Characterization of Whole Fibril-Forming Collagen Proteins of Types I

Fibril-forming collagen proteins of the types I, III, and V were extracted from fetal calf skin, purified by differential salt precipitation, and anal...
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Anal. Chem. 2004, 76, 3482-3491

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Characterization of Whole Fibril-Forming Collagen Proteins of Types I, III, and V from Fetal Calf Skin by Infrared Matrix-Assisted Laser Desorption Ionization Mass Spectrometry Klaus Dreisewerd,*,† Andreas Rohlfing,† Beatrice Spottke,† Claus Urbanke,‡ and Werner Henkel§

Institute of Medical Physics and Biophysics and Institute for Arteriosclerosis Research, Westfa¨lische-Wilhelms-Universita¨t, Mu¨nster, Germany, and Department of Biophysical Chemistry, Medizinische Hochschule, Hannover, Germany

Collagen proteins comprise more than 25% of the total protein content in mammals. They are localized primarily in the extracellular matrix. Fibril-forming collagens of types I, III, and V are made up of linear R-chains of ∼100 kDa in molecular weight, which form closely coiled triple-stranded helical molecules (γ). Type I and V triple helices are essentially held together by hydrogen bridges. Type III triple helices are further stabilized by disulfide bridges between cysteine residues located in the Cterminal region of the R-chains. The formation of further sub-

structures [dimer (β)] and supramolecular structures [tetramer (τ), hexamer (η)] is possible by covalent cross-linking via aldimine or disulfide bonds. Traditionally, SDS disc electrophoresis is used to identify fibrillar collagens, their subunits, and cyanogen bromide peptides of the chains. Mass spectrometry, which offers the potential advantage of a higher accuracy and sensitivity, as well as that of reduced analysis times, has so far hardly been applied to the analysis of whole collagen molecules. In fact, their large molecular weights and, in the case of the fibril-forming types, marked linear dimensions pose particular demands on any desorption/ionization system. The detection of type III R-chain proteins from calf skin employing UV-MALDI mass spectrometry and sinapinic acid as MALDI matrix has been reported by Kim et al.1 However, the triple helix was not detected, and the R-chains were only detected after thermal denaturation of the sample. This group also monitored the succinylation reaction of lysine residues as a potentially important reaction in the production of collagen-based biomaterials. The detection of even noncovalently bound triple helices was reported by Zaia et al.2 These authors investigated chicken collagen proteins of types I, II, and XI. IR-MALDI-MS with succinic acid and UV-MALDI-MS with sinapinic acid as solidstate matrixes were both employed successfully. The present article describes experiments that were performed with glycerol as liquid IR-MALDI matrix. Glycerol has been recognized before as being well-suited for the analysis of particularly large or labile biomolecules. For example, proteins with molecular weights exceeding several hundred kDa3 and large DNA molecules4 have been analyzed. Types I, III, and V collagen proteins were extracted from fetal calf skin with pepsin and purified by differential salt precipitation. The mass spectrometric analysis was supplemented by denatur-

* To whom correspondence should be addressed. Phone: +49-251-8356726. Fax: +49-251-8355121. E-mail: [email protected]. † Institute of Medical Physics and Biophysics, Robert-Koch-Str. 31, D-48149 Mu ¨ nster, Germany. ‡ Department of Biophysical Chemistry, Carl-Neuberg-Str. 1, D-30623 Hannover, Germany. § Institute for Arteriosclerosis Research, Domagkstr. 3, D-48149 Mu ¨ nster, Germany.

(1) Kim, S. H.; Lee, J.-H.; Yun, S.-Y.; Yoo, J. S.; Jun, C.-H.; Suh, H.; Chung, K.-Y.; Suh, H. Rapid Commun. Mass Spectrom. 2000, 14, 2125. (2) Zaia, J.; Hronowski, X. L.; Costello, C. E. In Proceedings of the 45th ASMS Conference of the American Society for Mass Spectrometry and Allied Topics, Palm Springs, CA, June 1-5, 1997; ASMS: Santa Fe; p 367. (3) Berkenkamp, S.; Menzel, C.; Karas, M.; Hillenkamp, F. Rapid Commun. Mass Spectrom. 1997, 11, 1399. (4) Berkenkamp, S.; Kirpekar, F.; Hillenkamp, F. Science 1998, 281, 260.

