane PMF Analysis of Glycoproteins. Peptide mass fingerprinting (PMF) coupled with direct on-membrane MALDI-TOF MS using chemical inkjet printing technology has been successfully demonstrated.18,25 To expand on this method for on-membrane glycoproteomics, we looked at PMF analysis of the same position that was used for on-membrane analysis of N-linked glycans on the four standard glycoproteins. However, enough peaks for identification of the protein could not be detected from the positions used for N-linked glycan

analysis. In this case, we added a trypsin solution onto the spot covered with a MALDI matrix (2,5-DHB), which is likely to lead to a low pH in the reaction system. The failure of PMF can be explained by slow reaction in such low pH conditions which are far from the optimal pH for the trypsin employed in this work. To avoid this, we washed the matrix from the blotted spot. We then tried trypsin digestion followed by PMF analysis of the same position used for N-linked glycan analysis. However, there was no obvious improvement in the PMF analysis. It may be that blotted proteins come off the PVDF membrane upon addition of the matrix solution which contains 30% acetonitrile, and are washed away together with the matrix. In further analysis, the region adjacent to the position which was printed with PNGase F was used for PMF analysis. The amount of the glycoprotein in the printing position was estimated to be approximate 4 pmol which is enough for onmembrane PMF analysis.18 All tested glycoproteins could be successfully identified with high scores from the adjacent region (data not shown). Compared to a conventional in-gel digestion for PMF analysis in which a gel piece of at least 1.2-1.5 mm in diameter is required, on-membrane PMF using the chemical inkjet printer requires only a tiny area 0.5 mm in diameter. This allows five different analyses of a single protein band which is 1.5 mm in diameter. Thus, the chemical inkjet printing technology could be applied to glycoproteomics by using adjacent regions in the same protein band for the digestion with the different two enzymes (trypsin and PNGase F). Furthermore, this method could be expanded to include independent analysis of a single protein band via a maximum five different reagents. On-Membrane Glycoproteomic Analysis of 2-DE Separated Glycoproteins of Human Serum. Serum or plasma proteomics is one of the most important techniques for the discovery of clinical biomarkers.9 Most proteins in serum with the exception of albumin are glycosylated, and characteristic changes of glycosylation associated with disease have been reported.26-29 Therefore, glycoproteomics of human serum has attracted attention as an alternative approach for the study of clinical biomarkers. To demonstrate the efficiency of the on-membrane glycoproteomic approach, human serum from which albumin and IgG were depleted was subjected to 2-DE and transferred to a PVDF membrane (Figure 5). The seven major spots on the membrane were analyzed by the on-membrane glycoproteomic

Table 3. Summary of N-Linked Glycans Detected by On-Membrane Glycoproteomic Approach with Human Seruma

a

peak no.

glycoprotein

m/z (obsd)

m/z (theor)

composition

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

R2-macroglobulin R2-macroglobulin R2-macroglobulin R2-macroglobulin R2-macroglobulin R2-macroglobulin transferrin transferrin transferrin transferrin transferrin transferrin IgA1 IgA1 IgA1 IgA1 IgA1

1257.52 1298.53 1460.68 1663.71 1809.81 1976.78 1136.47 1298.50 1663.5 1809.66 1976.67 2289.77 1663.67 1704.66 1809.81 1976.72 2012.84

1257.42 1298.45 1460.50 1663.58 1809.64 1976.66 1136.40 1298.45 1663.58 1809.64 1976.66 2289.74 1663.58 1704.61 1809.64 1976.66 2012.72

