Universal Isolation of Cross-Linked Peptides: Application to

Xiaohui Chen, Devayani Eswaran, Mark A. Smith, George Perry, and Vernon E. Anderson*. Department of Biochemistry, Case Western Reserve University, ...
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Bioconjugate Chem. 1999, 10, 112−118

Universal Isolation of Cross-Linked Peptides: Application to Neurofibrillary Tangles Xiaohui Chen,†,‡ Devayani Eswaran,†,‡,| Mark A. Smith,§ George Perry,§ and Vernon E. Anderson*,‡ Department of Biochemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106-4935. Received August 11, 1998; Revised Manuscript Received October 19, 1998

A universal procedure to isolate cross-linked peptides has been demonstrated. The procedure relies on initially converting the -amino groups of lysine to dimethyl lysine by reductive methylation with sodium cyanoborohydride and formaldehyde. The lysine-modified protein is proteolytically cleaved and the resulting R-amino groups derivatized with methoxypoly(ethylene glycol)5000 succinyl hydroxysuccinimide. Any unintentional derivatization of tyrosine side chains can be reversed by incubation under mildly alkaline conditions. The cross-linked polypeptides contain two poly(ethylene glycol)5000 chains while non-cross-linked peptides contain a single poly(ethylene glycol)5000 chain. The two populations of peptides can be separated by gel filtration chromatography. This procedure has been shown capable of isolating cross-linked peptides using glutathione, lysozyme, chemically cross-linked hemoglobin, and neurofibrillary tangles isolated from the brain of a case of Alzheimer’s disease.

The determination of the sites of cross-links of proteins has been a central feature of protein structure determination from the initial work on insulin (Ryle et al., 1955). The location of disulfide cross-links has played a central role in the study of protein folding (Darby and Creighton, 1995, 1997). The use of cross-linking reagents, both specific (Wells and Yount, 1979) and nonspecific, e.g., Cervoni et al. (1994), has provided valuable information on the tertiary and quaternary structure of proteins. A wide variety of pathological processes including atherosclerosis and Alzheimer’s disease have been characterized by the occurrence of proteinaceous deposits. It has been suggested that one cause of deposit formation and/or existence may be the generation of cross-linked proteins resulting in a decreased solubility and resistance to proteolytic removal of the aggregate (Munch et al., 1997). Previous methods for isolating cross-links have relied on two separate approaches. The first is a prior knowledge of the chemical structure or property of the crosslink. Thus, fluorescent cross-linkers have been utilized (Mornet et al., 1985) to assist in the recovery of crosslinked peptides. Alternatively, careful characterization of a proteolytic peptide map can be used to identify new peptides following treatment with a cross-linker (Giorgianni et al., 1997). Both of the classical approaches of isolating cross-links will fail when both the chemical mechanism of crosslinking is uncertain and the identity of the cross-linked proteins uncertain or derived from impure biological matrixes rendering the peptide maps inherently irreproducible. In this contribution, we describe a method and * To whom correspondence should be addressed. E-mail: [email protected]. Phone: 216-368-2599. Fax: 216-368-3419. † These two authors contributed equally to the experimental work and primary authorship of this paper. ‡ Department of Biochemistry, Case Western Reserve University School of Medicine. | Current address: 182 St. Mary’s Rd., Apt 1A, R.A. Puram, Chennai 600028, India. § Department of Pathology, Case Western Reserve University School of Medicine.

demonstrate its implementation at isolating an ensemble of cross-linked peptides to permit their further characterization by other analytical methods. This procedure, shown in concept in Scheme 1, relies on the obvious premise that cross-linked peptides will contain two R-amino groups while all other peptides will contain at most one R-amino group. To prevent the -amino groups of lysine from confounding the analysis, these amino groups must be derivatized prior to the proteolytic generation of the cross-linked peptides. A conceptually similar approach was taken by Jue and Doolittle (1985) to label the N-terminus of peptides. The N-terminal derivatization suggested in Scheme 1 has three essential requirements: (1) the derivatization must be close to quantitative; (2) the product must permit the separation of peptides that contain one derivative from the cross-linked peptides which contain two of the derivatives, independent of what amino acid side chains are present in the peptide; and (3) the chemistry must result in the derivatization of only the R-amino group. In this contribution, we demonstrate that the N-hydroxysuccinimidyl ester of poly(ethylene glycol) succinate fulfills these three requirements and permits the universal isolation of cross-linked peptides. MATERIALS AND METHODS

