Data for analysis of mannose-6-phosphate glycans labeled with fluorescent tags

Mannose-6-phosphate (M-6-P) glycan plays an important role in lysosomal targeting of most therapeutic enzymes for treatment of lysosomal storage diseases. This article provides data for the analysis of M-6-P glycans by high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The identities of M-6-P glycan peaks in HPLC profile were confirmed by measuring the masses of the collected peak eluates. The performances of three fluorescent tags (2-aminobenzoic acid [2-AA], 2-aminobenzamide [2-AB], and 3-(acetyl-amino)-6-aminoacridine [AA-Ac]) were compared focusing on the analysis of bi-phosphorylated glycan (containing two M-6-Ps). The bi-phosphorylated glycan analysis is highly affected by the attached fluorescent tag and the hydrophilicity of elution solvent used in HPLC. The data in this article is associated with the research article published in “Comparison of fluorescent tags for analysis of mannose-6-phosphate glycans” (Kang et al., 2016 [1]).


Subject area
Biology, Chemistry More specific subject area

Glycobiology, Analytical biochemistry
Type of data Image, Figure (HPLC profile and Mass spectra), Graph How data was acquired M-6-P glycans labeled with fluorescent tags were analyzed by HPLC and MALDI-TOF mass spectrometry Data format Analyzed Experimental factors Mannosylphosphoryalted N-glycans obtained from the glyco-engineered yeast were converted to M-6-P glycans by the uncapping process using mild acid hydrolysis Experimental features M-6-P glycan peaks in HPLC analysis were identified by measuring the masses of the collected peak eluates.

Data source location
Daejeon, Republic of Korea

Data accessibility
The data are supplied with this article

Value of the data
The M-6-P glycan analysis data, which were obtained by HPLC and mass spectrometry after the labeling of three commonly used fluorescent tags (2-AA, 2-AB and AA-Ac), can be used for the comparison of their performances.
The hydrophilicity-optimized elution solvent in HPLC analysis can be used for proper detection and quantification of the bi-phosphorylated glycan (containing two M-6-Ps).
Careful analysis and interpretation are required when analyzing mannosylphosphorylated glycans by using the MALDI-TOF mass spectrometry because the acidic matrix preparation condition can convert some of them to M-6-P glycans.

Data
After the optimization of HPLC condition for the analysis of bi-phosphoyrlated glycan (containing two M-6-Ps), the 2-aminobenzoic acid (2-AA)-labeled glycan peaks were identified by measuring the masses of the collected peak eluates using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (Fig. 2). In contrast to 2-AA labeling, 2-aminobenzamide (2-AB) and 3-(acetyl-amino)-6-aminoacridine (AA-Ac) labelings enabled the detection of bi-phosphorylated glycan in HPLC without hydrophilicity optimization of elution solvent (Fig. 3). Fig. 4 shows the data of MALDI-TOF mass spectrometry analysis for the conversions of mannosylphosphorylated glycans to M-6-P glycans after the labeling of three fluorescent tags in order to compare their performances increasing detection sensitivity.

