IR-MALDI-Mass Analysis of Electroblotted ProteinsDirectly from the Membrane: Comparison ofDifferent Membranes, Application to On-MembraneDigestion, and Protein Identification by DatabaseSearching

Detlev Schleuder, Franz Hillenkamp, and Kerstin Strupat*

Institute for Medical Physics and Biophysics, University of Munster, 48149 Munster, Germany

A systematic membrane study investigating differentneutral, cationic derivatized, and hydrophilic PVDF mem-branes for their suitability to carry out on-membranetryptic digestions and to obtain infrared-matrix-assistedlaser desorption/ionization (IR-MALDI) mass informationon the proteolytic fragments directly from the membranewas performed. Clearly, the Immobilon CD membrane(Millipore) showed the most reproducible results over aprotein mass range from 12 to 66 kDa. Typical proteinload to SDS-PAGE was in the 1-2 µg range. The proteinamount used for enzymatic treatment was estimated tobe in the low picomole range. Now both the intact proteinmass and the masses of the specific proteolytic fragmentsare available directly from the membrane. Protein data-bases can be searched via search algorithms on theInternet using the information on the intact protein massand the masses, e.g., of its tryptic fragments. Investiga-tions were performed to search for neutral, enzyme-compatible IR matrixes which allow the enzymatic treat-ment (on-membrane digestion) while the membrane ismatrix-incubated. Thiourea could be tolerated duringenzymatic cleavage in solution in concentrations of 15 g/Land resulted in high-quality spectra of intact proteinsignals and turned, therefore, out to be the most promis-ing candidate.

One- and two-dimensional polyacrylamide gel electrophoresis(SDS-PAGE1,2 and 2D-PAGE3,4) are widely used techniques forthe separation and molecular weight determination of proteins inmolecular biology, biochemistry, and medicine.5-7 For furtherinvestigations, the proteins can be electroblotted from the gel toan immobilizing polymer substrate, such as nitrocellulose or poly-

(vinylidene fluoride) (PVDF) membranes, in order to make themacromolecules accessible for amino acid sequencing (Edmandegradation), enzymatic degradation, or immunological tests. Onthese inert polymer substrates a protein of interest remainsseparated from the other ones in the mixture and is free of anychemical additives, such as buffers, detergents, or salts. Enzymaticor chemical digestion can be done either in the gel8-15 or on theblot membrane.9,16-19 With the advent of soft ionization techniques,mass spectrometry is increasingly used to further and morespecifically characterize proteins by determining the exact massof the intact protein and/or of chemically or enzymaticallygenerated fragments. Whereas fast atom bombardment (FAB) andelectrospray ionization (ESI) mass spectrometry (MS) require theelution of the proteins or fragments from the gel or the blotmembrane, matrix-assisted laser desorption/ionization mass spec-trometry (MALDI-MS) offers the option either to desorb the ionsof interest directly from the gel or the membrane or to performthe analysis by standard preparation of solutions after elution. UV-MALDI-MS, i.e., desorption with wavelengths in the ultravioletregion, has been used successfully by several groups to analyzeenzymatic digests of either dot-blotted20-22 (spotted onto the

* Corresponding author: (e-mail) strupat@uni-muenster.de; (phone) 0049-251-835-5108; (fax) 0049-251-835-5121.(1) Shapiro, A. L.; Scharff, M. D.; Maizel, J. V., Jr.; Uhr, J. W. Proc. Natl. Acad.

Sci. U.S.A. 1966, 56, 216-220.(2) Laemmli, U. K. Nature 1970, 227, 680-685.(3) Klose, J. Humangenetik 1975, 26, 231-243.(4) O’Farrell, P. H. J. Biol. Chem. 1975, 250, 4007-4021.(5) Wilkens, M. R.; Sanchez, J. C.; Williams, K. L.; Hochstrasser, D. Electro-

phoresis 1996, 17, 830-838.(6) Anderson, N. G.; Anderson, N. L. Electrophoresis 1996, 17, 443-453.

(7) Gorg, A.; Boguth, G.; Obermaier, C.; Posch, A.; Weiss, W. Electrophoresis1995, 16, 1079-1086.

(8) Eckerskorn, C.; Lottspeich, F. Chromatographia 1989, 28, 92-94.(9) Patterson, S.; Aebersold, R. Electrophoresis 1995, 16, 1791-1814.

(10) Kawasaki, H.; Emori, Y.; Suzuki, K. Anal. Biochem. 1990, 191, 332-336.(11) Rosenfeld, J.; Capdevielle, J.; Guillemot, J. C.; Ferrara, P. Anal. Biochem.

1992, 203, 173-179.(12) Eckerskorn, C.; Grimm, R. Electrophoresis 1996, 17, 899-906.(13) Plaxton, W. C.; Moorhead, B. G. Anal. Biochem. 1989, 178, 391-393.(14) Shevchenko, A.; Wilm, M.; Vorm, O.; Mann, M. Anal. Chem. 1996, 68,

850-858.(15) Cohen, S. L.; Chait, B. T. Anal. Biochem. 1997, 247, 257-267.(16) Aebersold, R. H.; Leavitt, J.; Saavedra, R. A.; Hood, L. E.; Kent, S. B. H.

Proc. Natl. Acad. Sci. U.S.A. 1989, 84, 6970-6974.(17) Bauw, G.; Van Damme. J.; Puype, M.; Vanderkerckhove, J.; Gesser, B.; Ratz,

G. P.; Lauridsen, J. B.; Celis, J. E. Proc. Natl. Acad. Sci. U.S.A. 1989, 86,7701-7705.

(18) Fernandez, J.; DeMott, M.; Atherton, D.; Mische, S. M. Anal. Biochem.1992, 201, 255-264.

