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and apply gentle vacuum to pull the methanol through the PVDF membrane. Rinse each sample well three times with 250 |J,L water, pulling the water through the membrane after each rinse with gentle vacuum.

3.2. Reduction and Alkylation (see Note 2)

1. Rinse each sample well twice with 50 |J,L RCM buffer, pulling the buffer through the membrane after each rinse with gentle vacuum. Add 50 |J,L RCM buffer to each sample well. Add 50 |J,g glycoprotein sample to the appropriate well, and pull the solution through the membrane with gentle vacuum. The maximum volume of a well is approx 250 |J,L, so if the sample volume exceeds 200 |J,L, this process should be repeated, starting from adding 50 |J,L RCM buffer until all the volume of the sample has been applied to the PVDF membrane.

2. After the samples are applied, rinse each sample well twice with 50 |J,L RCM buffer, pulling each rinse through the PVDF membrane with gentle vacuum. Wipe the top and bottom of plate with a clean Kimwipe (see Note 3).

3. Freshly prepare a 0.1 M DTT solution in the RCM buffer. Add 50 |lL DTT solution to each sample well. Place the covered plate in a 37°C incubator for 1 h. Place an open vessel of water in the incubator to humidify the atmosphere and prevent evaporative loss of liquid from the sample wells.

4. After the reduction step, the DTT solution is removed by placing the plate onto the vacuum apparatus and applying gentle vacuum to pull the solution through the membrane. Rinse each sample well three times with 250 |J,L water, pulling the water through the membrane following each rinse with gentle vacuum. Wipe the top and bottom of plate with a clean Kimwipe (see Note 3).

5. Freshly prepare a 1 M IAA solution in 1 M NaOH. Dilute this stock solution 1:10 with RCM buffer to obtain a working solution of 0.1 M IAA.

6. Add 50 |J,L 0.1 M IAA solution to each sample well. Place the covered plate in the dark for 30 min at ambient temperature.

7. After the alkylation step, remove IAA by placing the plate onto the vacuum apparatus. Apply a gentle vacuum to the plate to pull the solution through the membrane. Each sample well should be rinsed three times with 250 |J,L water, pulling the water through the membrane after each rinse with gentle vacuum. Use a clean Kimwipe to wipe the top and bottom of plate (see Note 3).

3.3. Blocking the PVDF Membrane

1. Add 100 |J,L 1% aqueous solution of PVP-360 to each sample well. Incubate the covered plate for 30 min at ambient temperature. This step blocks any remaining protein-binding sites on the PVDF and prevents adsorptive losses of the PNGase F enzyme.

2. After blocking, remove PVP-360 by applying a gentle vacuum to the plate and pulling the solution through the membrane. Rinse each sample well three times with 250 |J,L water, pulling the water through the membrane after each rinse with gentle vacuum. Wipe the top and bottom of plate with a clean Kimwipe (see Note 3).

3.4. PNGase F Digestion

1. Prepare a 20 IUB milliunit/mL PNGase F solution in 10 mM Tris acetate buffer, pH 8.3 (see Note 4).

2. Add 25 |J,L PNGase F solution to each sample well. Place the covered plate in the 37°C humidified incubator for 3 h.

3. After digestion, transfer each sample into a labeled 500-^L microcentrifuge tube (see Note 5).

3.5. Acidification of Released Oligosaccharides

1. Prepare a 1.5 M acetic acid solution in water.

2. Add 2.5 |J,L acetic acid solution to each microcentrifuge tube (150 mM final acetic acid concentration), and mix the sample by pipet action. Incubate for 2 h at ambient temperature (see Note 6).

3.6. Removal of Cations From Released Oligosaccharides

1. Assemble the compact reaction columns by firmly inserting the filter into the column using the plunger provided with the columns. One column is needed for each sample.

2. Pipet 700-800 |J,L cation-exchange resin into a compact reaction column (see Note 7). Rinse each column with approx 5 mL water using a 60-mL syringe equipped with the luer-lock lid adapter. After rinsing, expel as much of the water in the columns as possible using the 60-mL syringe filled with air. Place each of the columns in a 1.5-mL microcentrifuge tube, and spin the columns in a microcentrifuge for 1-2 min at 16,000g to remove all residual water from the cation-exchange resin.