Chemicals. The methoxypoly(ethylene glycol)5000 succinyl hydroxysuccinimide (PEG-O-succinyl-O-Su)1 was purchased from Shearwater Polymers Inc. Sodium cyanoborohydride, succinic anhydride, chymotrypsin, Pronase, fluorescamine, sodium sulfite, bismuth nitrate, 5,5′dithio bis(2-nitrobenzoic acid) (DTNB), trinitrobenzenesulfonate (TNBS), dithiothreitol (DTT), bovine insulin, and all amino acids, peptides, and buffers were purchased from Sigma Chemical Co. Potassium iodide 1 Abbreviations: DTNB, 5,5-dithio bis(2-nitrobenzoic acid); DTT, dithiothreitol; MDC-Hb2, β1-Lys82-N trimesyl β2-Lys82N 5-(N′-dansyl cadaverine) cross-linked hemoglobin; NTB, 2-nitro-5-thiobenzoate; NTSB, 2-nitro-5-thiosulfobenzoate; PEGO-succinyl-O-Su, methoxypoly(ethylene glycol)succinyl hydroxysuccinimide; SDS, sodium dodecyl sulfate.

10.1021/bc980098v CCC: $18.00 © 1999 American Chemical Society Published on Web 12/18/1998

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Scheme 1

was purchased from Aldrich Chemical Co. Hydroxylamine hydrochloride, acetic anhydride, ammonium acetate, sodium phosphate, sodium sulfate, sodium sulfite, sodium borate, sodium hydroxide, concentrated HCl, formaldehyde, acetone, and acetonitrile were purchased from Fisher Scientific. 2-Nitro-5-thiosulfobenzoate (NTSB) was synthesized as previously decribed (Thannhauser et al., 1987). Superdex 200 PG gel and PD-10 desalting columns were purchased from Pharmacia Biotech. Brain samples of cases of Alzheimer’s disease case were from the CWRU brain bank. Hemoglobin was obtained from Dr. K. Magnus (Department of Biochemistry, CWRU). Hemoglobin cross-linked with a fluorescent cross-linker, MDC-Hb2 shown in Scheme 2, was a generous gift from Hemosol Inc. (Etobicoke, Canada) (Jones et al., 1993; Kluger et al., 1992; Schumacher et al., 1995). Insoluble Paired Helical Filaments and Control Fractions. Hippocampus and temporal cortex with meninges and white matter removed from a case of Alzheimer’s disease (77 year old) was homogenized in 5

vol of 50 mM Tris-HCl, pH 7.6 (buffer A), with 0.1 g of sodium dodecyl sulfate (SDS)/g of wet tissue. After centrifugation at 10000g for 4 h at 22 °C, the supernatant was removed, and the pellet was rehomogenized in buffer A with 1% SDS. After centrifugation at 25000g for 1 h, the pellet was again homogenized in buffer A with 1% SDS and 10% sucrose, layered over a gradient of 1.0, 1.2, and 2.0 M sucrose/buffer A, and centrifuged for 1 h at 72000g. The insoluble paired helical filament fraction at the 1.2-2.0 M interface was used in further experiments. Gel Filtration Chromatography. A 420 mL Superdex 200 PG gel in a Pharmacia XK 26/100 column was used for most separations. In some early experiments, a 320 mL Pharmacia 26/60 HiLoad Superdex 200 PG gel filtration column was used. Constant flow was maintained with a Pharmacia P-500 FPLC system and the column effluent monitored with a 20 µL flow cell in an HP 8452A diode array spectrophotometer and constant volume fractions collected in an Isco Retriever II fraction collector.