(19) Bai, J.; Qian, M. G.; Liu, Y.; Liang, X.; Lubman, D. M. Anal. Chem. 1995,67, 1705-1710.

(20) Vestling, M.; Fenselau, C. Anal. Chem. 1994, 66, 471-477.(21) Zaluzec, E. J.; Gage, D. A.; Allison, J.; Throck Watson, J. J. Am. Soc. Mass

Spectrom. 1994, 5, 230-237.

Anal. Chem. 1999, 71, 3238-3247

3238 Analytical Chemistry, Vol. 71, No. 15, August 1, 1999 10.1021/ac9810720 CCC: $18.00 © 1999 American Chemical SocietyPublished on Web 06/30/1999

membrane) or electroblotted proteins.20,23-26 The fragments weredesorbed either from the membrane directly or from solutionpreparations after elution of the proteolytic fragments. The massspectrometric determination of the mass of the whole protein byUV-MALDI directly from gels,27,28 from membranes after elec-troblotting,20,29-32 or after elution from the gel with organicsolvents15,33 has been only moderately successful so far. Encourag-ing results have, however, been reported for the direct desorptionof intact proteins from membranes after gel separation andelectroblotting by IR-MALDI-MS, i.e. by desorption with wave-lengths in the infrared region.32,34-36

The large amount of information available from protein andcDNA databases, which is still increasing at a very fast rate, hasled to an improvement in protein analysis. In the majority of cases,full sequencing of proteins has been replaced by the acquisitionof only partial information, sufficient for an unambiguous identi-fication of a protein in the databases. The principal strategy ofthe proteome research approach is documented in the literaturein detail, e.g. in the papers by Blackstock and co-workers37 andby Yates.38

For the identification of an individual protein, the masses ofproteolytic fragments are the most important information to startwith. Early mass analyses of fingerprints by FAB-MS39 have, morerecently, been replaced by ESI-MS and MALDI-MS. In manycases, the fingerprint information alone is not specific enough foran unambiguous identification of a given protein. In such cases,partial sequence information (tag sequencing) regarding one ortwo of the enzymatic fragments as well as the mass of the intactprotein is important and often sufficient additional data. Often tagsequencing combined with the fingerprint information allows anunambiguous identification of the protein under investigation. The

power of tag sequencing derives from the fact that EST (expressedsequence tag) databases can be searched, even if sequenceinformation is the only information available. The principles oftag sequencing with MALDI-MS and ESI-MS were published byMann and co-workers.40 The mass of the intact protein is a thirdparameter of great importance since it provides direct informationabout posttranslational processing, truncations in particular. It can,for example, distinguish preproinsulin from proinsulin and insulinor trypsinogen from trypsin.

In this paper, results for the IR-MALDI-MS of intact proteinsas well as proteolytic fragments directly from membranes after1D or 2D elelectrophoretic separation and electroblotting arereported. In the past, emphasis had been placed on the desorptionof intact proteins with succinic acid (or the related compoundadipic acid) as the matrix of choice for such IR-MALDI analyses:32,34-36 In those experiments, the membrane was incubated in anearly saturated aqueous succinic acid solution immediately afterthe blotting procedure. Desorption of proteolytic fragments,directly from membranes after an on-membrane digestion, is morecomplicated in that both the enzymatic cleavage and the desorp-tion/ionization process of the fragments must be optimizedsimultaneously. On-membrane digestions of dot-blotted and elec-troblotted proteins followed by desorption of the proteolyticfragments directly from the membrane have been performed inthe past, and some experimental procedures for a subsequent UV-MALDI-mass analysis have been published.20-22 For the on-blotenzymatic digestion followed by a desorption directly off the blot,experimental conditions had to be found that met at least the basicrequirements for the digestion as well as the desorption/ionization.A systematic study of different membranes was undertaken, tofind a membrane optimal for both the tryptic digestion and thecollection of high-quality IR-MALDI-mass spectra. The criteria forthe evaluation were the quality of ion signals in terms of single-shot intensities, peak widths, and signal-to-noise ratios, as well asthe reproducibility from spot to spot and from preparation topreparation. Another important criterion for evaluating the qualityof the analysis of proteolytic fragments obtained by mass spec-trometry is the sequence coverage. Obviously, the higher thesequence coverage, the more reliable is the protein identificationby its proteolytic fragments. For these investigations, proteinsdiffering in mass and membranes differing in their physical andchemical parameters were tested.

Originally, experimental conditions for the desorption of theintact proteins and of proteolytic fragments were optimizedseparately. As a final goal, the sequential analysis of first the intactprotein, followed second by proteolytic digestion and massspectrometry, all directly on or from the blot membrane, is highlydesirable. Succinic acid, if used as matrix for the desorption ofthe intact protein, would, however, fully suppress any followingenzymatic digestion, e.g. by trypsin or Lys-C, because of its acidpH (pH 2 in solution). All attempts to fully wash it out of themembrane after the desorption of the intact protein but beforethe digestion failed. Alternative physiological matrixes were,therefore, tested for their performance in the desorption/ionizationof the intact proteins as well as their enzymatic fragments andwith respect to their compatibility with the activity of proteolyticenzymes.

(22) Blackledge, J. A.; Alexander, A. J. Anal. Chem. 1995, 67, 843-848.(23) Liang, X.; Bai, J.; Liu, Y. H.; Lubman, D. M. Anal. Chem. 1996, 68, 1012-

1018.(24) Henzel, W. J.; Billeci, T. M.; Stults, J. T.; Wong, S. C.; Grimley, C.; Watanabe,

C. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 5011-5015.(25) Patterson, S. D.; Thomas, D.; Bradshaw, R. A.; Electrophoresis 1996, 17,

877-891.(26) Mann, M.; Højrup, P.; Roepstorff, P. Biol. Mass Spectrom. 1993, 22, 238-

345.(27) Orgorzalek Loo, R. R.; Stevenson, T. I.; Mitchel, C.; Loo, J. A.; Andrews, P.