Fibril-forming collagen proteins of the types I, III, and V were extracted from fetal calf skin, purified by differential salt precipitation, and analyzed by infrared matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (IR-MALDI-TOF-MS). Glycerol was used as liquid IRMALDI matrix. Noncovalently bound triple helices of the types I and V were detected from the NaCl precipitate. After heating at 43 °C for 10 min, resulting in the dissociation of the triple helix, the single r-chain subunits were detected. For type I, mass spectra acquired from molecular sieve chromatography fractions revealed the presence of further substructures of dimeric type and of supramolecular complexes up to the tetramer. Triple helices of type III, stabilized by covalent disulfide bonds, were detected from the total protein precipitate also after heat treatment. For type III, even hexamer and nonamer structures with molecular weights close to 600 and 900 kDa were recorded. For comparison, ultraviolet (UV-)MALDI-MS measurements with 2,5-dihydroxybenzoic acid as matrix were carried out with some of the samples. Here, only the single r-chains were detected with significantly reduced sensitivity.

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Figure 1. Schematic drawing of the IR-MALDI time-of-flight mass spectrometer. Ions were accelerated in a two-stage Wiley-McLarentype ion source with a low extraction field of ∼20 V/mm in the first stage. The total acceleration voltage was 19.4 kV. Ion signals were detected in reflector mode with a secondary electron multiplier (SEM). This detector is equipped with a conversion dynode for efficient production of secondary ions in order to enhance detection of large mass ions.

ation studies employing thermal unfolding and chemical reduction of disulfide bonds along with chromatographic separation methods. UV-MALDI-MS was performed on a limited number of samples. EXPERIMENTAL SECTION Time-of-Flight Mass Spectrometer. All IR-MALDI-MS experiments were carried out with an in-house-built single-stage reflectron time-of-flight mass spectrometer (TOF-MS) of 3.5-m equivalent drift length (Figure 1). Measurements were performed in reflector mode. The back pressure in the mass spectrometer was ∼2 × 10-6 mbar. Samples were observed with a CCD camera chip at an optical resolution of ∼20 µm. An Er:YAG laser (Speser, Spektrum GmbH, Berlin) emitting at a wavelength of 2.94 µm was used for desorption/ionization. The focal spot size of the laser beam was ∼150 µm in diameter (1/e2-definition) with an approximately Gaussian intensity distribution. The temporal laser pulse profile was also approximately Gaussian and had a fwhm of ∼150 ns. The laser fluence was adjusted by means of a dielectric attenuator. Pulse energies were measured by placing a high precision commercial energy meter (Laser Precision, Corp., Yorkville, NY) directly into the beam line. The shot-to-shot pulse energy stability was ∼5% (standard deviation). Laser fluences were calculated by dividing the pulse energy, corrected for transmission losses, by the above spot size. Unless otherwise stated, a laser fluence within 2-3 times the ion detection threshold fluence F0 [F0 ∼ (3000-3500) J m-2] was used throughout the measurements. A two-stage Wiley-McLaren-type ion source with planar grids at distances from the sample plate of 6 and 18.5 mm, respectively, was employed for ion extraction. Experiments were performed using static ion extraction with a voltage of +19.4 kV applied to the sample plate and the first grid voltage set to +19.3 kV, thus providing a very low extraction field strength of ∼20 V/mm. This turned out to be essential for suppressing an extensive collagen ion fragmentation, which was observed for more common MALDITOF-MS extraction conditions. Application of delayed extraction was not found to improve the performance. The second grid was held at ground potential. A venetian-blind secondary electron

