Created: 26th February 1997, last updated: 23rd September 1998 © 1998 ABRF
Correspondence to: bill_kopaciewicz@Millipore.com
A variety of approaches are used to link the unparalleled resolution of SDS PAGE with biochemical methods appropriate for protein structural analysis. Efforts in most recent years have been relatively successful in identifying proteins from 1D and 2D gels following enzyme digestion from nitrocellulose, PVDF and polyacrylamide. Selection of these supports is dictated by the type of analyses, throughput and data quality. Minimal sample loss is avoided with gel digestion, however background contaminants can obscure high sensitivity analysis in the low pmol range. Routine sample work-up has been reported with gel digestion protocols in the low pmol range with approximately 5-15 % recovery using detection by either Coomassie Blue or silver staining. In to continue refinement of this procedure and increase overall recovery, preparation of excised gel for subsequent digestion has been modified to increase both adsorption of enzyme into gel and substrate diffusion. The requirement to concentrate sample free of salt or detergent for obtaining high quality HPLC and mass spectrum is addressed.
The necessity to obtain samples in high concentration and free of contaminants has become the rate-limiting step in protein structural characterization (1,2) . Although the use of gel electrophoresis to isolate and characterize low-abundant proteins has become the method of choice, sample recovery is reported from 15 to 20 %. Gel recovery of oligonucleotides has been shown to be more efficient with recoveries in the range of 50-70 %. In the case of proteins or peptides, high background noise compromise data quality and interpretation while DNA samples contain salts which interfere with downstream procedures. Sample contaminants that contribute to background noise include most buffers (i.e. tris, phosphates, glycine) or surfactants (i.e. detergents, urea, guanidine) (3,4,5). A preparative membrane can be used to prepare in-gel samples salt-free in high yields that would permit harsh digestion and extraction conditions. An ideal adsorptive membrane should exhibit efficient binding for all analytes with high capacity and quantitative elution while removing most contaminants. To meet these requirements, a styrene sulfonate cation-exchange membrane has been incorporated into the Amicon Microcon® centrifugal device. A styrene sulfonate functional group offers a broad range of selectivity for the adsorption of polypeptides and other biomolecules.
A quantitative review of membrane performance shows rapid kinetics for adsorption with excellent analyte binding. The Microcon-SCX format offers a wide range of applications for concentrating complex peptide or oligonucleotide mixtures. Examples of in-gel digestion of polyacrylamide-containing-proteins are presented with recoveries in the range of 40 - 60 %. In-gel recovery of oligonucleotides for DNA cloning is greater than 70% and salt free. Isolated oligo samples are also shown to be functional by polymerase extension. The flow characteristic of the membrane permits binding and desorption during 15-30 sec centrifugations in a table-top centrifuge to accommodate 0.1 - 250 µg of sample. Applications include analyses by sequencing, HPLC, CE, mass spectroscopy, pulsed amperometric detection and preparation of gel-containing samples.
A.Use of Microcon-SCX
The membrane is wetted by adding methanol to the device, emptying and repeating with DIW. Samples are applied in volumes < 500 µl at a pH below the pI or pKa of the analyte, with optimal binding at salt concentrations < 0.1 M. For unknown samples, the pH is routinely lowered to about 2 with 5&endash;10 µl of glacial acetic acid or diluted in 20 mM HCl to protonate free amines of peptide or analytes in sample. After loading sample into Microcon-SCX (Figure 1), binding occurs during a 30 sec centrifugation. Sample adsobs to membrane when protonated amines selectively exchange with [H+] of sulfonic acid. An optional wash step is performed by using 500 µl of 10 mM HCl and 10% MeOH.
B.Sample Elution
Following analyte binding and washing, a clean vial is placed into the unit, with the addition of 25-50 µl of desorption reagent to sample bound membrane, spun at 14,000 x g for 15 sec and repeated with another 25-50 µl of desorption reagent. The salt-free eluted sample can be either used directly for analyses, neutralized or speedvac dried. As described in the following section, selection of desorbant will depend on both the application and the analysis.
C.Sample recovery from gel with Microcon-SCX
IN-GEL PROTEIN DIGESTION:
1. For reduction-alkylation before electrophoresis, prepare sample in "cracking buffer", add 10 mM DTT, boil 3min, cool to RT, add 20 mM iodoacetamide and incubate RT for 20 min.
2. Run gel, stain/destain and excise bands containing the protein of interest which are cut into 1 mm slices and placed into the supplied Eppendorf tube. Wash in 50 % acetonitrile (ACN), 200 mM NH4HC03 to remove residual SDS and Coomassie Blue, save wash and completely dry gel sample in speedvac (9).
3.The gels are swollen in either trypsin (Sequencing grade modified Trypsin, Promega) or lysyl endopeptidase (Achromobacter Protease I, (Wako Chemicals, USA) for 30 min in an appropriate volume at a concentration of 10 ng/ml in 0.1 M Tris-HCl, 5 mM CaCl2,, pH 8 (10) with or without 0.1 % C18 Zwittergent (Calbiochem).