C. Anal. Chem. 1996, 68, 1910-1917.(28) Orgorzalek Loo, R. R.; Mitchel, C.; Stevenson, T. I.; Martin, S. A.; Hines,

W. M.; Juhasz, P.; Patterson, D. H.; Peltier, J. M.; Loo, J. A.; Andrews, P. C.Electrophoresis 1997, 18, 382-390.

(29) Schreiner, M.; Strupat, K.; Lottspeich, F.; Eckerskorn, C. Electrophoresis1996, 17, 954-961.

(30) Blais, J. C.; Nagnan-le-Meillour, P.; Bolbach, G.; Tabet, J. C. Rapid Commun.Mass Spectrom. 1996, 10, 1-4.

(31) Patterson, S. D. Electrophoresis 1995, 16, 1104-1114.(32) Sutton, C. W.; Wheeler, C. H.; Sally, U.; Corbett, J. M.; Dunn, M. J.

Electrophoresis 1997, 18, 424-431.(33) Ehring, H.; Stromberg, S.; Tjernberg, A.; Noren, B. Rapid Commun. Mass

Spectrom. 1997, 11, 1867-1873.(34) Strupat, K.; Karas, M.; Hillenkamp, F.; Eckerskorn, C.; Lottspeich, F. Anal.

Chem. 1994, 66, 464-470.(35) Eckerskorn, C.; Strupat, K.; Schleuder, D.; Sanchez, J. C.; Hochstrasser,

D.; Lottspeich, F.; Hillenkamp, F. Anal. Chem. 1997, 69, 2888-2892.(36) Strupat, K.; Eckerskorn, C.; Karas, M.; Hillenkamp, F. In Proceedings of the

Third International Symposium on Mass Spectrometry in the BiologicalSciences; Burlingame, A. L., Carr, S. A., Eds.; Humana Press: Totowa, NJ,1996; pp 203-216.

(37) Humphery-Smith, I.; Cordwell, S. J.; Blackstock, W. P. Electrophoresis 1997,18, 1217-1242.

(38) Yates, J. R. J. Mass Spectrom. 1998, 33, 1-19.(39) Barber, M.; Bordoli, R. S.; Sedgwick, R. D.; Tyler, A. N. J. Chem. Soc., Chem.

Commun. 1981, 325-327. (40) Mann, M.; Wilm, M. Anal. Chem. 1994, 66, 4390-4399.

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EXPERIMENTAL SECTIONMaterials. Bovine trypsin (acetylated) for enzymatic cleavage,

horse heart cytochrome c, horse heart myoglobin, chicken eggwhite lysozyme, human haptoglobin (R2-chain), bovine carbonicanhydrase, chicken ovalbumin, yeast alcohol dehydrogenase, andbovine serum albumin were obtained from Sigma-Aldrich (Dei-senhofen, Germany). All sequences of the examined proteins weretaken from the SwissProt database. Ammonium hydrogen carbon-ate (NH4HCO3) buffer and succinic acid were purchased fromFluka (Neu-Ulm, Germany). Thiourea was obtained from JanssenChimica, poly(vinylpyrrolidone) PVP-40 from Sigma, and Coo-massie Blue R from Pharmacia (Uppsala, Sweden).

All tested membranes were made of poly(vinylidene fluoride)(PVDF) material: Immobilon PSQ, Immobilon P, hydrophobic andhydrophilic Durapore, and Immobilon CD were obtained fromMillipore (Eschborn, Germany). Transblot was obtained from Bio-Rad (Munich, Germany), Roti-membrane No. A147 from Roth(Karlsruhe, Germany), PVDF membrane No. 66542 from GelmanSciences (Dreieich, Germany), and ProBlot No. 400994 from PEApplied Biosystems (Foster City, CA). The membranes differ intheir physical and chemical features, in particular in their averagepore size and surface structure. The information, provided by themanufacturers, was very limited, particularly with respect to thesurface structure. Most of the membranes had hydrophobicsurfaces, presumably unmodified PVDF. Durapore and ImmobilonCD membranes were derivatized to have a hydrophilic surface,the former as a result of neutral, polar surface groups and thelatter by carrying cationic surface groups. The features of themembranes, mostly supplied by the manufacturers, are sum-marized in Table 1. Transblot, Roti No. A147, PVDF No. 66542,and ProBlot No. 400994 membranes were only tested for dot-blotting; all other membranes were used for dot- as well aselectroblot-experiments.

Gel Electrophoresis. SDS-PAGE (1D-PAGE) was preparedaccording to the method described by Laemmli.2 The thicknessof the gels was 1 mm. All proteins except the haptoglobin (seebelow) were separated by SDS-PAGE. The protein load for all SDS-PAGE experiments was 1-2 µg/5 mm (sample well width).Vinylpyridine-modified haptoglobin (mass after vinylpyridine treat-ment: 16 683 ( 2 Da, as determined by ESI-MS35) was separatedby 2D-PAGE according to Hochstrasser and co-workers.41

Blotting. Proteins were immobilized on the different mem-branes either by dot-blotting or by electroblotting. Dot-blottingwas done as follows: Prior to the protein immobilization theunderivatized PVDF membranes were prewetted in methanol for5-10 s and washed in bidistilled water for 30-60 s to remove themethanol; 3 µL of a 1 mg/mL protein solution was then spotteddirectly onto a circular 12 mm2 piece of the PVDF membrane. Itwas distributed evenly over the full area of the membrane pieceand was allowed to dry.

