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MPI für Biochemie  

Proteomics and Signal Transduction
Matthias Mann

N-Glyco-FASP

 

N-Glyco FASP protocol as pdf

N-Glyco-FASP: Protocol for Generation of Deglycosylated Peptides from Tissue Samples for Mass Spectrometric Analysis

1. FASP (Filter Aided Sample Preparation) 

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) 
 fasp
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.