2
Experimental Conditions
Chemicals and Reagents
• Deionized (DI) water, 18.2 M
Ω
-cm resistivity
• Acetonitrile (CH
3
CN), HPLC grade (Fisher Scientific
™
,
AC610010040)
• LC/MS grade formic acid (Fisher Scientific, A117-50)
• Ammonium formate (Fisher Scientific, AC40115-2500)
• Thermo Scientific Premium 2 mL vial convenience kit,
60180-600
• PNGase F (New England BioLab, P0705L)
• Bovine fetuin (Sigma-Aldrich
®
, F2379)
• Thermo Scientific
™
Hypercarb
™
cartridge, 6 mL,
60106-403
• Trifluoracetic acid (Fisher Scientific, 28904)
• Sodium cyanoborohydride (Fisher Scientific,
AC16855-0500)
• Anthranilamide (2AB) (Fisher Scientific, AC10490-5000)
• Glacial acetic acid (Fisher Scientific, AA36289AP)
• Dimethylsulfoxide (DMSO) (Fisher Scientific,
D128500LC)
• Sodium hydroxide (NaOH) (Fisher Scientific,
S318-100)
• Ammonium acetate (Fisher Scientific, A637-500)
• SEC column, 0.9 x 50 cm Sephadex
®
(GE Healthcare,
G-10-120)
• GlykoClean
™
G Cartridges, Prozyme, GC250
• 2-mercaptoethanol (Fisher Scientific, O3446I-100)
Equipment
• Thermo Scientific
™
Dionex
™
UltiMate
™
3000 system,
including pump: LPG-3400RS, thermal compartment:
TCC-3000RS, pulled-loop well plate auto sampler:
WPS-3000TRS, fluorescence detector with Dual-PMT:
FLD3400RS, and 2µL micro flow cell: 6078.4330
• Q Exactive hybrid quadrupole-Orbitrap mass
spectrometer
• Thermo Scientific
™
SpeedVac
™
Concentrator
• Thermo Scientific Lyophilizer (Labconco
®
FreeZone
®
-105 ºC 4.5 L benchtop freeze dry system) 16-080-207
• Thermo Scientific 24-Port SPE vacuum manifold,
60104-233
Buffer Preparation
• Ammonium formate (80 mM, pH 4.4):
Dissolve 5.08 ± 0.05 g of ammonium formate (crystal)
and 0.60 g of formic acid in 999.6 g of DI water.
Sonicate the resulting solution for 5 min.
• 0.1 M sodium phosphate buffer, pH 7.25:
Add 102.24 mg of Na
2
HPO
4
and 38.14 mg of
NaH
2
PO
4
to 10 mL of DI water. Vortex to mix the
solid completely. Verify that the pH of the solution is
7.25 ± 0.02.
Release of
N
-Glycans from Proteins
1. Dissolve 1 mg of the bovine fetuin protein in 500 µL
of 0.1 M sodium phosphate buffer, pH 7.2 ± 0.05, in
an Eppendorf tube.
2. Add 0.5 µL of 2-mercaptoethanol to this solution.
3. Finally, add 50 U (units) of PNGase F and incubate
total solution at 37 ºC water bath for 18 h.
4. Cool to room temperature and purify the released
glycans as described in the next section.
Purification of
N
-Glycans
Purify free glycans after digestion using a Hypercarb
cartridge as follows:
1. Attach a single Hypercarb cartridge per reaction to a
designated port in the SPE manifold.
2. Slowly, and with a consistent flow rate, pre-treat each
cartridge with the following volumes of reagents in the
order described: 15 mL of 1MNaOH, 15 mL of HPLC
grade water, 15 mL of 30% acetic acid, 15 mL of HPLC
grade water.
3. Prime the cartridge with 15 mL of 50% acetonitrile/0.1%
trifluoroacetic acid (TFA), followed by 15 mL of 5%
acetonitrile/0.1% TFA.
4. Load the entire sample volume into the cartridge and let
it permeate into the resin by pulsing the vacuum on and
off quickly.
5. Rinse the reaction tube with ~50 µL of HPLC grade
water, transfer into the cartridge, and pulse the vacuum
again.
6. Wash the cartridge with 15 mL of HPLC grade water,
followed by 15 mL of 5% acetonitrile/0.1% TFA.
7. Elute the glycans with 4 x 2.5 mL of 50% acetonitrile/
0.1% TFA into a labeled 15 mL conical tube.
8. Immediately freeze samples on dry ice and then lyophilize
to dryness (16–24 h).
9. After lyophilization, dissolve the solid in 1 mL of water,
dry the samples again in a 1.5 mL Eppendorf tube, and
store at -20 ˚C.