Electrophoretically separated proteins were electroblotted ontomembranes with 2-amino-2-methyl-1,3-propanediol dissolved inbidistilled water (to a pH 8.5) as transfer buffer; 20% methanol(v/v) was added to yield a 50 mM overall concentration. A constantcurrent of 1 mA/cm2 was applied for 2-4 h in a semidry transfer

cell as described previously by Eckerskorn et al.42 In general, inan individual SDS-PAGE run, two neighboring, identical lanes weregenerated, electroblotted, and cut into two pieces parallel to theformer migration direction of the proteins in the gel. The lanewhich was later used for the proteolytic digestion and/or massspectrometric analysis remained unstained. The other (parallel)lane was stained with Coomassie Blue R (0.1% in methanol/water/acetic acid, 4:5:1), followed by destaining for 30 min in the samesolvent with several changes of solvent, therefore serving as areference for the unstained lane.

Immobilon PSQ membranes from a lot obtained several yearsago exhibited a dramatically different and better (in terms ofnumber and quality of fragment signals) behavior in the proteolyticdigestion experiments compared to the ones purchased recently(see below). Old Coomassie Blue R stained electroblots ofhaptoglobin (spot at pI 5.7), which had been kept dry on the shelffor over half a year, were, therefore, used in a few referenceexperiments. They had been separated, electroblotted, and stainedon the earlier lot of membranes, as described by Eckerskorn etal.35

On-Membrane Digestion. Slightly different preparation pro-cedures were used depending on the separation and/or blottingprocedures, the type of membrane, and the laser desorptionmatrix. In preparation for the enzymatic digestion of proteinsloaded onto underivatized PVDF membranes by dot-blotting, themembranes were wetted in methanol and washed in water foronly 1-2 s to remove the methanol, to prevent unspecificadsorption of the proteins and protein/protein interactions at themembrane surface (preparation I). A 4 µL portion of a 50 mMNH4HCO3 buffer, pH 8.5, was then added. For the digestion ofelectroblotted proteins after SDS-PAGE, the whole membraneswere washed twice in bidistilled water directly after electroblottingto remove the transfer buffer. The derivatized hydrophilic CDmembranes were additionally washed in a 50 mM NH4HCO3

buffer (pH 8.5), before ca. 2 × 5 mm2 pieces with the protein ofinterest were excised with a blade (preparation IIa). UnderivatizedPVDF membranes (PSQ, P, hydrophobic Durapore) were ad-ditionally coated by incubation in an aqueous solution of PVP-40(concentration: 2 g/L, 5 × 10-5 M, 0.2%) before excision of theca. 2 × 5 mm2 pieces with the protein of interest (preparationIIb). For the old electroblots, ca. 2 × 5 mm2 pieces, containingthe 2D-PAGE-separated and stained haptoglobin, were cut out ofthe PSQ membrane and washed in methanol for 5 min to partlyremove the stain, followed by washing with bidistilled water threetimes, 3 min each washing step (preparation IIc).

To demonstrate the enzyme compatibility of the matrix,membranes were incubated in an aqueous solution of thiourea(20 g/L) immediately after electroblotting as described elsewherefor succinic acid (preparation III).34

For the proteolytic digestion, ca. 5 µL of a 50 mM NH4HCO3

buffer and 2 µL of a 0.05 g/L trypsin solution in the same buffer(protein-to-enzyme ratio ca. 20:1) were added to the membranepieces, still wet from the preparations, similar to the proceduredescribed by Vestling and Fenselau.20 The wet pieces were placed

(41) Hughes, G.; Frutiger, S.; Paquet, N.; Ravier, F.; Pasquali, C.; Sanchez, J.-C.;James, R.; Tissot, J. D.; Bjellqvist, B.; Hochstrasser, D. Electrophoresis 1997,13, 707-714.

(42) Eckerskorn, C.; Lottspeich, F. Electrophoresis 1993, 14, 831-838. Ecker-skorn, C. Electroblotting. In Microcharacterization of Proteins; Kellner, R.,Lottspeich, F., Meyer, H. E., Eds.; VCH Verlag: Weinheim, Germany, 1994;p 75.

3240 Analytical Chemistry, Vol. 71, No. 15, August 1, 1999

in a polycarbonate Petri dish, covered with a lid (to avoid too fastevaporation of the solvent), and stored at 37 °C for up to 2 h.During this time, bidistilled water (up to 3 µL) was addedoccasionally to keep the membranes from drying. Samplespreincubated in the thiourea MALDI matrix (preparation III) werethen allowed to dry. All other samples were incubated in anaqueous solution of succinic acid (40 g/L) immediately followingthe digestion while the membrane pieces were still wet. The pieceswere then allowed to dry, placed on a stainless steel support byconductive double-sided adhesive tape, and introduced into themass spectrometer.34

Several search algorithms, available on the Internet for theidentification of proteins in databases, were used: (a) RockefellerUniversity (New York), ProFound, Internet address http://www.chait-sgi.rockefeller.edu; (b) EMBL (Heidelberg, Germany),

PeptideSearch, Internet address http://www.mann.embl-heidel-berg.de; (c) UCSF (San Francisco, CA), MS-Fit, Internet addresshttp://www.rafael.ucsf.edu/MS-fit.html.

Mass Spectrometry. The mass spectrometric analysis wasperformed with two different time-of-flight instruments, theprototype of a Vision 2000 (Finnigan MAT, Bremen, Germany)and an in-house-built instrument. Both instruments have beendescribed in the literature.43,44 Taking the differences in someinstrumental parameters into account, the results obtained withthe two instruments were comparable in all respects.

