The source of the skin keratins remains to be identi-

189NOTES & TIPS

amide gel was prepared according to Laemmli (4).Sample buffer containing 0.0625 M Tris–HCl (pH 6.8),2% (w/v) SDS, 10% (v/v) glycerol, 5% (v/v) 2-mercapto-ethanol, and 0.001% (w/v) bromophenol blue wasloaded into the wells of the gel and allowed to stand for5 min. Subsequently, electrophoresis was performed inthe reverse direction at a constant current of 40 mA for5 min to recover the sample buffer, and this samplebuffer was removed from the wells. Fresh samplebuffer and protein standards were loaded in the wellsof the gel, and then SDS–PAGE was performed usingthe Laemmli method (4) at a constant current of 40mA. The gel was silver stained according to the instruc-tions of the manufacturer.

Results and discussion. Sample buffer was loadedonly into the odd-lane wells of a polyacrylamide gel.The gel was treated as described above. The result isshown in Fig. 1. Artifactual bands were observed in theeven lanes, whereas few artifactual bands were ob-served in the odd lanes (Fig. 1). Artifactual bands werealso detected in the gel used to analyze the samplebuffer collected from the odd lanes after reverse elec-trophoresis (Fig. 2). These results indicate that theartifactual bands were derived from the polyacryl-amide gel, not from sample buffer or electrode buffer,and could be eliminated effectively by this pretreat-ment method. The electrophoresis pattern of the pro-tein standards also indicates that electrophoresis inthe reverse direction does not affect the quality of thesubsequent electrophoresis separation.

Judging from the molecular weight of the artifactualbands, they are probably skin keratins (1–3). No arti-factual bands were detected in the individual reagentsused for making polyacrylamide gels (data not shown),suggesting that contamination by the skin keratinsoccurs during the preparation of polyacrylamide gels.

FIG. 2. Silver-stained SDS–polyacrylamide gel. The sample buffercollected from the odd lanes after reverse electrophoresis (Fig. 1) wasseparated by electrophoresis using a commercial minigel system andsilver stained. Lane 1, protein standards; lane 2, fresh sample buffer;lane 3, sample buffer collected from the odd lanes.

fied, although they are probably contaminated fromcombs, glass plates, glassware used for preparation ofgel solution, or dust. Artifactual bands are sometimesobserved when using commercial precast gels, andthese artifactual bands are often eliminated by thismethod (data not shown). We consider the method aneasy and useful way for removal of keratins from poly-acrylamide gels.

Conclusion. Artifactual bands due to contaminationof polyacrylamide gels by skin keratins can be eliminatedby pretreatment of polyacrylamide gels using the follow-ing method: load sample buffer containing no protein inthe wells, conduct electrophoresis in the reverse direc-tion, and remove the sample buffer. This procedure offersan easy and practical method for eliminating artifactualbands from polyacrylamide gels.

Acknowledgments. We thank Y. Murata and H. Yamaguchi fortheir thoughtful discussions and Mr. S. Johnson for editing theEnglish manuscript.

REFERENCES

1. Ochs, D. (1983) Protein contaminants of sodium dodecyl sulfate–polyacrylamide gels. Anal. Biochem. 135, 470–474.

2. Berube, B., Coutu, L., Lefievre, L., Begin, S., Dupont, H., andSuillivan, R. (1994) The elimination of keratin artifacts in im-munoblots probed with polyclonal antibodies. Anal. Biochem.217, 331–333.

3. Shapiro, S. Z. (1987) Elimination of the detection of an artefac-tual 65 kDa keratin band from immunoblots. J. Immunol. Meth-ods 102, 143–146.