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Chen et al.

Scheme 2

Modification of the Amino Group of the CrossLinked Protein. Initial protection of protein lysine -amino groups as shown in Scheme 1 was accomplished by acetylation, succinylation, or reductive methylation, which proved to be most useful. Acetylation was accomplished by infusion of acetic anhydride at a flow rate of 0.9 µL/min using a syringe pump, into 1 mL of solution containing 1-3 mg of protein in 0.1 M phosphate buffer at pH 8.0 until a final concentration of 150 mM was achieved. The infusion procedure permitted the pH to be held constant at 8.0 by the manual dropwise addition of 1 M NaOH. Succinylation, after the method of Kooistra et al. (Kooistra et al., 1979), was achieved by addition of 1 mg of solid succinic anhydride to a 1 mL of solution containing 1-2 mg of protein in 0.1 M phosphate buffer at pH 8.0 with constant stirring. The pH of the solution dropped to 5.5, after 12 min, it was increased to 8.0 by titration with 1 M NaOH. Then, another addition of succinic anhydride was performed. This process was repeated for a total of five additions. The pH was increased to 8.0 at the end of each 12 min interval. Reductive methylation followed the procedure of Jentoft and Dearborn (1983). Formaldehyde and sodium cyanoborohydride were both added to a 1 mg/mL protein solution in 50 mM sodium phosphate buffer, pH 7.0, to final concentrations of 10 mM, and the was reaction allowed to proceed at ambient temperature for 24 h. The mixture was then dialyzed extensively against 0.2 M sodium phosphate buffer, pH 8.0, or alternatively desalted by elution with water through a PD-10 desalting column, preequilibrated with water. The extent of the amino group modification was determined by detection of residual primary amines using a TNBS assay (Snyder and Sobocinski, 1975) or in later experiments with the more sensitive fluorescamine assay (Udenfriend et al., 1972). Protease Digestion. The N-modified and desalted protein was then cleaved with the proteases chymotrypsin and Pronase to generate free amino termini. Except where noted, chymotrypsin was added first in a 1:50 (w:w) protease-to-protein ratio. The cleavage reaction was incubated for 5 h at 37 °C. Pronase was then added to the protein solution in a 1:50 (w:w) proteaseto-protein ratio and incubated for 24 h at 37 °C. For the brain sample solution, only Pronase was added in a 1:25 (w:w) protease-to-protein ratio and incubated for 30 h at 37 °C. Derivatization of the Proteolytically Liberated Peptide N-Termini with PEG-O-succinyl-O-Su. Following proteolysis, solid PEG-O-succinyl-O-Su was added into the above peptide solution with constant stirring to

produce a concentration of 10 mg/mL. After 15 min, approximately 4 µL of 1 M NaOH was added to raise the pH back to 8.0. Then, an additional 10 mg/mL of PEGO-succinyl-O-Su was added. This process was repeated to a total of seven PEG-O-succinyl-O-Su additions (final concentration of 70 mg/mL). The pH of the solution was increased to 8.0 by titration with 1 M NaOH at the end of each 15 min reaction. Selective Hydrolysis of PEG-O-succinyl-O-Tyr. The modification of tyrosine with PEG-O-succinyl-O-Su revealed that the phenoxyl group of the side chain is derivatized to a small extent, forming the PEG-Osuccinyl-O-Tyr phenyl ester. To unambiguously identify the doubly labeled peptides as cross-linked peptides, it is imperative to remove the PEG-O-succinyl tag from the side chain of tyrosine residues. After derivatizing the peptides with PEG-O-succinyl-O-Su, as described above, the phenyl ester linkage in PEG-O-succinyl-O-Tyr moiety was selectively hydrolyzed by titration to pH 11 with saturated trisodium phosphate. The reaction mixture was maintained at pH 11 for 30 min at ambient temperature and the reaction stopped by reducing the pH to 8.0 with acetic acid. This mild alkaline hydrolysis additionally hydrolyzes PEG-O-succinyl-S-Cys thiolesters but unfortunately will also cleave disulfide bonds. However, it does not hydrolyze the PEG-O-succinyl oxygen ester. Separation of Peptides Containing either One or Two PEG5000 Chains. The PEG-O-succinyl-O-Su derivatized peptides were separated on a Pharmacia XK26/ 100 Superdex 200 PG gel filtration column, preequilibrated with 50 mM ammonium acetate, pH 5.0, and eluted with the same buffer. The PEG-O-succinyl-O-Su derivatized peptides (1 mL) were injected, and eluted at 1 mL/min, collecting 4 mL fractions. The gel filtration column was calibrated with PEG-O-succinyl-O-Su derivatized tryptophan and lysine nitroanilide which served as models for peptides containing either one or two PEG5000 chains, respectively. The tryptophan and lysine nitroanilide derivatives could be identified by their UV absorbance at 280 and 318 nm, respectively. Quantitation of Disulfide Bonds. Fractions from the Superdex gel chromatography could be analyzed for disulfide bonds employing a 2-nitro-5-thiosulfobenzoate (NTSB) assay (Thannhauser et al., 1987). From each fraction, 1 mL was taken to dryness in a centrifugal vacuum concentrator, redissolved in 20 µL of water, and added into a 300 µL of NTSB assay solution, consisting of 250 µM NTSB, 50 mM glycine, 100 mM sodium sulfite, and 3 mM EDTA. The concentration of disulfide bonds