All spectra shown in this paper were obtained in the positive-ion mode using an Er-YAG laser (Spektrum GmbH, Berlin) with

(43) Stahl-Zeng, J. Ph.D. Thesis in Physics, University of Munster, 1997.(44) Berkenkamp, S.; Menzel, C.; Karas, M.; Hillenkamp, F. Rapid Commun.

Mass Spectrom. 1997, 11, 1399-1406.

Table 1. Results of a Systematic Membrane Study Comparing Different Neutral and Charged PVDF Membranes forTheir Suitability To Perform On-Membrane Tryptic Digestion Followed by IR-MALDI-MS Directly from the Membrane

Analytical Chemistry, Vol. 71, No. 15, August 1, 1999 3241

an emission wavelength of 2.94 µm and a pulse duration of 90 ns.The laser light was focused to a spot diameter of 100 µm. Typically,up to 20 spectra were summed. Neither “pulsing out” of low-massions nor data processing, such as smoothing, was applied to thespectra shown. A diluted mixture of known peptides was addedto the digests on the membrane for an internal mass calibration.

RESULTS AND DISCUSSIONSystematic Membrane Studies. In these studies, different

PVDF membranes were compared with respect to their suitabilityfor enzymatic digestions followed by matrix incubation and IR-MALDI-mass analysis directly from the blots. The membranesdiffered in their surface structure, pore size, and protein-bindingcapacity. In a first set of screening experiments, the membraneswere tested for dot-blots. Electroblots were then looked at forthose membranes which gave promising results for the dot-blots.The results of these experiments are summarized in Table 1.Spectral quality (signal intensity, mass resolution, and reproduc-ibility from shot to shot and from preparation to preparation) andsequence coverage were used as evaluation criteria. Differentspectral qualities were evaluated by “+ +”, “+”, “+ -”, “-”,“- -” representing very good, good, indifferent, poor, or noresults. E.g., very good results differ from good results in an evenhigher reproducibility from shot to shot, preparation to prepara-tion, and protein to protein. For indifferent results, we obtainedlow single-shot intensities and smaller amounts of fragmentsignals, together with poorer but acceptable reproducibility,compared to poor results.

The results summarized in Table 1 clearly demonstrate adependence on both the pore size diameter and the surface stateof the individual membranes. On one hand, the shown spectraindicate higher signal intensities, better reproducibility, and largeramounts of fragment signals with increase of the pore sizediameter. This is clearly demonstrated by comparing, for example,the PSQ membrane (pore size 0.1 µm) to the Immobilon P (poresize 0.45 µm) and the Durapore membrane (pore size 0.65 µm).Larger pore sizes seem to allow a better accessibility of theenzyme to the surface-bound proteins and/or less interferencewith its activity. On the other hand, the results obviously showthat a hydrophilic surface is more conducive to an efficient trypticdigestion than a hydrophobic surface. This is in agreement withresults reported earlier by Patterson and Aebersold.9

For dot-blots, the superior performance of hydrophilic mem-branes was demonstrated for the neutral hydrophilic Duraporemembrane as well as for the CD membrane with its hydrophilizedor cationic derivatized surface. However, the Durapore membranehas a very low protein-binding capacity, a factor of ca. 40 belowthat of the CD membrane. Its performance was, therefore, notfurther pursued with electroblots. Examples of these tests areshown in Figure 1 for cytochrome c. The strongly differentabsolute fragment ion intensities are indicated by different ordinatescales, all intercomparable on an absolute level. All hydrophobicmembranes gave higher and more reproducible signals aftercoating as described above (preparation IIb). This is in agreementwith results reported earlier by Aebersold et al.16 It is interestingto compare these results with those obtained earlier for the IR-MALDI desorption of the intact proteins directly from electroblotsafter incubation with the same succinic acid matrix.34,35 For

the intact proteins, all attempts to obtain reproducible and high-quality mass spectra directly from CD membranesstaking ourIR-MALDI results for uncoated, hydrophobic PVDF membranesas a standard in this contextsresulted in quite poor quality massspectra. The reason for this different performance needs furtherinvestigation; however, it is in reasonable agreement with the work

Figure 1. IR-MALDI-mass spectra of on-membrane digestion ofelectroblotted cytochrome c using different PVDF membranes.Spectra were obtained directly from the membranes. A succinic acidmatrix was applied after the digestion was performed. Sums of 15single-shot spectra each were obtained. Key: /, peaks not identified;CT, chymotryptic fragments; AL, trypsin autolysis; ES, electronicinterference signal; (a) Immobilon PSQ (coated); (b) Immobilon P(coated); (c) Immobilon P (uncoated); (d) Durapore (hydrophobic;coated); (e) Immobilon CD. All ordinates are intercomparable on anabsolute scale.

3242 Analytical Chemistry, Vol. 71, No. 15, August 1, 1999

of Patterson,31 who reported UV-MALDI results using a CDmembrane with electroblotted â-casein and R-cyano-4-hydroxy-cinnamic acid as a matrix. The results reported there also showan increase in peak width and a decrease in signal-to-noise ratiocompared to those of a standard preparation.31 Currently thisobservation limits the options for a desorption of first the intactprotein and then the digest fragments from the same piece ofblot membrane. Possibly, the charged surface groups on the CDmembrane interact in an undesirable way with the succinic acidmatrix. So, it can be speculated that, during the electroblottingprocedure, the charged surface groups of the CD membrane bindproteins so strongly that they cannot, afterward, interact suf-ficiently with the matrix for an efficient desorption. Trypticfragments, in contrast, are mobilized again by the cleavage to thenbecome partly or fully incorporated into the matrix.