multiplier (SEM; EMI 9643, Electron Tubes Ltd., Ruislip, U.K.) equipped with a conversion dynode mounted 10 mm in front of the first dynode of the SEM was used as ion detector. The postacceleration potential applied to the conversion dynode was set to -20 kV for an efficient generation of secondary ions, enhancing the detection sensitivity for primary molecular ions in the mass range above ∼10 kDa.5,6 SEM signals were amplified with a custom-built fast amplifier and digitized and stored with a digital oscilloscope (LeCroy 9345A, Chestnut Ridge, NY). The digitized data were transferred to a PC for subsequent processing with a custom-made software program. UV-MALDI-MS measurements were performed with a Reflex III instrument (Bruker Daltonik, Bremen, Germany). The linear mode of the instrument was used. In contrast to the low-field conditions used in the glycerol IR-MALDI experiments, “normal” delayed extraction (DE) parameters were applied with electrical field strengths in the first extraction region on the order of ∼1 kV/mm (after DE switching) and an overall acceleration voltage of 25 kV. An SEM-conversion dynode detector of the same type as used for IR-MALDI-MS was employed for increased detection efficiency. Materials. Glycerol, 2-hydroxy-5-methoxybenzoic acid, and bovine serum albumin (BSA) were purchased from Fluka, 2,5dihydroxybenzoic acid (2,5-DHB) from Sigma-Aldrich, and pepsin from Serva. Aqueous solutions of 10 mg/mL 2,5-DHB and 10 mg/ mL 2-hydroxy-5-methoxybenzoic acid (each containing 10% ethanol) were mixed 10:1 (v/v) to produce DHBs. Preparation of Collagens. Purification of the collagen proteins was achieved following a modified method of Burgeson et al.7 A 460-g portion of fetal calf skin tissue was pre-extracted with 0.5 M sodium acetate and then with water. The extracted tissue (250 g) was suspended in 2.5 L of 0.5 M formic acid and proteins solubilized by adding 1.75 g of pepsin and stirring the suspension for 48 h at 4 °C, conditions that do not cause degradation of the triple-helical regions. Type I and III collagens were separated from type V by differential salt precipitation at pH 2. By adding NaCl to a concentration of 4.1%, collagens I and III were precipitated from the acid pepsin extract. Type V collagen was precipitated from the remaining solution by increasing the NaCl concentration to 7%. Collagens of types I and III were separated from each other by differential salt precipitation at pH 7.5. The precipitate obtained at 4.1% NaCl was dissolved in 0.1 M acetic acid, and the solution was adjusted to pH 7.5 by dialysis against Tris buffer (0.05 M, pH 7.5, 1 M NaCl). Collagen fractions were obtained by dialysis against the same buffer, progressively increasing the concentration of NaCl to 1.5, 1.8, 2.1, and 2.5 M. The fractions precipitating at 1.5 and 1.8 M, representing type III collagen, and at 2.1 and 2.5 M, containing type I collagen, were reprecipitated at the specific salt concentrations. From 460 g fetal calf skin, 15.254 g of collagen type I, 3.979 g of type III, and 406 mg of type V were obtained as lyophilized proteins. Molecular Sieve (Gel Filtration) Chromatography. Separation of different molecular species from the collagens of types I (5) Kaufmann, R.; Kirsch, D.; Rood, H. A.; Spengler, B. Rapid Commun. Mass Spectrom. 1992, 6, 98. (6) Dreisewerd, K.; Schu ¨ renberg, M.; Hillenkamp, F. In Proceedings of the 50th ASMS Conference of the American Society for Mass Spectrometry and Allied Topics, Orlando, FL, June 2-6, 2002; ASMS: Santa Fe; A02367. (7) Burgeson, R. E.; El Adli, F. A.; Kaitila, J. J.; Hollister, D. W. Proc. Natl. Acad. Sci. U.S.A 1976, 73, 2579.