Universal Isolation of Cross-Linked Peptides

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Scheme 3

was calculated from the increase in the 2-nitro-5-thiobenzoate (NTB) absorbance at 412 nm, 412 ) 13 600 M-1 cm-1. Hydroxaminolysis of PEG-O-succinyl-N-peptide. To permit further analysis of the fractions containing peptides with two PEG5000 chains, it is useful to be able to remove the N-terminal PEG-O-succinyl label. The treatment of the PEG-O-succinyl-labeled peptides with hydroxylamine, cleaves the PEG-O-succinyl ester, and the HONH-succinyl-N-peptide subsequently cyclizes releasing N-hydroxysuccinimide and the peptide with an unblocked amino terminus as shown in Scheme 3. This cyclization of succinyl hydroxamate with cleavage of an amide is a known facile reaction (Notari, 1969). The reaction was monitored by following the release of ionized N-hydroxysuccinimide, λmax ) 260 nm. To effect this cleavage, N,N′-(PEG-O-succinyl)2glutathione was isolated by chromatography and solid hydroxylamine was added to a final concentration of 0.8 M. The pH was adjusted to 9.5 with 1 M NaOH, and the reaction mixture held at 40 °C for 2 h (Notari, 1969). To analyze the time course of reaction, aliquots of the reaction at 30 min intervals up to 2 h were analyzed by gel filtration HPLC using a 30 cm Phenomenex Biosep SEC S2000 column eluted with 100 mM ammonium acetate buffer, pH 5, at a flow rate of 1 mL/min. The eluate was monitored at 280 nm and each 1 mL fraction analyzed for the presence of PEG by the addition of an equal volume of Dragendorff’s reagent (0.11 M potassium iodide and 0.6 mM bismuth nitrate in 3.5 M acetic acid) (Rubia and Gomez, 1977) which generates an orange solution (λmax ) 524 nm) or precipitate in the presence of PEG. Desalting Cross-Linked Peptides. Following hydroxaminolysis, the reaction mixture contained newly regenerated peptides, ammonium acetate, PEG5000, hydroxylamine, and N-hydroxysuccinimide. To remove the PEG5000 and the excess hydroxylamine prior to further analysis, the solution was fractionated using a 1 g C18 solid-phase extraction column washed with 10 mL of 1 M sodium sulfate and 2 mL of water and eluted with 50% acetonitrile. Solid sodium sulfate was added to the peptide solution to a final concentration of 1 M prior to being loaded onto the solid-phase extraction column. Hydroxylamine and PEG5000 were removed by the wash, and the peptides were eluted in an elution volume of 1.5-2 mL of 50% acetonitrile. RESULTS