Digestions of Different Proteins on CD Membranes.Tryptic on-membrane digests of lysozyme, myoglobin, carbonicanhydrase, alcohol dehydrogenase, ovalbumin, and serum albu-min, electroblotted onto CD membranes, were analyzed by IR-MALDI with a succinic acid matrix. Preparation procedure IIa wasused for these experiments. The results are shown in Figure 2.High-quality fragment spectra (high single-shot intensities, highreproducibility from preparation to preparation) could be obtainedfor all the different proteins. None of the samples failed, indepen-dent of the protein mass. The mass accuracy of the signals wasin the range of 0.03%. As can be seen from Figure 2, fragments

deriving from all along the protein chain were obtained in theIR-MALDI-mass spectra. In our evaluation of the results, noattention was paid to the hyrophobicity of the individuallydesorbed peptides; the aim of our study was more to investigatethe principal feasibility and some general performances to useCD membranes as a support for on-membrane digestion followedby mass analysis off the membrane.

Scheme 1 demonstrates the sequence coverage obtained forthe on-membrane digestion of myoglobin (17 kDa). In the caseof myoglobin, a quite high sequence coverage was obtained. Ithas to be noted, however, that the sequence coverage decreaseswith increasing protein mass. For example, the observed sequencecoverage for proteins such as alcohol dehydrogenase (37 kDa)or bovine serum albumin (66 kDa) is lower (less than 50%). It isnot clear if this is related to the protein mass or to another proteinfeature such as hydrophobicity of the individual fragments.Different reasons for this observation can be discussed: A limitedcleavage rate due to the enzyme accessibility to the protein inthe membrane could restrict the MS results (those fragmentswhich are missed in the mass spectra were not produced at allby cleavage), or the physicochemical features (hydrophobicity)of the individual protolytic fragments could limit the results(those fragments which are missed in the mass spectra wereproduced by cleavage and exist in the membrane, but they arenot desorbed/ionized due to insufficient interaction with thematrix in the membrane or due to sticking to the membrane). To

Figure 2. IR-MALDI-mass spectra of on-membrane digestion of electroblotted proteins using CD membranes. Spectra were obtained directlyfrom the membranes. A succinic acid matrix was applied after the digestion was performed. Sums of 15 single-shot spectra each were obtained.Key: /, peaks not identified; (a) lysozyme; (b) myoglobin; (c) carbonic anhydrase; (d) alcohol dehydrogenase; (e) ovalbumin; (f) serum albumin.All ordinates are intercomparable on an absolute scale.

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our knowledge and as far as the proteins under investigationare concerned, sequence coverage from digestion in solutiondepends less on the protein mass than on the individual featuresof the protein. However, we wish to emphasize that although theamount of protein loaded is distributed over an area of ca. 5 × 2mm2 on the electroblot, only parts (ca. 5 mm2) of these areassufficed for the tryptic digestions and desorption of fragments witha high signal-to-noise ratio in all examples under investigation.

Local Fidelity. From the analytical point of view, it isnecessary to consider the question of local fidelity of proteinbands/spots on the electroblot. The quality of local fidelityswhichdescribes the quality of the immobilization of the protein or itsproteolytic fragments after matrix incubation or after on-membranedigestionsis another important analytical question to answer.Therefore, differently treated membranes were scanned by theIR laser. The demand of preservation of local fidelity is, obviously,more difficult to fulfill for the digested proteins than for the intactproteins. The preservation of local fidelity by the matrix incubationof the membrane and mass-analyzing the intact proteins isdiscussed further down in the text.34-36 Two experiments werecarried out to answer the question of whether the local fidelity ispreserved in on-membrane digestion. In the first experiment,cytochrome c and lysozyme were applied to a standard SDS gel.These two proteins comigrated under the experimental conditionsused and appeared as one band in the Coomassie-stained refer-ence blot. Tryptic digestion and mass spectrometry were per-formed from an Immobilon CD membrane as described above.Scanning across the piece of membrane (ca. 5 × 5 mm2 ) revealedtryptic fragments of both proteins independent of the desorptionsite (data not shown). In the second experiment, only cytochromec was applied to a standard SDS gel in the same manner with theexception that an Immobilon P membrane (coated with PVP-40after the blotting procedure) was used. Scanning across a largepiece of membrane (ca. 8 × 8 mm2) yielded tryptic fragmentsfrom the complete membrane piece; fragments did not staylocalized in the band area (data not shown). This demonstratesthat local fidelity is not preserved for the tryptic fragments underthe experimental conditions used. This behavior was observedfor the hydrophilic Immobilon CD membranes with chargedsurface groups as well as for the underivatized, hydrophobic

Immobilon P membrane. However, the loss of separation seemsto be more pronounced for the hydrophilic CD than for thehydrophobic PVDF membrane. The at least partial loss ofseparation for the tryptic fragments contrasts to results obtainedearlier for intact proteins desorbed from underivatized PVDFmembranes. In those experiments, a band of â-casein and carbonicanhydrase, also not separated under the chosen conditions, wasscanned by an IR-laser after succinic acid matrix incubation.34

Mass spectra taken from adjacent sites, separated by 300 µm(scanning from the slow to the fast migration side of the lane),revealed clearly that there was indeed a local separation withinthe seemingly homogeneous band (as judged from the stainedreference band) which was preserved even after the matrixincubation. The preservation of local fidelity of intact proteins wasalso recently demonstrated for electroblots of 2D-PAGE separa-tions of human blood proteins35,45 and for Lys-C digestion products,separated by HPLC and eluted (dotblotted) onto PVDF (Immo-bilon PSQ) membranes, followed by succinic acid incubation andIR-MALDI analysis.46 These observations lend credibility to thehypothesis that intact proteins are bound to the membranesurfaces by the electroblotting and remain so even under matrixincubation, whereas proteolytic fragments, produced on-mem-brane, are released upon the enzymatic bond cleavage and/or thefollowing matrix incubation.

