SUPPLEMENTARY DATA
Investigating the Role of Artemin Glycosylation
Qiu Danwen1, Christian Code1, Chao Quan2, Bang-Jin Gong2, Joseph Arndt2, Blake Pepinsky2, Kasper D. Rand1*, and Damian Houde2*
1 Department of Pharmacy, University of Copenhagen, Denmark.
2 Biogen, Cambridge, MA, USA
* To whom correspondence should be addressed: and/or
Intact mass characterization of ART
After enzymatic or chemical preparation, all variant ART proteins were characterized by mass spectrometry. Approximately 10 µg of each ART isoform was denatured with 6M guanidine HCl and reduced with DTT for 1 hour at 37°C. The samples were subsequently loaded onto a protein macro trap from Michrom BioResources (Auburn, CA, USA) and eluted using a step gradient of acetonitrile and water with 0.1% formic acid and 0.01% TFA. An Agilent 1100 HPLC system (Foster City, CA, USA) coupled to a Qstar (ESI-q-TOF) mass spectrometer (Applied Biosystems, Framingham, MA, USA) was used for mass measurements. The TOF analyzer was calibrated using standard peptides (insulin fragment B). The mass spectra were acquired from m/z 2000 to 4000 in positive ion mode. The mass accuracy of the measurements was on average ± 0.005%. The intact ART electrospray mass spectra are shown in Supplementary Data Figure S1. The native form of this particular ART is routinely expressed with a heterogeneous N-terminus (about 50% of the protein is missing an N-terminal Ala residue). As well, ART is expressed with a heterogeneous glycan composition (Figure 1S, top panel). Mass spectrometry verified that all enzymatic reactions employed to achieve removal of the glycan preceded to > 99% conversion. All glycans were removed in the desialylated form (Figure S1, middle panel) and from the deglycosylated form (Figure S1, bottom panel).
Figure S1. Reduced mass spectra of wild type ART, wt, (top trace), desialylated ART, desia, (middle trace), and deglycosylated ART, degly, (bottom trace).
Released glycan analysis of ART
The analysis of ART release was performed in a 50 mM ammonium bicarbonate at 37°C for 24 hrs using N-glycosidase F (PNGaseF, Prozyme, GKE5006). Upon digestion, released glycans were isolated via reversed-phase solid phase extraction (RP-SPE). Hydrophilic/lipophilic blend (HLB) resin in 96 well blocks (Waters, WAT058951) was washed with 50% methanol and conditioned with 10% methanol prior to sample application. Samples were drawn through the HLB blocks by vacuum, and the sample flow-throughs containing the glycans were collected. The HLB wells were washed with 0.5 mL of 10% methanol, and the washes pooled with the sample flow-through. The effluents from RP-SPE were evaporated to dryness in a vacuum centrifuge. Released glycans were labeled with 2-aminobenzoic acid (2-AA) as per the method described by Bigge and Patel (1). The reaction temperature was changed to 45°C to preserve glycan sialylation. Excess labeling reagents were removed by paper chromatography and eluates were evaporated to dryness in a vacuum centrifuge. Labeled and dried glycans were reconstituted in water and briefly vortexed prior to chromatographic analysis. Labeled glycan mixtures were separated by anion-exchange chromatography on an AsahiPak NH2P 4D carbohydrate column developed with a linear gradient of ammonium acetate at pH 4.8. Column effluent was monitored by fluorescence using an excitation wavelength of 330 nm, and an emission wavelength of 420 nm (emission bandwidth = 40 nm). Glycan identification was made by comparing glycan profile (i.e., retention times) with those of individual glycan standards.
Figure S2. Chromatographic trace of released and 2AA labeled NBN glycans. Glycans were detected by fluorescence
Far-UV circular dichroism
Circular dichroism (CD) measurements were performed using a Chirascan Plus multi-functional spectrophotometer (Applied Photophysics, UK) with the following aperture and optical cells for far-UV CD: 0.2 cm aperture, 0.01 cm optical path length, and black walls flow-through quartz cuvettes. 0.5 mg/mL of sample was introduced into a fixed measuring compartment within the CD system using the Chirascan Plus autosampler technology (this allows the cuvette to remained fixed inside instrument’s sample holder, thus avoiding an array of cell handling errors). The sample is temperature equilibrated (30 ses) before scans were recorded and then averaged. Samples were run in triplicate and analyzed with a Measurement range of 200 to 300 nm and a bandwidth of 2 nm. The data averaging time was 2 sec and the analysis temperature was set to 20°C. A continuous flow of N2 (total 5 L/min) was used to flush the sample, lamp, and monochromator area. CD analysis was performed on both ART and degly-ART and no significant difference was observed, see Figure S3.
Figure S3. Far-UV CD analysis of ART (black solid line) and degly-ART (blue dotted line).
Hydrogen deuterium exchange mass spectrometry of ART
As described in the Materials and Methods section of this paper, ART was digested online using an immobilized pepsin cartridge and peptides were analyzed by LC-MS. Identification of the peptic fragments was accomplished through standard workflow using a combination of exact mass analysis and MSE using Protein Lynx Global Server software (Waters Corp). The peptide coverage map is shown below, Figure S4.
Figure S4. The peptide coverage map for ART. The blue and red bars represent those peptides detected in all samples analyzed and there is no significance to the color scheme. Also, the peptide numbering is off by 9 amino acids in this image, thus the peptide 28-37 in the text is represented in this figure as 19-28. This discrepancy is a function of the data analysis software used.
REFERENCES
1. Bigge J, Patel T, Bruce J, Goulding P, Charles S, Parekh R. Nonselective and efficient fluorescent labeling of glycans using 2-amino benzamide and anthranilic acid. Analytical biochemistry. 1995;230(2):229-38.
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