Protection of E-Amino Groups. Quantitative derivatization of lysine -amino groups has been accomplished by many procedures. In our hands, the best procedure was reductive methylation. This procedure had the benefits of retaining the positive charge for solubility while only chemically altering the protein amines, leaving Cys, His, and Tyr residues unmodified. To demonstrate that the conditions chosen successfully derivatized all of the -amino groups, lysozyme was chosen as a test protein. Following reductive methylation, less than 1% of the initially present amino groups was detected by

fluorescamine assay. In all protein methylations monitored, the reaction with fluorescamine decreased by at least 99%. In the presence of aggregates, the potential exists that -amino groups could be inaccessible to both methylation and fluorescamine. However, extensive reaction times and elevated temperatures should prevent this from being a difficulty. Derivatization of Amino Termini with PEG-Osuccinyl-O-Su. The derivatization of the proteolytically generated R-amino groups was moderately successful. The aqueous derivatization with a hydroxysuccinimide ester represents a competition between hydrolysis and aminolysis. The pH of this reaction is the crucial parameter, lowering the pH makes the reaction more specific for amines and decreases the rate constant for hydrolysis at the price of increasing the required reaction time. We adopted a repetitive addition, titration scheme to neutralize the acid generated during hydrolysis of the excess reagent. In favorable cases, this repetitive procedure derivatized over 90% and, in the worst case, 70% of the amino groups, corresponding to a predicted complete derivatization of at least half of all cross-linked peptides. Hydrolysis of PEG-O-Suc-O-Tyr. Control reactions with tyrosine indicated that some O-esterification of tyrosine with PEG-O-succinyl-O-Su occurred at pH 8.0. To define conditions required to hydrolyze PEG-Osuccinyl-O-Tyr, the peptide YGGFL was intentionally Nand O-derivatized by raising the pH of the derivatization reaction. The O-Tyr ester hydrolysis can be monitored by following the appearance of tyrosine phenoxide at 292 nm (Edelhoch, 1967) and was shown to be complete in 30 min at pH 11. This mild alkaline hydrolysis effectively cleaves both phenyl and thiolesters while leaving the PEG-O-Suc-N-peptide ester and amide linkages uncleaved. Any histidine derivatization would generate an unstable acyl-imidazole that hydrolyzes at neutral pH. Protein Hydrolysis. Proteolytic treatment with Pronase generates very small cross-linked peptides as it has endopeptidase activity as well as exopeptidase peptidase activities (Narahashi and Yanagita, 1967) which permits small modified peptides to be isolated (Catley et al., 1969). The use of more specific proteases such as trypsin will generate larger peptides that retain more sequence information. In our standard procedure the initial cleavage with chymotrypsin was introduced to enhance the efficacy of the Pronase cleavage. Separation of Doubly and Singly Derivatized Peptides. The gel filtration separation of lysine nitroanilide and tryptophan derivatized with PEG-O-succinyl-O-Su on this column is shown in Figure 1. Lysine nitroanilide derivatized with two PEG5000 chains eluted between 239 and 277 mL and the tryptophan derivative containing a single PEG5000 chain eluted between 277 and 332 mL on the Superdex XK26/100 column. The elution volumes for peptides containing either one or two PEG5000 chains were relatively consistent and had a resolution of 1.2-1.3. These elution volumes compare to that of a globular protein with an Mr of 5-10 times the nominal molecular weight of the polymer. Insulin with three amino groups,

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Figure 1. Gel filtration chromatogram of PEG-O-succinyl-NTrp and NR,N-bis(PEG-O-succinyl-)lysine nitroanilide as singly and doubly labeled calibrators on an XK 26/100 Superdex 200PG column. The elution volumes of the tryptophan and lysine derivatives define the volumes for the elution of peptides containing either one or two PEG5000 chains, respectively. UV analysis of the fractions indicated that the first peak contained only the lysine derivative, the second peak contained the tryptophan derivative and the third peak was largely hydroxysuccinimide.