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and III was carried out essentially as described by Fujii and Ku¨hn.8 A 30-mg portion of collagen proteins was dissolved in 3 mL of elution buffer containing 8 M urea and heated to 43 °C for 10 min. After centrifugation at 14 000 rpm for 5 min at room temperature, the colorless supernatant was applied to a Bio-Rad Bio-Gel A-15 column (3.0 × 135 cm, 200-400 mesh size) and eluted with Tris buffer (0.05 M, pH 7.5, 1 M CaCl2) at room temperature and a flow rate of 33 mL/h. Anion-Exchange Chromatography. To separate the R1- and R2-chains of collagen V triple helices [R1(V)]2 R2(V), a modified method of Mann was applied.9 A 2-mg portion of collagen proteins, precipitated at 7% NaCl, was dissolved in 500 µL of Tris buffer (0.02 M, pH 7.5, 2 M urea), and triple helices unfolded at 50 °C for 15 min. After removing insoluble proteins by centrifugation at 14 000 rpm for 10 min at 4 °C, the clear supernatant was applied to a Mono Q HR5/5 column (Pharmacia-LKB) equilibrated in the same buffer. After washing for 6 min, the bound R-chains were eluted in the Tris buffer using a gradient from 0.1 to 0.2 M NaCl in 39 min at a flow rate of 0.5 mL/min at room temperature. The fractions containing the separated chains were desalted in 0.1 M acetic acid, and the solution was concentrated to a volume of 300 µL, in each case by evaporation of the solvent. Reduction of Disulfide Bonds and S-Carboxymethylation. The method was performed according to Gurd.10 A 20 mg portion of collagen III was dissolved in 2 mL of Tris (0.4 M, pH 8.4, 4 M urea, 0.2% disodium EDTA), which had been flushed with nitrogen. After addition of 100 µL of 2-mercaptoethanol (1.42 mM), the solution was again saturated with nitrogen, then heated at 43 °C for 15 min and allowed to stand at room temperature in a lightprotected vessel for at least 1 h. After this time, 300 mg of sodium iodoacetate (1.44 mM) was added. After 15 min, another 100 µL of 2-mercaptoethanol was added. The solution was stirred for 2 h at room temperature and S-carboxymethylation was finished by lowering the pH to a value of 3 by addition of 400 µL of acetic acid. After centrifugation at 14 000 rpm for 5 min at 4 °C, the clear supernatant was applied to the Bio-Gel A-15 column as described above. Sample Preparations for MALDI-MS and Measurement Protocol. Glycerol samples were prepared by mixing a glycerol droplet of ∼1 µL in volume on-target with ∼1 µL of analyte solution to produce a molar analyte-to-matrix (A/M) ratio of ∼10-5 to 10-4. Prior to transfer into the high vacuum of the mass spectrometer, most of the water in the preparation was gassed off in the prevacuum (∼10-2 mbar) of the transfer stage, in which the sample plate was kept for ∼5 min. Evaporation of glycerol in the high vacuum led to continuous shrinkage of the droplet and increased analyte concentration with time. Typically, a droplet would have evaporated completely within 3-4 h. However, measurements were generally finished within 0.5-1 h after target transfer. Samples were irradiated at the apex of the drop at a repetition rate of 2 Hz. Depending on signal intensity, between 10 and 200 singleshot mass spectra were accumulated. External mass calibration was achieved with singly charged bovine serum albumin molecular ions (MW, 66.4 kDa) and BSA gas-phase oligomers up to the tetramer. Mass values were determined as the centroid of the (8) Fujii, T.; Ku ¨ hn, K. H.-S. Z. Physiol. Chem. 1975, 356, 1793. (9) Mann, K. H.-S. Z. Physiol. Chem. 1992, 373, 69. (10) Gurd, F. R. N. Methods in Enzymology XI; Academic Press: New York, 1967; p 532.

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signal peaks. Therefore, any nonresolved tailing of the signals, for example, by adduct formation, contributed to the so-determined mass. The accuracy (reproducibility) of the mass determination was determined exemplarily for γ-ions of types I and III from a total protein precipitate to ∼0.4% (single standard deviation). Sedimentation Analysis. A Beckman XL-A analytical ultracentrifuge equipped with a photoelectric scanner and an 8-place An50 Ti rotor with double sector centerpieces was used for all sedimentation velocity experiments. Because the collagen proteins contain only few aromatic residues, carbonyl absorption at 225 nm was used to measure the sedimentation profiles [A(x, t)]. These were transformed to a sedimentation coefficient distribution [A(s)] using the formalism described by Grale´n and Lagermalm,11

A(s) ) A

(∫ )( ) ln(x/xm)

x t 2 x ω dt′ m 0

2

(1)