4. Laemmli, U. K. (1970) Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London) 227,680–685.

Solid-Phase Labeling with a Fluorescent Reagentto Fingerprint Nonradioactive Proteins1

Andrea Gatti,2 Kevin C. Menes,and Jolinda A. TraughDepartment of Biochemistry, University of California,Riverside, California 92521

Received December 21, 1999

An established method originally designed by Gor-man (1) to label soluble, cysteine-containing proteins

1 This work was supported by grants from the U.S. Public HealthService (GM26738) and NSF (BIR-9601810).

2 To whom correspondence should be addressed at current address:Skirball Institute of Biomolecular Medicine, New York UniversitySchool of Medicine 540 First Avenue, New York, NY 10016. Fax:212-263-0723. E-mail: gatti@saturn.med.nyu.edu.

Analytical Biochemistry 280, 189–192 (2000)doi:10.1006/abio.2000.4519

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with a fluorescent reagent was adapted to label nitro-

a

Before being labeled with 5-I-AEDANS, the pro-

bl

Z2

190 NOTES & TIPS

cellulose-bound proteins, and combined with a previ-ously described two-dimensional peptide mapping sys-tem (2) to fingerprint nonradioactive proteins.

Different peptide mapping techniques can be char-acterized by the degree of proficiency in addressingmultiple objectives, including peptide fractionation, de-tection of the fractionated peptides, and suitability ofthe isolated peptides for preparative applications (e.g.,sequencing). To efficiently serve all these purposes, wedesigned a novel experimental strategy based on 2-Dpeptide PAGE of digests from 32P-labeled proteins,which is utilized for sequence analysis and identifica-tion of phosphorylation sites on the fractionated phos-phopeptides (2). One apparent limitation of such aprocedure is the requirement for the peptides to beradiolabeled in order to be detected after 2-D peptidePAGE, given that conventional staining procedures re-sult in rapid diffusion of the fractionated peptides.Indeed, little data are available on peptide mappingsystems that use any means other than radiolabelingto fingerprint proteins with high sensitivity (3).

Here we describe a labeling procedure by which ni-trocellulose-immobilized proteins are rendered fluores-cent prior to proteolytic digestion and peptide mappingby 2-D peptide PAGE. This procedure provides theoption of generating two-dimensional fingerprints fromproteolytic digests of isolated, nonradioactive proteinsand allows fluorescent peptides to be sequenced afterfractionation. As model proteins, we employed bovineserum albumin (BSA)3 and the glutathione-S-trans-ferase (GST) fusion protein of the g-isotype of p21-ctivated protein kinase (g-PAK).BSA has 35 cysteineyl residues and could thus be

readily labeled by cysteine-reactive fluorescent agents,such as 5-N-[(iodoacetamidoethyl)amino]naphthalene-1-sulfonic acid (5-I-AEDANS). g-PAK is a serine/threo-nine protein kinase, whose autophosphorylation andactivation states are stimulated by binding of the smallG-protein Cdc42 or by cleavage with caspase 3 (4, 5).The choice of g-PAK was based on our recent interestin analyzing the autophosphorylation of g-PAK by two-dimensional phosphopeptide mapping (6). GST-g-PAKhas a total of nine cysteineyl residues: four in the GSTmoiety and five in the g-PAK sequence. In this study,GST-g-PAK was rendered fluorescent after being sub-jected to in vitro autophosphorylation in the presenceof [g-32P]ATP. The use of a 32P-labeled phosphoproteinallowed us to rule out the possibility that fluorescentlabeling could interfere with the procedural steps em-ployed for phosphopeptide mapping.

3 Abbreviations used: 5-I-AEDANS, 5-N-[(iodoacetamidoethyl)-amino]naphthalene-1-sulfonic acid; BSA, bovine serum albumin;DTT, dithiothreitol; GST, glutathione S-transferase; PAK, p21-acti-vated protein kinase.

teins were either directly spotted onto nitrocelluloseor transferred to nitrocellulose following conven-tional SDS–PAGE. After reduction of disulfidebonds, the cysteineyl residues of the nitrocellulose-immobilized proteins were rendered fluorescent byalkylation with 5-I-AEDANS. The choice of 5-I-AE-DANS to integrate solid-phase labeling with an es-tablished strategy for peptide mapping and identifi-cation was based on previously reported properties(1) of this fluorescent reagent: (i) 5-I-AEDANS iswater soluble; (ii) the detection limit for 5-I-AE-DANS-labeled proteins is in the subpicomolar range;(iii) 5-I-AEDANS labeling does not interfere with theautomated amino-terminal sequencing of residuespreceding the modified cysteine.