(Hex)2+(Man)3(GlcNAc)2 (Hex)1(HexNAc)1+(Man)3(GlcNAc)2 (Hex)2(HexNAc)1+(Man)3(GlcNAc)2 (Hex)2(HexNAc)2+(Man)3(GlcNAc)2 (Hex)2(HexNAc)2(Deoxyhexose)1+(Man)3(GlcNAc)2 (NeuAc)1(Hex)2(HexNAc)2+(Man)3(GlcNAc)2 (HexNAc)1+(Man)3(GlcNAc)2 (Hex)1(HexNAc)1+(Man)3(GlcNAc)2 (Hex)2(HexNAc)2+(Man)3(GlcNAc)2 (Hex)2(HexNAc)2(Deoxyhexose)1+(Man)3(GlcNAc)2 (NeuAc)1(Hex)2(HexNAc)2+(Man)3(GlcNAc)2 (NeuAc)2(Hex)2(HexNAc)2+(Man)3(GlcNAc)2 (Hex)2(HexNAc)2+(Man)3(GlcNAc)2 (Hex)1(HexNAc)3+(Man)3(GlcNAc)2 (Hex)2(HexNAc)2(Deoxyhexose)1+(Man)3(GlcNAc)2 (NeuAc)1(Hex)2(HexNAc)2+(Man)3(GlcNAc)2 (Hex)2(HexNAc)3(Deoxyhexose)1+(Man)3(GlcNAc)2

All observed ions correspond to [M + Na]+.

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technique described above. Using direct on-membrane PMF analysis or peptide sequence tag search with a MALDI-QITTOF mass spectrometer,30 the selected proteins were identified as (I) R2-macroglobulin, (II) ceruloplasmin, (III) serotransferrin, (IV) serum albumin, (V) IgA1, (VI) vitamin D-binding protein, and (VII) apolipoprotein A-1 (Table 2). PNGase F was then microdispensed onto the region adjacent to the area digested by trypsin, and direct on-membrane glycan analyses were performed. While peaks of N-linked glycans were detected from R2-macroglobulin, transferrin, and IgA1 (Figure. 5b), no distinct signals were observed from serum albumin, apolipoprotein A-1, and vitamin D-binding protein. The three proteins not giving glycan signals have not been shown to be glycosylated. Table 3 summarizes the glycan signals detected in these MS spectra. Ceruloplasmin is a serum glycoprotein bearing N-linked glycans.31,32 However, N-linked glycans from spot 2 could not be detected in this work even in combination with neuraminidase treatment (data not shown). Since the disadvantage of this method is relatively low sensitivity, the lack of detection of N-glycans from ceruloplasmin might be due to low concentration of this protein. Thus, the novel approach using microdispensing technology coupled with direct on-membrane MALDITOF MS analysis was successfully applied to the profiling of N-linked glycans and identification of proteins when analyzing 2-DE separated glycoproteins of human serum.

Conclusion We describe a novel approach for glycoproteomics using onmembrane direct MALDI-TOF MS analysis coupled with microdispensing of multiple enzymes onto glycoproteins immobilized on membrane. To be compatible with on-membrane direct MALDI-TOF MS analysis, enzyme reactions were carefully optimized. Using the optimized format, N-linked glycans were effectively released from glycoproteins immobilized on membrane and successfully analyzed by on-membrane direct MALDI-TOF MS without any interfering signals. Parallel treatments of a single protein spot with PNGase F and trypsin enabled both N-linked glycan profiling and protein identification of standard glycoproteins immobilized on membrane from 1D SDS-PAGE of standard glycoproteins as well as glycoproteins from 2-DE of human serum. Sequential treatment with neuraminidase and PNGase F provided an option for effective detection of glycans from highly sialylated glycoproteins. In an analogous way the combination of parallel and sequential treatments with exoglycosidases and PNGase F can be applied to the structural analysis of N-linked glycans using stepwise digestion and mass spectrometry.33 O-Linked glycans, another category of glycoprotein glycan, cannot currently be analyzed by this approach due to lack of an appropriate endo-glycosidase for this type of glycan. Discovery of endo-glycosidases which have wide range specificity for O-linked glycans will enlarge the applicability of our glycoproteomic approach. It is important to note, however, that the sensitivity of onmembrane direct MALDI MS detection of glycans still remains to be improved. Application of sensitizers of glycan detection, such as Cy-hydrazide,34 in the microdispensing technology might be one solution to this difficulty. Sensitive detection of glycans on membrane would allow the determination of the detailed structures including branching, linkage, and anomericity of glycans on glycoproteins using multistage tandem MS analysis.35,36 Thus, the glycoproteomic approach using microdispensing technology might be useful for exploring qualitative differences between normal states and those in patients.

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