where x is the radial position in the cell, xm is the position of the meniscus, ω is the angular velocity of the rotor, and A is the optical absorption. A(s) values obtained at different times in a single experiment were averaged for smoothing and were corrected to 20 °C and pure water as solvent (S20,W). Calculation of Theoretical Mass Values. Theoretical molecular weights of the R-chain backbones were derived from amino acid sequence and cDNA data. Posttranslational modifications, such as hydroxylation and glycosylation, were taken from cyanogen bromide peptide, hydrolysis, and amino acid composition studies, documented in the literature, and were performed on the different collagen species. Pepsin cleavage sites were determined by correlating cDNA and cyanogen bromide peptide data. In the case of the R1(V)- and R2(V)-chains, for which no entries could be found in the databases, human R1(V)- and R2(V) pro-collagen data were used, instead. R1(I). The calculated mass of R1(I) is based on amino acid sequence12 and cDNA data.13 Taking the site between glycine 171 [residue no. 171 of R1(I)] and isoleucine 172 as N-terminal and between leucine 1198 and proline 1199 as C-terminal pepsin cleavage point, the pepsin-digested R1(I)-chain extends from isoleucine 172 to leucine 1198 of the pro-R1(I)-chain. Considering the hydroxylation of proline and lysine14 and the presence of two glucosylgalactosyl disaccharides as the most likely modification, linked to the hydroxylysine residues 262 and 1105, the theoretical mass of R1(I) amounts to 93 915 Da. R2(I). The calculated mass of R2(I) is based on cDNA data.15 Assuming as pepsin cleavage points the sites between phenylalanine 77 and alanine 78 and between glycine 1109 and phenylalanine 1110, the pepsin-derived R2(I)-chain covers the sequence range from residue 78 to residue 1109 of the pro-R2(I)-chain. Assuming further that the degree of hydroxylation of proline and lysine is the same for R2(I) and R1(I), a mass value of 94 262.7 Da is obtained for R2(I). Estimating that R2(I), as R1(I), is also two times diglycosylated, a final mass of 94 910.7 is derived. (11) Grale´n, N.; Lagermalm, G. J. Phys. Chem. 1952, 56, 514. (12) Fietzek, P. P.; Ku ¨ hn, K. Mol. Cell. Biochem. 1975, 8, 141. (13) https://www.ncbi.nlm.nih.gov; entry AAB94054. (14) Rauterberg, J.; Ku ¨ hn, K. Eur. J. Biochem. 1971, 19, 398. (15) Shirai, T.; Hattori, S.; Sakaguchi, M.; Inouye, S.; Kimura, A.; Ebihara, T.; Irie, S.; Nagai, Y.; Hori, H. Matrix Biol. 1998, 17, 85.

R1(III). The pepsin-derived R1(III)-chain covers the amino acid sequence range from 168 to 1196 of the pre-pro-R1(III)-chain16 and, in addition, a C-terminal extension of eight residues within the nonhelical region (1197-1204). The pepsin-sensitive cleavage site at the N terminus coincides with the N terminus of the triple helical part of R1(III).17 At the C-terminal end, it is between alanine 1204 and isoleucine 1205.18 The hydroxylation of proline and lysine was taken from ref 19. Considering the presence of glucosylgalactosyl disaccharides linked to hydroxylysine 263 and 1106, a total mass of 95 373 Da is obtained for R1(III). R1(V). Pepsin-digested R1(V)-chains contain the complete triple-helical region extending from the amino acid residue 559 to 1572 of the pre-pro-R1(V)-chain. This corresponds to a theoretical mass of 96 327 Da if posttranslational hydroxylation of the proline and lysine residues is included.20,21 Due to the pepsin cleavage point between the residues leucine (555) and alanine (556), an extension of three residues (556-558) within the N-terminal nonhelical region must be considered with a mass of 340.5 Da.21 Assuming that the C-terminal pepsin cleavage point coincides with the C terminus of the triple helix, the mass of the pepsin-derived R1(V)-chain (556-1572), based on cDNA analysis, amounts to 96 667 Da. Unfortunately, the degree of glycosylation is not known for bovine type V collagen. A study by Chung et al. on R1(V) from human skin indicated that in this case, 13% of the 39 hydroxylysine residues were modified with galactose and 75% with glucosylgalactose.22 Assuming a similar extent of glycosylation for the 41 hydroxylysine residues in the bovine type, the theoretical mass of the R1(V)-chain would increase by 10 826 Da to 107 493 Da. R2(V). The pepsin-derived R2(V)-chain includes the sequence range from 213 to 1229 of the pro-R2(V)-chain.23 If hydroxylation of the proline and lysine residues is taken into account, this amounts to 95 550 Da for the nonglycosylated chain.24 The chain is flanked by two pepsin cleavage points, one coinciding with the N terminus of the triple-helical region and the other being localized between the residues of alanine 1229 and leucine 1230 within the C-terminal nonhelical region.23 In R2(V), 22.5 hydroxylysine residues are, on average, available for glycolysation.24 Again, the extent of the modification is not known yet. Taking the values determined for R2(V) from human skin,22 ∼23% of hydroxylysines were diglycosylated, and 13.6% were modified with a single galactose residue. This would result in a theoretical increase in mass by 2181 Da to 97 731 Da. RESULTS AND DISCUSSION Type I. Triple helices of type I collagen are of the two-chain form [R1(I)]2 [R2(I)]. Figure 2a displays an IR-MALDI mass (16) Ala-Kokko, L.; Kontusaari, S.; Baldwin, C. T.; Kuivaniemi, H.; Prockop, D. J. Biochem. J. 1989, 260, 509. (17) Fietzek, P. P.; Allmann, H.; Rauterberg, J.; Henkel, W.; Wachter, E.; Ku ¨ hn, K.; H-S. Z. Physiol. Chem. 1979, 360, 809. (18) Allmann, H.; Fietzek, P. P.; Glanville, R. W.; Ku ¨ hn, K. H.-S. Z. Physiol. Chem. 1979, 360, 861. (19) Rauterberg, J.; Allmann, H.; Henkel, W.; Fietzek, P. F. H.-S. Z. Physiol. Chem. 1976, 357, 1401. (20) Takahara, K.; Sato, Y.; Okazawa, K.; Okamoto, N.; Noda, A.; Yaoi, Y.; Kato, I. Biol. Chem. 1991, 266, 13124. (21) Rhodes, R. K.; Miller, E. J. J. Biol. Chem. 1979, 254, 12084. (22) Chung, E.; Rhodes, R. K.; Miller, E. J. Biochem. Biophys. Res. Commun. 1976, 71, 1167. (23) http://www.ncbi.nlm.nih.gov; entry P05997. (24) Rhodes, R. K.; Gibson, K. D.; Miller, E. J. Biochemistry 1981, 20, 3117.