Search for an Enzyme-Compatible Matrix for IR-MALDI-MS. As discussed above, the long-range goal for high-throughputMALDI-MS in proteomics would be to obtain the information onthe intact proteins and their proteolytic fragment information fromone and the same electroblot with a minimum number ofpreparation steps. This requires the availability of an enzyme-compatible matrix to be used in the first step of the desorption ofthe intact protein before the enzymatic digestion. Succinic acid,used so far, does not meet this criterion because its low pHsuppresses the enzyme activity and it cannot be washed from themembrane to a sufficient extent before the enzyme is applied. A

(45) Strupat, K.; Eckerskorn, C.; Karas, M.; Hillenkamp, F. Proceedings of the42nd ASMS Conference on Mass Spectrometry and Allied Topics, Chicago,IL, 1994; p 964.

(46) Eckerskorn, C.; Strupat, K.; Kellermann, J.; Lottspeich, F.; Hillenkamp, F.J. Protein Chem. 1997, 16, 349-362.

Scheme 1. Single Letter Code of the Horse Heart Myoglobin Amino Acid Sequence, AA 1-153a

a Fragments found after tryptic on-membrane digestion by IR-MALDI-mass analysis directly from a PVDF membrane are indicatedby the arrows. For the sake of simplicity, solid lines and differently dashed lines were used to assign different fragments detected byIR-MALDI-MS from the electroblot.

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variety of IR-matrix candidates have a neutral or slightly basicpH in aqueous solution and should therefore be tolerated bytrypsin. The matrix candidates trihydroxybenzene, urea, dimeth-ylurea, thiourea, dimethylthiourea, and pentaerythritol were firsttested by comparing the results of tryptic digestions in solutionunder standard buffer conditions to those with added matrix. Theintact proteins were also desorbed directly off electroblots withthe same matrixes in parallel experiments. Thiourea evolved asthe most promising matrix from these experiments. It is toleratedduring the tryptic digestion of proteins in solution up to concentra-tions of 15-20 g/L (ca. 100 mM) and also gives good signals forthe desorption of intact electroblotted proteins, directly from themembrane. By application of this knowledge to a tryptic cleavageof an electroblotted, thiourea-incubated carbonic anhydrase band,first the intact protein and then specific fragments of it wereobtained (Figure 3). Although mass accuracy (M + H)+ ) 29 000( 50 Da for the protein, external calibration) and spectral qualitystill need to be improved in terms of single-shot intensities andreproducibility, this result shows clearly the general feasibility ofthe approach. Further investigations are needed to improvesequence coverage and reproducibility as well as single-shotintensities.

PSQ Membranes. The poor behavior of the PSQ membranesfor the on-blot tryptic digestion and desorption directly off theblot came somewhat as a surprise, even though it seemingly fitsthe overall pattern of hydrophobic membranes performing infe-riorly to hydrophilic ones. In earlier experiments, vinylpyridine-modified haptoglobin had been separated by 2D-PAGE, electro-blotted, and analyzed by IR-MALDI with succinic acid as thematrix.35 Very good spectra of the R2-chain of human haptoglobin(spot at pI 5.7) had been obtained. Parallel to the desorption ofthe nonstained intact protein (Figure 4a), a preliminary experiment

with a tryptic digestion of the Coomassie Blue R stained proteinon the reference PSQ membrane, followed by matrix incubation,had, at that time, been conducted as well (Figure 4b). This on-membrane digestion resulted in a number of signals in the massrange from 600 to 5000 Da, most of them for specific trypticfragments of the protein (see ref 35). These experiments couldnot be reproduced under any of the conditions tested with thePSQ membranes purchased after December 1995 for the experi-ments described in this publication. To exclude an experimentalartifact, stained and dried reference blots which had been storedon the shelf for over half a year were reactivated. The haptoglobinwas digested by trypsin, incubated in succinic acid, and analyzedby IR-MALDI. Mass spectra of this experiment are shown inFigure 4b. All labeled peaks in the bottom spectrum are specificfor a tryptic cleavage. The sequence coverage of the tryptic on-membrane digestion of haptoglobin of about 100% is shown inScheme 2. Surprisingly, the fragments have no Coomassie Bluemolecules attached, in contrast to intact proteins. The reproduc-ibility of these experiments from spot to spot and from preparationto preparation was rather poor (3 out of 10 preparations workedas shown), but the results demonstrate that an on-membranedigestion on the PSQ membrane is possible in principle. In theseexperiments, the membrane was not even coated with PVP-40,and digestion and preparation took place after the membrane hadbeen dry for several months. We have obtained similar cleavageresults for two different standard proteins (â-lactoglobulin andcarbonic anhydrase) electroblotted onto an old PSQ membraneand stained by Coomassie Blue R using preparation IIb. So far,the change in properties of the PSQ membranes is not understood.Sample membranes from different lots, all purchased recently,failed to produce at least reasonable results for the on-blot trypticdigestion. Neither scanning electron microscopy nor secondaryion mass spectrometry (SIMS) revealed any difference between

Figure 3. IR-MALDI-mass spectra of electroblotted carbonic an-hydrase using the enzyme-compatible matrix thiourea. The membranewas incubated in thiourea directly after the electroblotting procedure.Immobilon P was used as the membrane. Sums of 15 single-shotspectra each were obtained. (a) IR-MALDI-mass spectrum of elec-troblotted carbonic anhydrase. Thiourea was used as the IR matrix.(b) IR-MALDI-mass spectrum of tryptic fragments of carbonic anhy-drase using the same piece of membrane as in Figure 3a. A succinicacid matrix was applied after the digestion was performed. Key: /,peaks not identified; AL, trypsin autolysis.

