Effect of Cationization Agent Concentration on Glycan Detection Using MALDI TOF-MS

: The effect of cationization agent concentration on glycan detection via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was investigated using Na + ions in the form of NaCl as the cationization agent. NaCl solution concentrations ranging from 1 mM to 1 M were investigated. Glycans from ovalbumin were mixed with the cationization agent solution and the 2,5-dihydroxybenzoic acid (2,5-DHB) matrix solution in a volume ratio of 1:1:1. The resulting mixture was loaded onto the MALDI plate. Two MALDI-TOF MS instruments (Voyager DE-STR MALDI-TOF MS and Tinkerbell RT MALDI-TOF MS) were used for detection of glycans. The best detection, in terms of the number of identified glycans, the peak intensity, and the signal-to-noise (S/N) ratio, was obtained with NaCl concentrations of 0.01–0.1 M for both MALDI-TOF MS instruments.


Introduction
Glycosylation is a common post-translational protein modification. 1 Glycans are products of glycosylation that play key roles in biological processes. 2,3 N-glycans are commonly released through deglycosylation of glycoproteins using peptide N-glycosidase F (PNGase F) 4 and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). 5 A cationization agent is commonly added to glycan samples to improve the ionization of glycans during positive-ion-mode MALDI-TOF MS analysis. Among cations, Na + is the most commonly used as a cationization agent in the form of sodium chloride 6-10 , sodium acetate [11][12][13][14] , sodium hydroxide 15 , or sodium iodide 16 . Na + ions have been used as a cationization agent in the concentration range of 1 mM 6,[11][12][13]15 to 20 mM 14 .
In this study, we investigated the effect of cationization agent concentration on the detection of glycans using MALDI-TOF MS. NaCl was selected as the cationization agent, and 2,5-dihydroxybenzoic acid (2,5-DHB) was used for the matrix solution. NaCl concentrations ranging from 1 mM to 1 M were prepared for the current investigation, and glycans were obtained via deglycosylation of ovalbumin. Mixtures of the matrix solution, glycan sample, and NaCl (1:1:1, v/v/v) were loaded onto a MALDI plate and analyzed using MALDI-TOF MS.
Ovalbumin (10 mg) was dissolved in 1 mL of 50 mM ABC buffer to prepare the ovalbumin stock solution. To release N-glycans from ovalbumin, PNGase F was added in a ratio of 1 unit per 200 µL of ovalbumin stock solution and mixed gently for 2 h at 37 o C (500 rpm). To prepare the matrix solution, 2,5-DHB (10 mg) was dissolved in 1 mL of 50% ACN/1% PA aqueous solution. 17 Na + ions were used as the cationization agent, and NaCl solutions were prepared at concentrations ranging from 1 mM to 1 M (1 mM, 0.01 M, 0.05 M, 0.1 M, and 1 M).
A MALDI plate target sample was prepared by loading 1.5 µL of a mixture of the matrix, glycan sample, and NaCl (1:1:1, v/v/v). MALDI mass spectra were recorded in positive-ion reflectron mode using a Voyager DE-STR

Results and discussion
To investigate the effect of cationization agent concentration on the detection of glycans, various concentrations of Na + were prepared and mixed with the glycan sample solution and the matrix solution in a ratio of 1:1:1 (v/v/v). A small amount of the mixture (1.5 µL) was then loaded onto the MALDI plate and analyzed using two different mass spectrometers (Voyager MALDI-TOF MS and Tinkerbell MALDI-TOF MS). Figure 1 shows the MALDI mass spectra of glycans from ovalbumin for various concentrations of Na + . The two MALDI-TOF MS instruments provided very similar glycan profiles, in which the most and second-most abundant peaks were observed at m/z 1,745.5 and 1,542.5, respectively. The maximum intensity was observed with 0.05 M Na + for the two instruments. Table 1 summarizes the glycans identified in the current study for the mass spectra shown in Fig. 1. The Voyager MALDI-TOF MS exhibited similar high numbers of identified glycans using Na + concentrations of 0-0.1 M, while the Tinkerbell MALDI-TOF MS provided similar high numbers of identified glycans using Na + concentrations of 1 mM to 1 M.   Figure 2 shows the change in the signal-to-noise (S/N) ratio for the three most abundant glycan peaks: Hex 3 (HexNAc) 6 Na at m/z 1,746.58, Hex 3 (HexNAc) 5 Na at m/z 1,543.39, and Hex 4 (HexNAc) 6 Na at m/z 1,908.73. For both MS instruments, 0.05 M NaCl provided the best S/N ratio for the Hex 3 (HexNAc) 6 Na peak at m/z 1,746.58 among the NaCl concentrations used in this study. For Na + concentrations that were too low (e.g., 0 M) or too high (e.g., 1 M), the S/N ratio and the number of identified glycan peaks decreased (Table 1).

Conclusion
The effect of cationization agent concentration on glycan detection using MALDI-TOF MS was investigated. The dried-droplet deposition method was used to load a mixture of equal volumes of cationization agent, glycan sample, and matrix solution. Based on the number of identified glycans and the S/N ratio, Na + concentrations ranging from 0.01 to 0.1 M were shown to optimize the detection of glycans.