Methods

Preparation and autophosphorylation of GST-g-PAK. Recombinant GST-g-PAK was expressed inbaculovirus-infected insect cells and purified by affin-ity chromatography (5). Autophosphorylation of therecombinant protein (10 mg; 110 pmol) was carried outwith [g-32P]ATP and terminated by the addition oftwice-concentrated sample buffer for conventionalSDS–PAGE, as previously described (6).

Binding of proteins to nitrocellulose. BSA was im-mobilized on nitrocellulose (0.45 mm, Schleicher &Schuell) by spotting a 10-ml aliquot of a 7.5 mg/ml stocksolution (1.12 nmol) onto nitrocellulose (0.5 3 0.5 cm)efore air-drying. After autophosphorylation, immobi-ization of GST-g-PAK on nitrocellulose was achieved

by conventional electrotransfer of the radiolabeled pro-tein after SDS–PAGE (6). Autoradiography allowedthe visualization and the excision of the relevant 32P-labeled protein.

Fluorescent labeling. The chosen model proteins,BSA and GST-g-PAK, were rendered fluorescent by asolid-phase labeling reaction with 5-I-AEDANS. Nitro-cellulose-bound BSA and GST-g-PAK were incubatedfor 2 h at room temperature in 100 ml of 0.1 MNH4HCO3 in the presence of 15 mM dithiothreitol(DTT) to reduce disulfide bonds. To this mixture, a50-ml aliquot of 300 mM 5-I-AEDANS in 0.25 MNH4HCO3 was added to a final concentration of 100mM. After an additional 4 h of incubation at roomtemperature in the dark, the solution was discardedand the nitrocellulose was extensively washed withwater to remove free 5-I-AEDANS.

Proteolytic digestion and peptide separation. Nitro-cellulose-bound protein was digested by incubationwith 50 ng/ml of trypsin in 50 mM NH4HCO3 and 1%

wittergent 3-16 for 24 h at 30°C in a volume of 100–00 ml. Aliquots (20–80 ml) of the digested protein were

subjected to nondenaturing isoelectric focusing in gel

tubes followed by electrophoresis on a 40% polyacryl-

cs(

191NOTES & TIPS

amide alkaline slab gel (40% PAGE), as previouslydescribed (6).

Peptide visualization. After the 40% gel was dried,the fluorescent peptides were detected by UV visualiza-tion with an EAGLE EYE II camera. 32P-labeled peptidesfrom GST-g-PAK were detected by autoradiography.

Amino acid sequencing. One of the fluorescent pep-tides resulting from tryptic digestion of BSA was iso-lated from the 2-D peptide gel and subjected to acleanup procedure through Sep-Pak cartridges (50 mg,Waters), as previously described (6). The peptide wasimmobilized on a disk of arylamine membrane (Seque-lon-AA, Millipore) and subjected to amino acid se-quencing (using a Procise 492 sequenator from AppliedBiosystems).

Results and Discussion

To determine whether a nitrocellulose-immobilizedprotein could be efficiently labeled with a water-solublefluorescent reagent, an aliquot of BSA dissolved inwater was spotted onto a nitrocellulose membrane.Nitrocellulose-bound BSA was rendered fluorescent byreduction of disulfide bonds with DTT, followed byalkylation with 5-I-AEDANS, essentially as describedby Gorman (1) for soluble BSA. After incubation withthe fluorescent reagent, the nitrocellulose was exten-sively washed with water to terminate the reaction andallow the removal of free 5-I-AEDANS from the mem-brane, thereby minimizing nonspecific binding of thefluorescent label. Nitrocellulose-immobilized fluores-cent BSA was cleaved with trypsin and the digest wassubjected to 2-D peptide PAGE to generate a 2-D pep-tide map, as previously described (6). As shown by UVvisualization of the 2-D peptide gel (Fig. 1A), manyfluorescent peptides were detected. BSA has a relativelyhigh cysteine content, 35 residues of a total of 582, whichmay exaggerate the sensitivity of this technique.