4. The supernantant is removed and replaced with 100-200 ml of 0.1 M Tris-HCl, 5 mM CaCl2, pH 8. The gel slices are homogenized at 3,000-4,000 RPM to a slurry with the Eppendorf fitting pestle-homogenizer (KT749520-0000 VWR pestle), using a high speed mixer or drill as shown in Figure 1 .
5. Following enzymatic digestion for 24 h at 37ºC, samples receive an equal volume of 20 % formic acid, 80 % acetonitrile and are allowed to extract for 60 min in a water bath sonicator. Using a cut pipette tip, transfer gel slurry digest into the Microcon-SCX unit.
6. Place unit into centrifuge as instructed and centrifuge for 5 min at high speed. Remove filtrate, save, and wash membrane with 0.5 ml of 20 mM HCl after vortexing and centrifugation at 13k x g for 5 min.
7. The digested peptides are recovered by adding 50 ml of 1.4 M NH4OH in 50 % methanol to the Microcon-SCX unit containing a clean collection vial and repeated at least once, using the same vial.
8. Samples can be either directly injected for MS or HPLC analysis or speedvac dried.
RECOVERY OF OLIGONUCLEOTIDES FROM POLYACRYLAMIDE:
1. Samples are electrophoresed in TBE-urea gels and visualized by UV shadowing while the bands of interest are excised.
2. The gels are cut into 1 mm slices and immersed in 20 mM Tris-HCl, 6 M urea, pH 8 (100-200 ml). Homogenization and extraction of sample from gel are performed as described above.
3. Binding of sample containing slurry to the SCX membrane occurs by lowering sample pH using an equal volume of 0.14 M HCl.
4. Washing and desorption from membrane are performed as described above.
RESULTS
Various peptide standards, protein digests and complex mixtures treated by Microcon-SCX are presented to demonstrate the efficiency of analyte binding, elution and selectivity.
1. Complex mixture of peptides from tryptic cytochrome c maps in Figure 2 for 6 separate samples show > 90% ± 2% recovery to control (untreated).
2. A complex glycopeptide mixture of endo lys c digested human immunoglobulin heavy chain (hIgG-HC) following SCX is shown in Figure 3 (trace above). Data shown in lower MS spectral scan was used to identify HPLC fractionated peptides in the upper trace in order to confirm peptide and glycopeptide recovery. MS results of SCX-LC samples confirmed that all peptides and glycopeptides were recovered.
3. Following reduction and alkylation, 25 pmol of BSA or hIgG-HC was electrophoresed and prepared for in-gel digestion and SCX desalting. Approximately 40 - 60 % of peptides were recoverd.
4. Microcon-SCX can be used to obtain oligonucleotides ranging from 18-45 base pairs and higher that are free of salt or reagents. Overall recovery of different oligomers was determined by reverse phase HPLC and shown to be approximately 90 %.
5. DNA functionality of SCX-treated 45-mer was synthesized and gel-purifed from a 10% TBE-urea polyacrylamide gel, desalted and annealed to ssM13mp19, extended, and digested with restriction enzymes. The gel-purified-SCX oligo product appears to be a longer extension product in higher yields than the unpurified, native extended product.
CONCLUSION
The fast kinetics for analyte binding occurs at low pH to allow for a brief 15 sec centrifugations. Since the membrane exhibits high selectivity toward protonated biomolecules, most salts and detergents in dilute concentrations are removed during the initial binding step. Desorption of sample is application-specific and performed at either low or high pH with volatile eluants for direct use or speed vac.
Microcon-SCX was affective for removing detergents, chaotropes and polyacrylamide from gel extracted samples. Agressive gel homogenization provides more accesibility of the enzyme to the protein as well as increases sample extraction efficiency in the 40-60 % range. While gel remains retained on the membrane, contaminants are removed in a wash step prior to peptide elution. HPLC analysis of in-gel digested BSA demonstrates efficient UV contaminant removal following homogenization, digestion and sample clean-up with Microcon-SCX. Excellent signal to noise ratio, ease of use, convenience and high turnover is achieved while maintaining sample fidelity.
REFERENCES
1.Kirchner, M., Fernandez, J., Shakey, Q.A., Gharahdaghi, F. and Mische, S.H.(1996) in Techniques in Protein Chemistry VII (Marshak, D.R. Ed.) pp 287-298, Academic Press, New York.
2.Merewether, L.A., Clogston, C., Patterson, S., Lu, H., (1995) in Techniques in Protein Chemistry VI. (Crabb, J. Ed) pp. 153-160, Academic Press, New York.
3.Vorm, O., Chait, B.I., and Roepstorff, P., (1993) Proc. 41st ASMS Conf., 654-655.
4.Pappin, D.J.C., Hojrup, P., and Bleasby, A.J., (1993) Current Biology 3, 327-332.
5.Swiderek, K.M., Klein, M.L., Hefta, S.A., and Shively, J.E. (1995) in Techniques in Protein Chemistry VI. (Crabb, J. Ed) pp. 267-275, Academic Press, New York.
