Enhanced Detection of Glycans by MALDI-TOF Mass Spectrometry Using a Binary Matrix of 2,5-Dihydroxybenzoic Acid and 2,6-Dihydroxybenzoic Acid

: Glycans released from ovalbumin by PNGase F were analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry using three different dihydroxybenzoic acid (DHB) matrix systems: 2,5-DHB, 2,6-DHB, and a 2,5-DHB/2,6-DHB binary matrix. Relative to the results obtained with the single-component matrices (2,5-DHB or 2,6-DHB), the 2,5-DHB/2,6-DHB binary matrix boasted lower background noise and higher sensitivity. A total of 16 glycan peaks were observed using the 2,5-DHB/2,6-DHB binary matrix, while only 10 and 9 glycan peaks were observed using the 2,5-DHB and 2,6-DHB matrices, respectively.


Introduction
Glycosylation is one of the most common post-translational modifications in biology. 1 Effective and reliable analytical methods for glycans are important because the products of glycosylation are often present in clinical biomarkers. 2 Recent developments in mass spectrometry (MS) have provided rapid and sensitive methods for the detection of a variety of glycans. 3 Among the different matrices used in matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, 2,5dihydroxybenzoic acid (2,5-DHB) is one of the most common and is frequently used for analyses of synthetic polymers, 4 peptides/proteins, 5 and oligosaccharides. 6 Recently, 2,6-DHB was introduced as an alternative matrix for the analysis of oligonucleotides, polysaccharides, 7 and synthetic polymers. 4 The combination of two matrix materials has been effective in several MALDI-TOF MS analyses. For example, a binary matrix of 2,5-DHB and α-cyano-4-hydroxycinnamic acid (CHCA) was effectively used to detect peptides and glycoproteins. 8 Recently, binary matrices of 2,5-DHB/CHCA and 2,5-DHB/sinapinic acid were reported in successful analyses of intact glycoproteins and glycans. 9 The current study investigated the effectiveness of a new binary matrix composed of 2,5-DHB and 2,6-DHB in analyses of glycans released from ovalbumin by enzymatic cleavage with PNGase F. The mass spectrum acquired with the binary matrix was compared with those obtained using the single matrix materials (2,5-DHB or 2,6-DHB).

Experimental
All chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified. To perform deglycosylation, 2 mg of ovalbumin (catalog number: A5503) was mixed with 1 unit of PNGase F (catalog number: P7367) in 200 µL of 50 mM ammonium bicarbonate buffer solution for 2 h at 37 o C with gentle mixing. To prepare the 2,5-DHB/2,6-DHB binary matrix solution, a mixture of 2,5-DHB (5 mg) and 2,6-DHB (5 mg) was dissolved in 1 mL of 50% acetonitrile (ACN) in H 2 O. For comparison, individual DHB matrices (2,5-DHB and 2,6-DHB) were also prepared to a concentration of 10 mg/ mL in 50% ACN. To prepare the sample targets for MALDI-MS analysis, 0.6 µL of the matrix solution was loaded and allowed to dry on a MALDI plate and covered with 1 µL of the mixed sample/matrix solution (1:1, v/v). MALDI spectra were obtained on a Voyager MALDI-TOF mass spectrometer (Applied Biosystems) in reflection positive ion mode.

Results and Discussion
Improvements realized with the 2,5-DHB/2,6-DHB binary matrix Figure 1 shows MALDI mass spectra of the glycans released from ovalbumin using PNGase F. Note that the highest number of glycans were detected using the binary matrix of 2,5-DHB/2,6-DHB. Table 1 summarizes the glycans identified in the current investigation along with the published results of other researchers. A total of 16 glycans were identified using the 2,5-DHB/2,6-DHB binary matrix, while only 10 and 9 glycans were identified when 2,5-DHB and 2,6-DHB were used independently, respectively. Harvey et al. identified 13 glycans from MALDI-TOF mass spectra *Reprint requests to Prof.Jeongkwon Kim E-mail: jkkim48105@cnu.ac.kr obtained from HPLC-separated ovalbumin with a 2,5-DHB matrix. 10 Snovida et al. used a 2,5-DHB/N,N-dimethylaniline (DMA) binary matrix to improve the MALDI detection efficiency of glycans. In that study, 11 glycans released from ovalbumin by PNGase F were identified, while no glycans were identified when using a 2,5-DHB matrix. 11 Overall, the number of glycans observed using the 2,5-DHB/2-6-DHB binary matrix was higher than those detected using any other published matrix systems, including 2,5-DHB, 2,6-DHB, and 2,5-DHB/DMA.

Effects of other additives
The addition of phosphoric acid has been reported to enhance the signal intensities of phosphopeptides 12 and nonphosphopeptides. 13 Therefore, the addition of phosphoric acid was also investigated with regard to glycan analyses. No improvements were observed in any of the matrix systems used in the current study (2,5-DHB, 2,6-DHB, and 2,5-DHB/ 2,6-DHB). This is thought to be due to the fact that the current glycan detection requires sodium ion adducts in order to be detected by MALDI-MS. The effect of adding trifluoroacetic acid (TFA) to the matrix solutions was also investigated; no improvement was observed with all three matrix systems. Because the sodium adducts are the detected entity in positive ion mode MALDI-MS analysis of glycans, sodium cations, as  a) Mixture of 2,5-DHB and 2,6-DHB b) Glycans were released from purified ovalbumin with PNGase F using an in-gel release technique. c) DMA: N,N-dimethylaniline NaTFA or NaCl, are often added to improve detection. 6 However, the addition of NaTFA or NaCl to the sample solutions herein did not improve detection.

Reasons for enhanced signals
Uniform sample distribution within the MALDI sample spot, due to homogeneous crystallization, 9 is believed to be the primary factor in realizing the observed improvement in glycan detection efficiency. In addition, synergistic effects of 2,5-DHB and 2,6-DHB, both of which have been reported as matrix materials in glycan analyses, are believed to enhance detection efficiency.