To investigate this issue, one of the detectable, butnot dominant, fluorescent peptides (indicated by thearrow in Fig. 1A) was isolated and subjected to auto-mated amino acid sequencing as previously described(6). The unequivocal identification of the isolated pep-tide (Fig. 1B), as the tryptic peptide of BSA startingwith Ser-285 and containing the modified Cys-287, val-idated the use of this procedure for sequencing fluores-cent peptides. Since two trypsin cleavage sites precedethe nearest downstream cysteine (Cys-314) in the BSAsequence, it can be concluded that a single modifiedcysteine is sufficient for peptide detection.

To assess further the sensitivity of this techniqueand to allow parallel mapping of fluorescent and 32P-labeled peptides derived from the same protein, wenext focused on GST-g-PAK, which contains a total of 9ysteineyl residues and has multiple autophosphoryla-

tion sites (6). Following autophosphorylation in thepresence of [g-32P]ATP, GST-g-PAK was subjectedto SDS–PAGE and conventional electroblotting (6).The nitrocellulose-bound GST-g-PAK was excised fromthe blot membrane, rendered fluorescent with 5-I-AEDANS, and subjected to on-membrane proteolyticdigestion, as described for BSA. The tryptic digest ofGST-g-PAK was then analyzed by 2-D peptide PAGEand the 32P-labeled and fluorescent peptides were vi-ualized by autoradiography and UV light, respectivelyFig. 2). Despite using significantly less protein (110

FIG. 1. Two-dimensional gel separation of tryptic peptides from5-I-AEDANS-labeled BSA. (A) A 75-mg aliquot of BSA was spottedonto nitrocellulose and the labeling reaction with 5-I-AEDANS wascarried out as described under Methods. After washing and subse-quent on-membrane digestion with trypsin, the released peptideswere fractionated by 2-D peptide PAGE. UV visualization of the 2-Dgel was photographically recorded and scanned. The resulting imagewas inverted to generate the figure. (B) The peptide indicated in Awas isolated, bound to a Sequelon-AA disk and subjected to micro-sequencing. The identified amino acid sequence is reported, togetherwith the corresponding sequence of BSA. X designates amino acidresidues that could not be identified by automated sequencing. Theunderlined residue corresponds to the cysteineyl residue modified by5-I-AEDANS labeling.

fluorescent peptides were efficiently released into solu-

5

flip2

192 NOTES & TIPS

pmol of GST-g-PAK vs 1.12 nmol of BSA) with fewercysteineyl residues (9 vs 35), several fluorescent pep-tides of GST-g-PAK were clearly detected.

The use of 5-I-AEDANS to render fluorescent the32P-labeled GST-g-PAK allowed us to rule out the pos-sibility that 5-I-AEDANS could interfere with on-mem-brane proteolytic digestion and release of the resultingpeptides into solution, as shown by the phosphopeptidepattern which is identical to that previously described(6). The finding that fluorescent and radiolabeled pep-tides of GST-g-PAK did not comigrate in the 2-D pep-tide PAGE is fully consistent with the complete lack ofcysteineyl residues in the major phosphopeptides ofautophosphorylated g-PAK (6). The 5-I-AEDANS-la-beled peptides are expected to migrate differently fromthe corresponding unlabeled peptides because of thechanges in molecular weight and isoelectric point re-sulting from derivatization.