6.Rohrer, J., Thayer, J.T., Avdalovic, N., and Weitzhandler, M (1995) Anal. Biochem. 170, pp 54-62.
7.Anumula, K.R. (1994) Anal. Biochem. 220, pp 275-283.
8. Kast, E., Pathmanabhan, N., Wong, J., O'connor, B., and Klein, M.L. (1996) Presented at the Tenth Symposium of the Protein Society, San Jose, CA, Abstract #464-T
9.Stone, K.L., LoPresti, M.B., Crawford, J.M., DeAngelis, R. and Williams, K.R. (1989) in: "A Practical Guide to Protein and Peptide Purification for Microsequencing". Ed: Matsudaira, P. Academic Press.
10.Rosenfeld J., Capdeville, J., Guillmot, J., and Ferrara, P. (1992) Anal. Biochem. 203, 173-179.
Figure 1. Homogenization and Microcon-SCX Orientation for Sample Binding and Recovery. Eppendorf fitting pestle-homogenizer for aggresive gel homogenization (left). In fixed-angle rotors, optimal recovery of sample in 50-100 µl requires consistent orientation in the rotor during adsorption and desorption (right).
Figure 2. Reversed Phase Chromatography of Trypsinized Cytochrome c Before and After Adsorption to Microcon-SCX. Approximately 250 µg of digest was diluted to a total volume of 500 µl and either injected directly onto the column (top) or bound and eluted from Microcon-SCX (below) as described in Methods. Separation was performed with an Amicon, C18-300-10sp (4.6 x 250 mm) column using a linear gradient of 5 % ACN to 55 % ACN (0.1 % TFA in DIW) in 20 minutes at 1 ml/min.
Figure 3. In Gel Digestion of BSA after Homogenization and Microcon-SCX Preparation. Approximately 10 pmol of BSA was reduced, alkylated and purified by an 8-12% Tris-Glycine acrylamide gel. Samples were prepared for SCX treatment as described in Experimental section. Following SCX elution, samples were speedvac dried to 10 ml, diluted with 40 ml Buffer A (0.1% TFA) and injected onto a 2.1 x 100 mm RP300 (Perkin Elmer) column. Peptides were separated using a 60 min linear gradient of 5 - 60 % acetonitile in 0.1 % TFA . (Upper trace) In-gel digested BSA peptides recovered after homogenization and SCX treatment. (Lower trace) Blank gel digested, homogenized and treated over SCX as experimental.
Figure 4. Identification of fragments by LC-MS of Endo Lys c Digested hIgG-HC following Microcon SCX. Digests were injected into an LC-MS system consisting of an HP1090 plumbed to a Finnigan TSQ7000 with an electrospray source. Approximately 45 mg of digest was loaded on a Nucleosil 300-5 C18 column (0.46 x 25 cm; Macherey-Nagel, Duren, Germany) and eluted with an ACN gradient at 0.7 ml/min; the effluent was analyzed on-line for both UV absorbance (top) and mass without flow splitting (below) (8).
Figure 5. In-gel Digestion of hIgG-HC after Homogenization and Microcon-SCX Preparation. Approximately 25 pmol of hIgG-HC was reduced, alkylated and purified on a Tris-Glycine acrylamide gel (8-12 %). Samples were digested with Endo lys c and prepared for SCX treatment as described in the Experimental section. Following SCX elution, samples were speedvac dried to 10 ml, diluted with 40 ml Buffer A (0.1% TFA) and injected onto a 2.1 X 100 mm RP300 (Perkin Elmer) column. Peptides were separated using a 75 min linear gradient of 5 - 60 % ACN in 0.1 % TFA after 5 min of 5 % ACN. (Upper trace) Approximately 20 pmol of reduced-alkylated Endo lys c digested HIgG-HC. (Lower trace) HIgG-HC excised from gel, Endo lys c digested, homogenized and treated over SCX as described.
Figure 6. Functionality of Oligonucleotides recovered from Acrylamide gel and Desalted with Microcon-SCX. A 45-mer of M13mp19 primer was either directly annealed or gel purified with Microcon-SCX and then annealed to M13mp19 single-stranded DNA (85oC and cooled to room temperature) (Invitrogen). Respective annealed primers were extended (37oC for 2h with AMV-RT and dNTP's) and digested with three different enzymes as shown in the corresponding lanes: 1) Native oligo (45 mer) annealed and extended 2) EcoR I digested native-extended 3) Kpn I digested native-extended 4) BsaB I digested native-extended 5) SCX gel purified oligo (45 mer) annealed and extended 6) EcoR I digested SCX purified anealed-extended 7) Kpn I-digested SCX purified anealed-extended 8) BsaB I digested SCX purified anealed-extended 9) M13mp19 s.s. 10) M13mp19 d.s. EcoR I and Kpn I sites within the M13mp19 priming site harbors one site each for EcoR I and Kpn I (one fragment in lanes 2,3,6,7). The M13mp19 priming site harbors one site each for EcoR I and Kpn I (one fragment in lanes 2,3,6,7). Two sites are generated in the region extended with AMV-RT (two fragments in lanes 4,8).
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