Sample Preparation Methods

Reliable methods for preparing samples for AAA

  • RP-HPLC in aqueous TFA/acetonitrile solvents.
  • Dialysis into an appropriate buffer or volatile solvent (e.g., water).

Useful Concentration Methods

Savant Speed-Vac

  • Useful for concentrating sample prior to hydrolysis.
  • Used extensively for the drying steps in hydrolysis and derivatization procedures.
  • Be sure to keep the unit clean.

Amicon Centricon

  • Useful for exchanging solvents as well as concentrating samples.
  • Generally faster than dialysis.
  • Be aware of exclusion limits and use the appropriate centricon size for your sample.

Precipitation

  • Organic solvents such as ethanol, acetone, chloroform/methanol and trichloroacetic acid are useful for precipitating proteins and removing salts and detergents.
  • An acetone precipitation procedure is attached.

Acetone Precipitation

This procedure is suitable for recovering proteins from most aqueous solvents and from SDS containing buffers. It is not recommended for proteins dissolved in urea or guanidine or for peptides. Practice the precipitation procedure with a readily available protein before attempting use with a precious sample.

1. The sample volume should be in the range of 20 µL - 2 ml. For less than 10 mg protein, the volume should be reduced to less than 500 ml. This can be accomplished by partial speed vac drying or by several extractions with excess ethyl acetate.

2. Add two volumes of acetone (Burdick and Jackson) and vortex. The precipitated protein will be visible, usually as a flocculent, white particulate material. A magnifying glass can help the non-believers or those with failing vision.

3. If no precipitate is apparent, add about 1/2 volume ethyl acetate, vortex again.

4. If still no precipitate is apparent, change pH slightly. If the starting solution is alkaline, add a few microliters of N-ethyl-morpholine. The protein will precipitate at its isoelectric pH.

5. Centrifuge and carefully remove the supernatant. Save the supernatant if the location of your sample is questionable.

6. When desalting for sequence and/or amino acid analysis, the pellet can be washed carefully with a small volume of 67% acetone, blown dry with a gentle stream of nitrogen and resuspended in 100% trifluoroacetic acid.

7. For proteolytic digestion, the pellet usually need not to be washed. This procedure has been particularly useful for removing SDS from samples after electrophoretic purification and electroelution.

Appropriate Solvents and Buffers for Amino Acid Analysis

  • Sample conditions for post-column amino acid analyzers are less restrictive than for pre-column analyzers. Post-column analyzers can tolerate a variety of salts and detergents in small amounts.

  • Salts, detergents and metal ions in samples for PTC amino acid analysis can interfere with accuracy. Dupont et al., has listed of the effects of common buffer salts and detergents in PTC-AAA. (1)

  • Volatile solvents are the solvents-of-choice for PTC-AAA. Below are some sample solvents that we find useful for PTC-AAA:

    Water (HPLC grade)
    30-50% methanol
    30-50% ethanol
    30-50% acetonitrile
    0.1-1% N-ethyl morpholine-acetate pH 8-9
    0.1N HCl
    0.1%-neat organic acid e.g. trifluoroacetic acid, acetic acid or formic acid
    1 mM MOPS pH 7
    4-morpholinepropanesulfonic acid

1 D.R. Dupont et al. (1989) In: Techniques in Protein Chemistry (T.E. Hugli, ed.) Academic Press, pp. 284-294.

Cysteine Determination

Requires chemical modification of Cys prior to analysis. Oxidation, alkylation, and disulfide exchange are three general approaches used. Use Cys-containing control proteins to calculate response factors.

Summary of Cystine/Cysteine Analyses (1)

Cys Method Total SitesAverage % Cys ErrorStd DevSites with < 10% Cys Error
a. Performance acid oxidation2415.811.510 (42%)
b. Dimethylsulfoxide oxidation919.416.34 (44%)
c. Pyridylethylation35.13.33 (100%)
d. Carboxymethylation255.4 -0
e. Dithiodipropionic acid641.148.73 (50%)
f. Dithiodiglycolic acid3 33.926.61 (33%)
g. Direct Analysis642.120.20

Performance of Cysteine Methodology, ABRF-91AAA and -92AAA Combined (1)

MethodTotal SitesSites with < 10% Cys Error
Oxidation 5622 (39%)
Alkylation96 (67%)
Disulfide Exchange13 7 (54%)
Total78 35 (45%)

1 D.J. Strydom et al. (1993) In: Techniques in Protein Chemistry IV (R.H. Angeletti, ed.) Academic Press, pp. 279-288.

Pyridylethylation (2)

1. Mix 1 vol. 1M Tris-HCl, pH 6.5 containing 4 mM EDTA with 3 vol. 8 M guanidine-HCl.
2. Dissolve or dilute sample with Ó50 µL of above solution.
3. Add 2.5 µL freshly prepared 10% 2-mercaptoethanol and incubate at room temperature under argon for 2 hours.
4. Add 2 µL 4-vinylpyridine (neat); mix and incubate as above.
5. Desalt immediately on a short C4 RP-HPLC column or extensively dialysis or Centricon preparation.
6. Hydrolyze and analyze for Pyridylethyl-Cysteine.

2 D. Hawke and P. Yuan (1987) ABI User Bulletin No. 28.

Quantification of In-Gel Samples

  • Gel pieces can be hydrolyzed for AAA on post-column instruments.

  • Coomassie stained polyacrylamide gel bands are excised, washed with water and a portion (e.g., 10%-20%) dried in a hydrolysis vial for liquid phase HCl hydrolysis. Following hydrolysis, centrifuge the tube and transfer the clear HCl hydrolysate to another tube and dry. Resuspend in appropriate buffer and apply to an ion-exchange post-column amino acid analyzer.

  • Compositional accuracy is typically low (~20% error) from in-gel samples but sufficient for obtaining approximate protein concentration. Such quantification is useful for determining the amount of protease to use for in-gel digestions.

S. Stein and L. Brink (1981) In: Methods in Enzymology, Vol. 79 (S. Pestka, ed.) Academic Press, pp. 25-27.
K.R. Williams and K.L. Stone (1995) In: Techniques in Protein Chemistry VI (J.W. Crabb, ed.) Academic Press, pp. 143-152.

AAA of PVDF Samples

  • General Method. PVDF immobilized proteins are hydrolyzed in liquid or vapor HCl; amino acids extracted with organic solvent and analyzed by pre- or post-column AAA methods.

  • AAA of PVDF samples can be problematic. Average compositional errors >20% have been observed in two recent collaborative studies (ABRF-90AAA3, n=30; ABRF-95AAA, n=77 participants).
  • Quantification of immobilized sample is usually approximate because recovery of extracted AAs is variable (e.g., 70-90% yield at best).
  • PVDF contains variable background amino acids. Methods for stripping away the background are evolving but the best approach is not yet apparent.
  • Blank PVDF may be analyzed to quantify background. Subtracting background from experimental data assumes that blank and experimental samples have similar background. Caution: this may not be true.

F. Gharahdaghi et al. (1992) In: Techniques in Protein Chemistry III (R.H. Angeletti, ed.) pp. 249-260.
G.E. Tarr et al. (1991) In: Techniques in Protein Chemistry II (J.J. Villafranca, ed.) Academic Press, pp. 139-150;
A.M. Mahrenholz et al. (1996) In: Techniques in Protein Chemistry VII (D.R. Marshak, ed.) Academic Press, in press.

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