Never mind.. I got the following N-Glyco FASP protocol from Max-Planck Institute of Biochemistry

Solutions and Reagents

1.40 mM NH4HCO3 in water (ABC)

2.0.1 M Tris/HCl pH 8.5

3.20% SDS in 0.1 M Tris/HCl pH 7.6

4.1 M DTT in (frozen stock)

5.Trypsin, stock 0.4 µg / µl (Promega)

6.0.55 M iodoacetamide in ABC

7.2 x binding buffer (2xBB): 40mM Tris/HCl pH 7.6, 2mM MnCl2, 2mM CaCl2, 1 M NaCl

8.Extraction buffer (EB): 4% SDS, 0.1 mM DTT in 0.1 M Tris/HCl pH 7.6

9.8 M urea solution in 0.1 M Tris/HCl pH 8.5 (UA)

10.0.05 M iodoacetamide in UA (IAA)

Note: EB, UA and IAA solutions must be freshly prepared and used within a day

Equipment

1.IKA Ultra Turrax blender (T 10 basic Ultra, IKA, Staufen, Germany)

2.Branson Sonifier 250 (Heinemann, Schwäbisch Gmünd, Germany)

3.Filter unit Vivacon 500(30,000 MWCO) (Sartorius Stedim Biotech; cat no. VN01H21)

4.Refrigenerated Bench-top centrifuge, temperature 20°C

5.Thermo-mixer

6.UV-Spectrophotometer, Quartz-cuvettes

1.1 Lysis and thiol-reduction

1.Add EB solution to tissue (fresh or frozen); Examples: Mouse brain + 4 ml EB; Mouse liver + 12 ml EB; Mouse kidney + 4 ml EB; Mouse heart + 2 ml EB; Mouse blood plasma (0.5ml) + 1 ml EB

2.Homogenize with a blender (20 s, max speed)

3.Incubate at 95°C for 3 min

4.Sonicate (outputcontrol 4, 20 sec)

5.Centrifuge at 16,000 x g for 10 min

Note : Avoid temperatures below 15°C and potassium salts to avoid precipitation of SDS.

1.2 Protein digestion

1.Mix up to 200–400 µg of a protein extract with 200 μl of UA in the filter unit, mix at 550 rpm in a thermo-mixer for 1 min and centrifuge at 14,000 x g for 15 min

2.Add 200 μl of UA to the filter unit and centrifuge at 14,000 x g for 15 min

3.Discard the flow-through form the collection tube

4.Add 100 μl IAA solution and mix at 550 rpm in a thermo-mixer for 1 min and incubate without mixing for 20 min in the dark

5.Centrifuge at 16,000 x g for 10 min

6.Add 100 μl of UA to the filter unit and centrifuge at 14,000 x g for 15 min. Repeat this step twice

7.7. Add 100 μl of ABC to the filter unit and centrifuge at 14,000 x g for 10 min. Repeat this step twice

8.Transfer the filter units to new collection tubes

9.Add 2 µg – 4 µg trypsin in ABC and mix at 550 rpm in thermo-mixer for 1 min

10.Incubate the units in a wet chamber at 37°C overnight

11.Centrifuge the filter units at 14,000 x g for 10 min

12.Add 40 μl 1xBB and centrifuge the filter units at 14,000 x g for 8 min. Repeat this step

Note : Alternatively other enzymes instead of trypsin can be used

1.3 Yield determination

1.Transfer peptide solution in Quartz-cuvette and record a spectrum from 240-340 nm

2.Transfer the peptide solution from the cuvette directly on a new filter unit

3.Incubate at 95°C for 3 min

4.Sonicate (outputcontrol 4, 20 sec)

5.Centrifuge at 16,000 x g for 10 min

Note :The spectrum should have a distinct peak with a maximum at 270-280 nm. A solution of (1mg/ml solution has 1.1 au)

2 Filter Aided Capture and Elution (FACE):N-glycopeptide enrichment, deglycosylation, 18O-labeling

Solutions and Reagents

1.H218O (97%, OLM-240-97, CIL)

2.40 mM NH4HCO3 in H218O (ABC18O)

3.100 U PNGase F (Roche) in 100 µl H218O

4.6 mg Concanavalin A (Sigma) in 1 ml 2xBB (ConA)

5.6 mg wheat germ agglutinin (Sigma) in 1 ml 2xBB (WGA)

6.18.89 mg / ml buffered aqueous RCA120 agglutinin solution (Sigma) (RCA)

7.CWR: 15 µl ConA, 15 µl WGA, 6 µl RCA. Prepare 36 µl per 1 sample

2.1. N-Glycopeptide enrichment and deglycosylation

1.Add 36 µl CWR to the filter units containing peptides, mix at 550 rpm in a thermo-mixer for 1 min and incubate without mixing for 60 min

2.Centrifuge the filter units at 14,000 x g for 10 min

3.Add 200 µl 1xBB and centrifuge the filter units at 14,000 x g for 8 min. Repeat this step twice

4.Discard the flow-through form the collection tube

5.Add 200 µl 1xBB and centrifuge the filter units at 14,000 x g for 8 min. Repeat this step

6.Add 50 µl ABC18O and centrifuge the filter units at 14,000 x g for 8 min. Repeat this step

7.Transfer the filter units to new collection tubes

8.Add 2 U PNGase in 40 µl ABC18O, mix at 550 rpm in thermo-mixer for 1 min

9.Incubate the units in a wet chamber at 37°C for 3 h

10.Centrifuge the filter units at 14,000 x g for 8 min

11.Add 40 µl ABC and centrifuge the filter units at 14,000 x g for 8 min. Repeat this step

Note : Alternatively 1 lectin per sample can be used. Instead 36 µl CWR mixture use 36 µl ConA, or 36 µl WGA (prepared in 0.1M Tris/HCl pH7.6) or 18 µl RCA

References:

1. Wisniewski et al. (2009) Universal sample preparation method for proteome analysis. Nat Methods, 6, 359

2. Zielinska et al. (2010). Precision Mapping of an In Vivo N-linked Glycoproteome Reveals Rigid Topological and Sequence Constraints. Cell 141, 5, 897-907

3. Ostasiewicz et al (2010) Proteome, N-Glycoproteome and Phosphoproteome are Quantitatively Preserved in Formalin-Fixed Paraffin-Embedded Tissue and Analyzable by High-Resolution Mass Spectrometry. J. Proteome Res. 9:3688-3700.

Did you manage to make this work? I've tried several times and I encountered some obstacles. First, yields from the initial FASP preparation is surprisingly low, according to Zielinska et al you need approx 100 µg, I never got close to that. Also, the lectins seem to behave weird... when I dissolve them, they seem ok, but after mixing with the peptides they seem to precipitate. I don't know if they are worth something after that. And lastly, 3 hours seem to me too short incubation time for PNGase F to work. I've tested it and after 3 hours, only a fraction of glycoproteins were shifted on SDS-PAGE, whereas ofter over-night incubation, the enzyme deglycosylated a major part.

Nice Contribution