Figure 2. Gel filtration chromatogram of NR,Na′-bis(PEG-Osuccinyl)glutathione by an XK 26/100 Superdex 200 PG column with the fractions monitored either by UV absorbance at 240 nm (0) or by using the NTSB assay for disulfide bonds ([).

was derivatized with PEG-O-succinyl-O-Su. The triply derivatized insulin eluted well before the lysine nitroanilide standard. This early eluting triply labeled species was pooled, reduced with DTT, and then rechromatographed on the same gel filtration column. Two peaks were observed, one from the doubly labeled β-chain (the amino terminus and carboxy terminal lysine would both be derivatized) eluted and the other from the singly labeled R-chain. The coelution of the PEG-O-succinylderivative of the 21 amino acid insulin R-chain with the singly labeled standards suggests that the size of the derivatized oligopeptide will not significantly alter the chromatographic behavior of the PEG-O-succinyl-peptides. Glutathione was used for the studies of both column recovery and derivatization efficiency. Glutathione was derivatized with PEG-O-succinyl-O-Su as described in the Materials and Methods. Derivatized glutathione was then chromatographed on the Superdex XK26/100 column. Using the NTSB assay for disulfide bonds, the recovery of cross-linked peptides was determined to be greater than 80%. The result with NTSB also showed that doubly labeled glutathione eluted between 236 and 276 mL and singly labeled glutathione eluted between 276 and 316 mL (Figure 2). The ratio of doubly labeled: singly labeled glutathione was 2.3:1. Thus, the efficiency

Chen et al.

Figure 3. Gel filtration chromatogram of reductively methylated lysozyme, proteolytically digested, and derivatized with PEG-O-succinyl-O-Su monitored at 240 nm (0) or by NTSB assay for disulfide bonds ([).

of derivatization of the glutathione amines was 85%. The pKa of the R-amino group of glutathione of 8.5 is higher than expected for the R-amino peptides, so that the efficiency of derivatization of the R-amino groups of peptides at pH 8.0 might be anticipated to be better. The incomplete derivatization results from the competitive hydrolysis of PEG-O-succinyl-O-Su by water. While more complete derivatization might be anticipated with additional PEG-O-succinyl-O-Su, higher concentrations of PEG5000 become counterproductive in the subsequent chromatographic separation. The requirement for excess derivatizing reagent made it difficult to use an affinity matrix with their attendant low capacities for the chromatographic resolution singly and doubly labeled peptides. Hydroxaminolytic Liberation of Cross-Linked Peptides from PEG-O-succinyl-N-peptide. Removal of PEG-O-succinyl moiety is desirable for planned sequencing studies of cross-linked peptides by Edman degradation or by mass spectrometry. Hydroxaminolysis was shown to remove the PEG-O-succinyl label from the derivatized peptides, as depicted in Scheme 3. The peptide MeOSuc-Ala-Ala-Pro-Val was used as a standard to determine the required reaction conditions. With peptides, the cleavage was additionally demonstrated by separating the liberated peptide from the polymeric PEG. Doubly labeled N,N′-(PEG-O-succinyl)2-glutathione was subjected to hydroxaminolysis, and the products separated by HPLC gel filtration. Following hydroxaminolysis, the PEG elution volume increased from 11 to 13 min, consistent with the release from the doubly labeled peptide with a nominal Mr in excess of 10 000 to the elution volume of a standard monomeric PEG5000 while the glutathione eluted in the included volume. Analyses at varying times were consistent with the spectrophotometric studies, indicating that the hydroxaminolysis was complete in under 1 h. Lysozyme. Lysozyme is cross-linked by four disulfide bonds. To demonstrate the method, peptides cross-linked by disulfides were isolated by the protocol outlined in Scheme 1. Lysozyme was methylated and desalted as described, digested with chymotrypsin and Pronase, and derivatized with PEG-O-succinyl-O-Su. The gel filtration chromatogram of the lysozyme derived peptides is shown in Figure 3. Each fraction was concentrated and then tested for disulfide bonds by using the NTSB assay. The ratio of underivatized: singly labeled: doubly labeled peptides was 2:2.1:1.5 based on the NTSB assay. The