3. Place each column in a labeled 1.5-mL microcentrifuge tube, and add 3-5 |J,L sample to the top of the cation-exchange resin. Spin the column/microcentrifuge tube combination in a microcentrifuge using the pulse spin button to slowly move the sample down the cation-exchange column into the microcentrifuge tube (see Note 8).

3.7. MALDI-TOF MS Matrix Preparation

1. Super DHB (sDHB) positive mode matrix.

a. Prepare 1 mM NaCl solution in 25% aqueous ethanol (solvent A).

b. Prepare 4 mg/mL DHB solution in solvent A.

c. Prepare 0.2 mg/mL 5-MSA solution in solvent A.

d. Combine equal amounts of the DHB and 5-MSA matrices to form the sDHB matrix (see Note 9).

2. THAP negative mode matrix.

a. Prepare a solution consisting of 13.33 mM ammonium citrate/acetonitrile, 75/25 (solvent B).

b. Prepare a 2 mg/mL THAP solution in solvent B (see Note 9).

3.8. Preparation of Sample Spots

1. Positive mode analysis for neutral oligosaccharides. Spot a 0.5-^L aliquot of each sample and the neutral standard mix onto the stainless steel MALDI plate, then apply a 0.5-^L aliquot of the sDHB matrix on top of each sample spot. Place the spotted stainless steel plate into a small vacuum desiccator, and apply vacuum to quickly dry all the spots.

2. Negative mode analysis for sialylated oligosaccharides. Spot a 0.5-^L aliquot of each sample and the acidic standard mix onto the stainless steel MALDI plate, then apply a 0.5-^L aliquot of the THAP matrix on top of each sample spot. Place the spotted stainless steel plate into a small vacuum desiccator, and apply vacuum to quickly dry all the spots (see Note 10).

3.9. MALDI-TOF MS Analysis of Released Oligosaccharides (see Note 11)

1. Neutral oligosaccharides are analyzed in the positive mode and are detected as the sodium adducts. First, acquire spectra from the neutral oligosaccharide calibration mixture. Smooth the neutral oligosaccharide spectra using the Savitsky-Golay 19-point algorithm, then calibrate the instrument using the two-point external calibration method (see Note 12). After the instrument is calibrated, analyze the unknown samples in the positive mode.

a. Instrument settings: Positive mode, linear configuration, and accelerating voltage set at 20 kV; delayed extraction on and set at 60 ns; grid voltage at 93.2%; and guide wire voltage at 0.05%. The laser power setting must be empirically determined for each instrument. For neutral oligosaccharides, apply enough laser power to obtain good signal intensities, along with an acceptable signal-to-noise ratio. Too much laser power broadens the peaks. Also, desialylation of sialylated oligosaccharides can alter neutral glycan distribution. Currently, we are using a laser power setting of 1600 for neutral oligosaccharides, but this value can change as the laser ages or after installation of a new laser (see Note 13).

b. To ensure consistent results between analysts and across assays, spectra are acquired in the summing mode for 240 laser shots for each sample in the positive mode (see Note 14).

2. Sialylated oligosaccharides are analyzed in the negative mode. Acquire spectra from the acidic oligosaccharide calibration mixture, then smooth the acidic oligosaccharide spectra using the Savitsky-Golay 19-point algorithm. Use the smoothed spectra to calibrate the instrument using the two-point external calibration method (see Note 12). Following calibration, analyze the unknown samples in the negative mode.

a. Instrument settings: Negative mode, linear configuration, and accelerating voltage set at 20 kV; delayed extraction on and set at 140 ns; grid voltage at 92.2%; and guide wire voltage at 0.05%. Again, the laser power setting for sialylated oligosaccharides must be empirically determined for each instrument (see Note 15). Sialylated oligosaccharides are much more sensitive to laser power than are the neutral oligosaccharides because desialyation can occur at higher laser powers. Currently, we are using a laser power setting of 1900 for sialylated oligosaccharides. Yet, this setting varies based on laser age or the installation of a new laser (see Note 13).

b. For consistent results between analysts and across assays, spectra are obtained in the summing mode for 256 laser shots for each sample in the negative mode.