While the method originally designed by Gorman (1)is used to label soluble proteins before their separationon conventional SDS–PAGE, here we used solid-phaselabeling to render proteins fluorescent prior to on-membrane digestion, combined with peptide fraction-ation on 2-D peptide PAGE. In addition to providingthe opportunity to selectively label proteins isolatedfrom complex mixtures by conventional gel separationand subsequent membrane blotting, this proceduregenerated high-resolution peptide maps from whichfluorescent peptides could be identified. Specifically,the idea of combining solid-phase labeling with a pre-viously established approach for peptide mapping andidentification was made possible because: (i) nitrocel-lulose-bound proteins were effectively rendered fluo-rescent; (ii) washing the nitrocellulose after fluorescentlabeling removed the vast majority of free label; (iii)

FIG. 2. Two-dimensional separation of 5-I-AEDANS-labeled and32P-labeled peptides after tryptic digestion of GST-g-PAK. A 10-mgaliquot of GST-g-PAK was autophosphorylated with [g-32P]ATP, an-alyzed by SDS–PAGE, and transferred onto a nitrocellulose mem-brane. Nitrocellulose-bound GST-g-PAK was excised and rendered

uorescent with 5-I-AEDANS as described under Methods. Follow-ng tryptic digestion, the resulting peptides were fractionated by 2-Deptide PAGE. UV visualization (A) and autoradiography (B) of the-D gel are shown.

tion and fractionated on 2-D peptide PAGE; (iv) fluo-rescent labeling did not interfere with the automatedEdman degradation of any residue other than the mod-ified cysteine.

The only significant limitation in the use of 5-I-AE-DANS is that proteins which do not contain cysteineylresidues cannot be labeled. However, a similar protocolusing other amino acid-selective fluorogenic reagentscould detect peptides that contain different derivatizedamino acids. Notably, the fluorescence of arginine-con-taining and tyrosine-containing peptides was reportedto be detectable at the subfemtomolar level (7).

The application of these procedures for isolating pro-teins from complex sample matrices and for selectivesolid-phase tagging, followed by on-membrane digestionand fractionation of the detectable peptides, should facil-itate the identification of a variety of biologically relevanttargets. Further, meaningful correlations between dis-tinctive protein fingerprints and dysfunctional molecularphenotypes (e.g., disease phenotyping) may be revealedand analyzed at the amino acid level.

Acknowledgment. We thank Barbara Walter for preparing re-combinant GST-g-PAK.

REFERENCES

1. Gorman, J. J. (1987) Anal. Biochem. 160, 376–387.2. Gatti, A., and Traugh, J. A. (1999) Anal. Biochem. 266, 198–204.3. Krull, S., Strong, R., Cho, B.-Y., Beale, S. C., Wang, C.-C., and

Cohen, S. (1997) J. Chromatogr. B 699, 173–208.4. Jakobi, R., Chen, C.-J., Tuazon, P. T., and Traugh, J. A. (1996)

J. Biol. Chem. 271, 6206–6211.5. Walter, B. N., Huang, Z., Jakobi, R., Tuazon, P. T., Alnemri, E.,

Litwack, G., and Traugh, J. A. (1998) J. Biol. Chem. 273, 28733–28739.

6. Gatti, A., Huang, Z., Tuazon, P. T., and Traugh, J. A. (1999)J. Biol. Chem. 274, 8022–8028.

7. Cobb, K. A., and Novotny, M. V. (1992) Anal. Biochem. 200,149–155.

Pepsin Inactivation of Deoxyribonuclease I

Yanusz Wegrowski,1 Corinne Perreau,and Francois-Xavier MaquartLaboratoire de Biochimie et Biologie Moleculaire, UPRESACNRS 6021, IFR 53-Biomolecules, Faculte de Medecine,1 rue Cognacq-Jay, 51095 Reims Cedex, France

Received January 5, 2000

Numerous routine procedures employing RNA needthe complete withdrawal of the remaining DNA. They

1 To whom correspondence should be addressed. Fax: (133)326.91.80.55. E-mail: yanusz@infobiogen.fr.

Analytical Biochemistry 280, 192–194 (2000)doi:10.1006/abio.2000.4520

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