Universal Isolation of Cross-Linked Peptides

Figure 4. Gel filtration chromatograms of cross-linked and uncross-linked hemoglobin by 26/60 Hiload Superdex 200 PG column, following reductive methylation, digestion with chymotrypsin and Pronase, and derivatization with PEG-O-succinyl-O-Su, followed by mild alkaline hydrolysis. The column fractions of the derivatized un-cross-linked hemoglobin peptides were monitored at 280 nm (×). The fractions of the derivatized cross-linked hemoglobin peptides were monitored by both UV at 280 nm (b) and the dansyl fluorescence at 550 nm (O) with excitation at 340 nm. The major fluorescent peak eluting around 215 mL elutes corresponds to the elution volume of peptides containing two PEG5000 chains by this column.

apparent lower yield based on disulfide bonds, when compared with that of glutathione, may result from (1) free thiols generation from the cleavage of disulfide bonds since NTSB assay detects both free thiol and disulfide and (2) incomplete proteolysis. An inherent requirement of the protocol is that the proteolysis generates a new R-amino group in the sequence prior to the amino acid whose side chain participates in the cross-link. In lysozyme, Cys-6 forms a disulfide bond with Cys-127. To isolate a peptide with this cross-link, the proteolysis must cleave the peptide backbone before Cys-6 and between Cys-115 and Cys-127. Failure to proteolytically cleave before Cys-6 would result in cross-linked peptide with only one R-amino group because the N-terminal of lysozyme has been blocked by dimethylation. Thus, the apparent low efficiency may be a function both of disulfide cleavage and incomplete proteolysis. Even with this difficulty, this chromatogram clearly demonstrates the potential separation of a pool of cross-linked peptides obtained from a protein with intramolecular cross-links. Hemoglobin. MDC-Hb2 (shown in Scheme 2) and control human hemoglobin were both treated according to Scheme 1 as follows: reductive methylation, desalting of protein, proteolytic digestion with chymotrypsin and Pronase, PEG-O-succinyl-O-Su derivatization, mild alkaline hydrolysis of labile PEG5000 esters, gel filtration chromatography, and fluorescent measurement of the collected fractions. The chromatograms of the crosslinked hemoglobin (3 mg) and un-cross-linked hemoglobin (3 mg) produced by monitoring the fluorescence at 550 nm with excitation at 340 nm and by monitoring the absorbance at 280 nm are shown in Figure 4. The peptides generated from proteolytic digestion of the crosslinked hemoglobin were separated by gel filtration column into three peaks, doubly labeled, singly labeled and underivatized peptides in the order of increased retention time. From the fluorescence assay of the fractions, it is obvious that the cross-linked peptide containing the dansyl group was concentrated in the first peak. This particular cross-linked protein was selected because (1) there is a single cross-link per 68 kDa hemoglobin tetramer and (2) the dansyl moiety on the cross-link can

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Figure 5. Gel filtration chromatogram of protein isolated from neurofibrillary tangles from Alzheimer disease that was reductively methylated, Pronase digested, PEG-O-succinyl-O-Su derivatized, and finally subjected to mild alkaline hydrolysis. The peptides eluted are monitored by absorbance at 280 nm. The peak eluting at 250 mL, elutes at the position expected for cross-linked peptides but not linear peptides. Unlike the previous examples this peak is small when compared to the monomeric fraction eluting at 300 mL because the cross-link is unknown and cannot be specifically detected.