Figure 2. (a) IR-MALDI mass spectrum of type I collagen proteins from total NaCl precipitate. Triple helices (γ) are detected as singly and multiply charged ions. (b) Mass spectrum from heat-treated sample (heating at 43 °C for 10 min prior to mixing with matrix). Noncovalently bound triple helix complexes dissociate upon the heat treatment, and the R-chain subunits are detected. Sum of (a) 40 and (b) 15 single-shot mass spectra.

spectrum acquired from a total NaCl protein precipitate of type I. For the approximately physiological conditions of the preparation, singly and doubly charged ions of the triple helix (γ) form the base peaks in the spectrum. The experimental mass of the triple helix of (283 300 ( 1300) Da agrees well with its theoretical value of 282 741 Da. The expected molecular weights of the R1(I)- and R2(I)-chains of 93 915 and 94 910.7 Da, respectively, are too close to one another to allow separation in the TOF mass spectrum. For the same reason, any heterogeneity in the triple helix composition would also not be differentiable. For some of the observed ion signals, assignment is also not without ambiguity. For instance, a triply charged γ-ion signal can potentially overlap with singly charged R-chain signals, whereas a cross-linked β-complex could overlap with nonspecific gas-phase dimers of R; potentially also with triply charged nonspecific gasphase dimers of the triple helix (2γ3+). Evidence of the specific character of the observed complexes and differentiation against such nonspecific oligomers was obtained by controlled dissociation of the supramolecular complexes and by combined chromatography/MS studies. Figure 2b displays the mass spectrum obtained from the total NaCl precipitate after the sample was heated at 43 °C for 10 min (prior to mixing it with matrix). As expected, the denaturation goes along with a strong reduction of the γ-ion and increase of the R-chain signal. As shown below, the residual signal at the mass of the triple helix can in part be attributed to trimeric complexes which are stabilized by cross-linking. Analytical Chemistry, Vol. 76, No. 13, July 1, 2004

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Figure 3. Molecular sieve chromatography of the collagen I species. Chromatography was performed on Agarose A-15 gel loaded with 30 mg of heat-treated (43 °C, 10 min) 2.4 M NaCl precipitate.

Figure 3 displays the molecular sieve chromatogram of the total NaCl protein precipitate. Fractions from the run were collected as indicated in the figure and subsequently analyzed by IR-MALDI-MS (Figure 4a-d). The denaturing conditions of the gel filtration allow only structures with covalent binding character to pass the run undegraded. Therefore, the mass spectra in b and c confirm the presence of cross-linked β- and γ-supramolecular structures (presumably of aldimine type). In addition, even a tetrameric complex (τ) is observed (Figure 4d). The cross-linked complexes are detected with a higher mass value than a simple multiplication of the R-chain mass according

