Zip tip c4

Some N-glycans were generated
by the degradation
of large N-glycan standards purchased from Omicron
Biochemicals, Inc., by using the enzyme α-mannosidase from Canavalia ensiformis and α-1–6 mannosidase.
The degradation reaction conditions were maintained according to the
manufacturer’s protocols. In brief, 1 μL of high-mannose N-glycan standard (1 mM) was added to the reaction buffer.
The reaction mixture for α-mannosidase from C.
ensiformis contained 15 μL of DI water, 2 μL
of GlycoBuffer 4 (10×), and 2 μL of zinc (10×), whereas
the reaction mixture for α-1–6 mannosidase contained
15 μL of DI water, 2 μL of GlycoBuffer 1 (10×), and
2 μL of BSA (100 μg/mL). The reaction mixture was incubated
at 37 °C with shaking at 800 rpm for 5 min before 0.1 μL
of α-mannosidase from C. ensiformis or α1–6 mannosidase was added. To obtain the desired
high-mannose N-glycans, mannosidase was removed from
the reaction mixture using 0.6 μL of ZipTip C4 (Merck, Ltd.,
Taipei, Taiwan) at different time points.

A sample taken out of the above storage solution was desalted prior to analysis:
A Zip Tip C4 (Merck) was rinsed with 10 µL H₂O (+0.1% trifluoroacetic acid), then H₂O, then H₂O/acetonitrile (3:2) and pushed dry. 10 µL of protein sample was loaded into the Zip Tip and washed with 10 µL H₂O. The sample was eluded with 10 µL H₂O/acetonitrile (3:2) directly onto the matrix. The sample was analysed by MALDI-TOF-MS (MALDI-TOF Autoflex Speed MS Bruker Daltonics Bremen, Germany) using 2′,6′-Dihydroxyacetophenone (DHAP) as matrix and a reflector method in positive ion mode.
The coupling efficiency of 9 ligands/protein was calculated by subtracting the mass of un-targeted GFP (27,094 Da) from the mass measured for t-GFP (31,500 Da) followed by division of the molecular weight of the ligand-linker construct (excluding the p-nitrophenyl leaving groups= 488 g/mol).

Protein masses were determined on purified solution samples. To this end, the proteins in 8 M urea containing buffer were loaded onto a G-25 column (Cytiva, Marlborough, MA, USA) and eluted with 10 mM ammonium acetate. After a concentration step using Zip Tip C4 (Merck Millipore Darmstadt, Germany), 1 μL of protein at ~1.5 mg/mL was mixed with 1 μL of sinapinic acid matrix solution in 0.3% TFA/CH₃CN (50:50 v/v). One μL of the mix was spotted on the target and analyzed by MALDI-TOF on a Ultraflex III spectrometer (Bruker Daltonics, Wissembourg, France) controlled by the Flexcontrol 3.0 package (Build 51) and operated in the linear mode, using a maximum accelerating potential of 25 kV and a 20,000–100,000 m/z range (LP_66kDa method). The laser frequency was fixed to 100 Hz and ~1000 shots per sample were cumulated. Four external standards (Protein Calibration Standard II, Bruker Daltonics) were used to calibrate each spectrum to a mass accuracy within 100 ppm. Peak picking was performed using the FlexAnalysis 3.0 software with an adapted analysis method. Parameters used were the centroid peak detection algorithm, S/N threshold fixed to 5 and a quality factor threshold of 30.