be identified by monitoring fluorescence, permitting the spectral identification of cross-linked peptides and their separation to be monitored. In the control experiment, un-cross-linked hemoglobin was treated according to the same procedure. The chromatogram monitored by UV absorbance at 280 nm showed only one peak at the same retention time of the peptides containing a single PEG5000 chain, indicating that the protocol does not produce false positive signals by incorporation of two PEG5000 chains by unanticipated derivatization of amino acid side chains. Insoluble Paired Helical Filaments of Alzheimer’s Disease. An enriched fraction of paired helical filaments, the component structures of neurofibrillary tangles, from a case of Alzheimer’s disease containing 0.4 mg of protein was treated by using the same procedure as for hemoglobin, except the fluorescence analysis of the chromatographic fractions was not required. The chromatogram obtained monitoring absorbance at 280 nm to detect peptides containing aromatic residues is shown in Figure 5. The peak with elution volume of 240-268 mL was that of the peptides containing two PEG5000 chains and are presumptively cross-linked peptides derived from neurofibrillary tangles. There were no disulfide bonds or free thiols detected in these fractions using the NTSB and DTNB assays, respectively. Therefore, the identification of peptides derivatized with two PEG5000 chains is suggestive of the existence of cross-link(s) in the neurofibrillary tangles. Further characterization of these putative cross-link(s) is being undertaken by mass spectrometry. Hydroxaminolysis will liberate the parent peptides and permit the reversed-phase HPLC-mass spectrometric characterization of this pool of peptides in our laboratory. DISCUSSION

Cross-linking has proven to be a powerful method of studying protein structure, by demonstrating that the two cross-linked groups must, for the chemical reaction to have occurred, have been in close proximity. While cross-linked peptides can be identified when a control peptide map exists or when the cross-linker contains a detectable marker, neither of these restrictions applies when cross-linked peptides are sought from a biological

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sample cross-linked by unknown processes. Scheme 1 presented a conceptual approach to isolating cross-linked peptides independent of the chemical nature or origin of the cross-link. All of the chemistry proposed is precedented and was substantiated in the studies reported. The main issue presented in Scheme 1 is the selection of an amine-derivatizing reagent that will quantitatively react with the R-amino groups, including the secondary amine of proline, liberated by the partial hydrolysis of the protein backbone. Our initial concept was to use myristoyl isothiocyanate, as the development of Edman degradation has substantiated that derivatization of the amino terminal group with isothiocyanates can be quantitative and yet easily removed by acid-catalyzed cyclization. We envisioned a separation based on reversed-phase HPLC. The difficulties of peptide solubility both before and after derivatization prevented the realization of this approach. The choice of PEG-O-succinyl-O-Su as the derivatizing reagent was in response to difficulties in maintaining peptide and reagent solubility. The PEG5000 chain is miscible with water and imparts enhanced aqueous solubility to derivatized peptides and minimizes interactions with the gel filtration matrix. Alternative derivatization conditions or reactive PEG5000 substituent may permit an increase in the efficiency of the derivatization conditions. Incorporation of a radioactive label into the PEG, e.g., using [14C]succinic anhydride to form the succinate ester, would permit the described assay to become a method of quantifying the isolated cross-links. The second major consideration in Scheme 1 is the choice of proteolytic conditions. As noted, one of the virtues of reductive methylation of the -amino groups is that this derivatization is stable to acid hydrolysis. Thus, quantitative hydrolysis with 6 N HCl or weak acid hydrolysis would be possible, allowing the described method to isolate all acid-stable cross-links. This is potentially an important virtue for handling insoluble protein aggregates. A similar method exists based on R-amino acids ability to chelate diphenylborinic acid (Graham and Gallop, 1994). However, this approach has the absolute requirement that the protein be hydrolyzed completely to its constituent amino acids, preventing any sequence information from being retained in the crosslinked peptides. Conclusion. A protocol has been proposed that permits the isolation of cross-linked peptides independent of the chemical nature of the cross-link. The validity of the key premise of this scheme, that the two amino termini of cross-linked peptides can be derivatized to permit separation from linear peptides, has been clearly demonstrated. Separation of PEG-O-succinyl-O-Su-derivatized peptides by gel filtration chromatography has been shown capable of isolating disulfide-bond cross-links from proteins of known sequence, a single fluorescent cross-linked peptide from the large number of proteolytic peptides of hemoglobin, and most provocatively, a poten-

Chen et al.

tial pool of cross-linked peptides isolated from neurofibrillary tangles obtained from an Alzheimer’s disease brain. ACKNOWLEDGMENT

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