3. Samples can also be acquired in an automated mode if this feature is available on the MALDI-TOF MS instrument.

4. Smooth the acquired spectra using the Savitsky-Golay 19-point algorithm, and print a copy of the labeled spectra.

3.10. Interpretation of MALDI-TOF MS Spectra

1. When analyzing the MALDI-TOF MS spectra of released oligosaccharides, it is important to be aware of factors that can affect the quality and interpretation of the spectral data. Regarding data quality, two attributes are generally considered to be of primary importance in assessing the value of a spectrum: resolution and signal-to-noise ratio (see Note 16). However, when the purpose of acquiring the spectrum is to determine the oligosaccharide distribution, spectral resolution is not usually considered a limiting factor. Actually, the act of processing a spectrum for integration smoothes the spectrum, thereby removing the resolution of isotopic forms of the oligosaccharide. Alternatively, the signal-to-noise ratio is a factor that can affect the quality of the data obtained when assessing the oligosaccharide distribution (see Note 17).

2. When interpreting MALDI-TOF MS spectra, the analyst must be aware that the spectra contain peaks that result not only from the sodium oligosaccharide adducts of expected structures but from oligosaccharides associated with either other components present in the sample or present in the matrices used in ionization. As previously discussed (see Note 8), in positive-mode spectra, a common ion observed is from an oligosaccha-ride related to a potassium ion, in addition to a sodium ion. Another typically observed ion in the positive mode is an ion that results from the association of a sialylated oligosac-charide with one or more sodium ions present in the sDHB matrix. Generally, a series of peaks whose associated masses differ from one another by 22 Daltons is observed (Fig. 1). In most cases, there are (n + 1) ions in the series, where n is the number of sialic acid residues present on the sialylated oligosaccharide.

3. In the negative mode, a commonly observed ion is one that results from the association of a neutral oligosaccharide with the citrate present in the THAP matrix. This ion is found in the negative mode at a mass that is equal to the ion observed in the positive mode plus 168 Daltons. The difference in mass is due to the variation between the mass of the oli-gosaccharide related to a sodium ion in the positive mode and the mass of the oligosac-charide associated with a citrate ion in the negative mode. For example, in Fig. 2, the citrate adduct of the 2120 neutral oligosaccharide is the peak at 1977 Daltons (marked with an asterisk). Fortunately, when present, this ion tends to be at relatively low intensity.

4. After acquiring acceptable spectra (see Note 19) from all samples, convert the unsmoothed .ms spectra into .prn or .txt files, which contain x-axis values (mass information) and y-axis values (relative abundance) from each of the sample spectra. The .prn or .txt files are used by the integration program to obtain peak area values.

Fig. 1. Positive-ion mode spectrum of neutral oligosaccharides. Peaks identified as sialylated oligosaccharides associated with sodium are labeled with an asterisk (*). For an explanation of oligosaccharide nomenclature used in the Figs. 1-3, see Tables 1 and 2 (see Note 18).

Fig. 1. Positive-ion mode spectrum of neutral oligosaccharides. Peaks identified as sialylated oligosaccharides associated with sodium are labeled with an asterisk (*). For an explanation of oligosaccharide nomenclature used in the Figs. 1-3, see Tables 1 and 2 (see Note 18).

Fig. 2. Negative-ion mode spectrum of sialylated oligosaccharides. Peak identified as neutral oligosaccharides associated with citrate are labeled with an asterisk (*).

Table 2

Sialylated Oligosaccharides Structures, Nomenclature, and Masses

Mass Daltons

Acidic oligosaccharide structures Name (M - H-)

NeuAc - < Man-GlcNAc-GlcNAc A1, 2021 1931.7

NeuAc - < Man-GlcNAc-GlcNAc A1F, 2121 2077.9

Man-GlcNAc-GlcNAc A2, 2022 2223.0

Man-GlcNAc-GlcNAc A2F, 2122 2369.2

NeuAc - Gal - GlcNAc \ Man-GlcNAc-GlcNAc

Man^

NeuAc - Gal - GlcNAc

GlcNAc - Man\ Fuc

NeuAc - Gal - < Man-GlcNAc-GlcNAc 2111 1915.8

GlcNAc - Man

GlcNAc - Man\ 3121 2281.1

Gal - GlcNAc \ Man-GlcNAc-GlcNAc

Gal - GlcNAc

Table 2 (continued)

Acidic oligosaccharide structures

Name

NeuAc - 4 Gal - GlcNAc\ Man-GlcNAc-GlcNAc

Gal - GlcNAc/Man

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