N-glycan preparation and fluorescent tag labeling
Most of the therapeutic enzymes for treatment of lysosomal storage diseases require M-6-P glycan, which are recognized by the M-6-P receptor on the plasma membrane for lysosomal targeting [2]. Although yeasts do not have M-6-P glycans in nature, some of their glycans containing mannosylphosphate residue can be converted to M-6-P glycans through the uncapping process to remove the outer mannose residue. Recently, we developed a glyco-engineered Saccharomyces cerevisiae och1Δmnn1Δ/YlMpo1 strain generating high content of mannosylphosphorylated glycans which can be converted to M-6-P glycans [3]. From this yeast, N-glycans were prepared as described previously [3]. Briefly, yeast cell wall mannoproteins were extracted by using hot citrate buffer and subsequent precipitation with ethanol. The obtained mannoproteins were digested to glycopeptides, followed by glycopeptide purification using a C18 Sep-Pak cartridge as previously described [4]. N-glycans were released from the purified glycopeptide by Peptide-N-glycosidase F (New England Biolabs, Ipswich, MA, USA) treatment at 37°C overnight and purified by solid-phase extraction using graphitized carbon (Alltech, Lexington, MA, USA) [5].
The purified glycans were fluorescently labeled with 2-AA, 2-AB, or AA-Ac; the structures of these fluorescent tags are represented in Fig. 1. 2-AA and 2-AB were purchased from Sigma-Aldrich (St. Louis, MO, USA), while AA-Ac was obtained from Ludger Ltd. (Oxfordshire, UK). The labeling reagent was freshly prepared by dissolving 6 mg of 2-AA, 2-AB, or AA-Ac in 100 μl of dimethyl sulfoxide/acetic acid (7:3, v/v) containing 1 M of sodium cyanoborohydride. Dried glycans were dissolved in 5 μl of each labeling reagent and mixed thoroughly. The reaction mixture for 2-AA or 2-AB labeling was incubated at 37°C overnight, while the one for AA-Ac labeling was incubated at 80°C for 30 min. The resulting 2-AA-, 2-AB-, or AA-Ac labeled glycans were purified from unreacted labeling reagents using cyano-SPE cartridge (Agilent Technologies, Santa Clara, CA) as described previously [5].

HPLC analysis of 2-AB-and AA-Ac-labeled glycans
M-6-P glycans fluorescently labeled with 2-AB or AA-Ac were analyzed by HPLC using the same conditions described in Section 2.2. Wavelengths for fluorescence detection were adjusted for 2-AB (Ex 330 and Em 420 nm) and AA-Ac (Ex 442 and Em 525 nm). Fig. 3 shows the profiles of 2-AB-and AA-Aclabeled glycans obtained from HPLC analysis using solvents A and B (without the increased hydrophilicity). Notably, the bi-phosphorylated glycan was detected at 58 and 23 min in 2-AB-and AA-Aclabeled glycan profiles in this condition, which is in sharp contrast with the result for 2-AA-labeled glycans requiring the use of elution solvent B-h (with the increased hydrophilicity) for the detection of biphosphorylated glycan (see the Fig. S1A and B in Ref. [1]). All 2-AB-and AA-Ac-labeled M-6-P glycans eluted later in the HPLC analysis using solvents A and B compared with the analysis using solvents A and B-h (see the Fig. S1C in Ref. [1]); especially, the elution times of bi-phosphorylated glycans labeled with 2-AB and AA-Ac on using solvent B were 8 and 2 min later than those on using solvent B-h (52 and 21 min).

MALDI-TOF mass spectrometry analysis of M-6-P glycan
Glycans were analyzed using a Microflex MALDI-TOF mass spectrometry (Bruker Daltonik, GmbH, Bremen, Germany) as previously described [5] with a slight modification. Briefly, the labeled glycans were spotted on the MALDI MSP96 polished steel chip (Bruker Daltonik) and then 6-Aza-2thiothymine (ATT)/2,5-dihydroxybenzoic acid (DHB) matrix solution was added, followed by drying in air. All mass spectra were acquired in a linear negative ion mode using the method recommended by the manufacturer because the mannosylphosphorylated and phosphorylated glycans have , and AA-Ac (C) labeling, the masses of mannosylphsphorylated N-glycans (upper panels) and M-6-P glycans generated through uncapping (lower panels) were analyzed. Notably, several glycans containing M-6-P were observed in the mass spectra of mannosylphsphorylated N-glycans. It seems that the matrix preparation condition for mass analysis, which is acidic, induces partial uncapping of mannosylphosphorylated N-glycans. Symbols are identical to those used in Fig. 2. negative charges in their phosphate groups. Due to the low resolution of linear negative mode, we experienced some deviations (up to $ 2 Da) from theoretical mass values. Fig. 4 shows the analysis results of 2-AA-, 2-AB-, or AA-Ac-labeled mannosylphosphorylated glycans and their uncapped forms containing M-6-Ps, which suggested that some of the mannosylphosphorylated glycans were uncapped during the acidic matrix preparation step.