Figure 4. IR-MALDI-mass spectra of electroblotted vinylpyridine-derivatized human haptoglobin (R2-chain) obtained directly from themembrane, with succinic acid used as the matrix and Immobilon PSQused as the membrane: (a) intact protein, sum of 10 single spectra;(b) tryptic fragments after on-membrane digestion, sum of 15 singlespectra (all labeled peaks are specific tryptic fragments; /, peaks notidentified).

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old and new PSQ membranes. Also, the manufacturer maintainsthat no changes in the raw material or the manufacturingprocedure have been introduced over the past few years. Furtherexperiments need to be conducted to find the cause for the changeand, hopefully, find a membrane equally suited for the desorptionof the intact proteins as well as their proteolytic fragments.

Search Results. Tests of search algorithms available via theInternet (for addresses, see above) for the recognition of frag-ments known to be specific for the tested proteins were carriedout to identify the proteins. For the search, we chose a mass errorwindow for peptide masses of 1 Da. All proteins could unambigu-ously be identified with all used search algorithms. Attentionshould be focused on several small restrictions: Knowledge ofthe mass of the intact protein as provided by mass spectrometrycan indeed be very valuable for the identification in databases.As an example, carbonic anhydrase (29 023 Da) was searched withthe search algorithm ProFound of Rockefeller University, settinga narrow mass window of only 28 900-29 100 Da. In most cases,only a few (less than five) additional intense fragment signals wereneeded to identify the protein unambiguously. It must be noted,however, that this approach is unsuccessful using the otheraccessible search algorithms. This failure is due to the fact thatthe masses of the ambiguous amino acids B (N or D) or Z (E orQ) are set to zero in these systems, which results in too smallfragment masses and, additionally, in too small mass for the intactprotein. More specifically, this is both a failure of the database(since the correct sequence of carbonic anhydrase is certainlyknown but not updated) and a failure of the search algorithm(EMBL and MS-Fit). Unfortunately, carbonic anhydrase comprisesfour of these not precisely specified amino acids. ProFound takesthis phenomenon into account by using average masses, i.e.setting B ) 114.5 Da and Z ) 128.5 Da. This approach should betaken by all search algorithms. The parameters to fill out theHTML form slightly differ for the three tested algorithms. All inall, MS-Fit at the University of California, San Francisco, gave thebest results for all proteins. Only this search algorithm allowstryptic as well as chymotryptic fragmentation and allows the use

of an increased number of missed cleavage sides in the form.These advantages (parameters to fill out the form) compared tothe features of the two other algorithms are the main reasons foran increased number of peptides fit, and this leads to a morereliable identification of the proteins.

In our view, additional important information should be madeavailable in the protein databases. This is information regardingcleavages of precursor proteins during processing, such aspropreinsulin to proinsulin and then to insulin and its R- andâ-chains. Direct access to these entries through the searchalgorithm would clearly enhance the value of the information onthe intact protein mass, since it is strictly related to the proteinfunction.

CONCLUSIONS

A new solid support for proteome analysis by IR-MALDI-massspectrometry has been described which avoids elution of samplesfrom the gel or the electroblot. The results reported show thatboth mass information on the intact protein and mass informationon the proteolytic fragments can be obtained by IR-MALDI directlyfrom the immobilizing membranes. The systematic membranestudy reveals superior results of fingerprint information taken from(cationic derivatized) Immobilon CD membranes.

Several performance features still need to be improved. First,a membrane needs to be identified which allows one to obtainhigh-quality information on the mass of the intact protein as wellas of the proteolytic fragments. This could be achieved by findingexperimental conditions for a better analysis of intact proteins offhydrophilic membranes such as CD or a better digestion onhydrophobic ones such as PSQ. Second, the search for otherenzyme-compatible matrixes should be continued to find theoptimal membrane-matrix combination for the goal of obtainingboth mass information from one and the same protein spot orband. Third, tag sequencing by PSD-MALDI directly from themembrane needs to be developed, to give access to the informa-tion in EST databases. The first study to obtain sequence

Scheme 2. Single Letter Code of the Human Haptoglobin r2-Chain Amino Acid Sequence, AA 1-142a

a See also ref 35. Fragments found after tryptic on-membrane digestion (some few chymotryptic fragments) by IR-MALDI-mass analysisdirectly from a PVDF membrane are indicated by the arrows. For the sake of simplicity, solid lines and differently dashed lines wereused to assign different fragments detected by IR-MALDI-MS from the electroblot.

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information by PSD-IR-MALDI was recently reported by Lude-mann et al.47

ACKNOWLEDGMENTThis work was done in partial fulfillment of the requirements

for the Ph.D. (Dr. rer. nat.) degree of D.S. at the University ofMunster. We thank Drs. Christoph Eckerskorn and Friedrich

Lottspeich, Max Planck Institute for Biochemistry, for helpfuldiscussions about on-membrane digestion and for providing thehaptoglobin samples. Financial support by the Bundesministeriumfur Bildung, Wissenschaft, Forschung und Technology (GrantBEO 9908170) is gratefully acknowledged.

Received for review September 28, 1998. Accepted April 9,1999.

AC9810720

(47) Ludemann, H. C.; Berkenkamp, S.; Hillenkamp, F. Proceedings of the 45thASMS Conference on Mass Spectrometry and Allied Topics, Palm Springs,CA, 1997; p 833.

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