to the degree of oligomerization would suggest. For example, the measured mass of the cross-linked γ-species of Figure 4c of (291 000 ( 1000) Da is larger by ∼7000 Da than that of the triple helix from the nonheated total NaCl precipitation (Figure 2a; see detailed discussion below). Type III. Triple helices of type III are of the single-chain form [R1(III)]3. They are stabilized by disulfide bridges between cystein groups, localized in the C-terminal part of the R-chains. Hence, mass spectra taken before (Figure 5a) and after heating at 43 °C for 10 min (Figure 5b) hardly differ from each other. A slight increase in intensity as well as in the width of the R-chain signal is, however, notable. Probably, two just not resolved species are composing the R-chain signal after the heat treatment. This is indicated by two labels in the figure. The determined mass of the triple helix of (288 300 ( 1300) Da is only slightly higher than its theoretical value of 286 119 Da. Because from the six cysteine residues in a triple helix (two cystein residues per chain) only four are needed to link the three chains, further supramolecular structures of hexamer (η) and nonamer (ν) type can be formed by “intermolecular” disulfide bonds between two or three triple helices.25 These structures are, indeed, detected in the form of multiply charged ions if a very low laser fluence close to the ion detection threshold is applied (Figure 5c) but not at elevated fluences. Some of the signals, ν5+, ν7+, ν8+, η5+, and η7+, are “unambiguous” because their m/z values do not overlap with any likely R-, β-, or γ-related signals. The m/z values of the νn+ series, thus, reveals a molecular weight of the nonamer of ∼870 000 Da; those of the ηn+ series, a molecular weight of the hexamer of ∼580 000 Da.

Figure 4. IR-MALDI mass spectra of type I collagen proteins from molecular sieve fractions, collected as indicated in Figure 3. The gel filtration is associated with denaturing conditions. Substructures of covalent character formed by cross-linking of presumably aldimine type are not destroyed and are detected by IR-MALDI-TOF-MS. Sum of (a) 45, (b) 15, (c) 55, and (d) 85 single-shot mass spectra. The mass spectrum in (d) has been smoothed by 9-point-FFT filtering. 3486

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Figure 6. Molecular sieve chromatography of the collagen III species. Chromatography was performed on Agarose A-15 gel loaded with 30 mg of heat-treated (43 °C, 10 min) 1.5 M NaCl precipitate. (a) Run with nonreduced precipitate; (b) run with reduced and carboxymethylated precipitate.

Figure 5. (a) IR-MALDI mass spectrum of type III collagen proteins from total NaCl precipitate. (b) Mass spectrum from heat-treated sample (heating at 43°C for 10 min prior to mixing with matrix). Type III triple helices, stabilized by disulfide bridges in the C-terminal part of the molecules, do not dissociate upon thermal unfolding of the strands. (c) Hexamer and nonamer supramolecular structures are detected for very low laser fluences of ∼3500 J m-2, close to the ion detection threshold. Sum of (a) 20, (b) 30, and (c) 185 single-shot mass spectra.

The η-complex is also found back in the molecular sieve chromatogram performed on the NaCl precipitate of type III (Figure 6a). A small, albeit unresolved, shoulder to the hexamer band likely reflects the ν-complex. That both species are, indeed, present in the fraction is confirmed by sedimentation velocity analysis, performed on the η-fraction. Figure 7a, displaying the sedimentation coefficient distribution, indicates a faster and a slower moving boundary with S20;w values of 2.5 and 7.5 S, respectively and, hence, the existence of two distinct supramolecular complexes. For comparison, the sedimentation coef-

Figure 7. Sedimentation velocity analysis at 32 °C and 37 000 rpm of molecular sieve fractions of Figure 6a. (a) η-Fraction at 1 M CaCl2. (b) γ-Fraction at 0.15 M NaCl and 0.15 M acetic acid. Sedimentation coefficient distributions were calculated as described in the Experimental Section and were corrected to 20 °C and pure water as solvent.

ficient distribution of the γ-fraction (Figure 7b) shows only a single species with a sedimentation coefficient S20;w of 5.0 S. Representative IR-MALDI mass spectra, acquired from the molecular sieve chromatography fractions with and without prior Analytical Chemistry, Vol. 76, No. 13, July 1, 2004

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Figure 9. (a) IR-MALDI mass spectrum of type V collagen proteins from total NaCl precipitate. Triple helices (γ) are detected as singly and doubly charged ions. Singly and doubly charged R1(V) and R2(V) ions are detected in addition. (b) Mass spectrum of heat-treated sample (heating to 43 °C for 10 min prior to mixing with matrix). Noncovalently bound triple helix complexes dissociate as a result of the heat treatment, and the R-chain subunits are detected. Sum of (a) 40 and (b) 30 single-shot mass spectra.

Figure 8. IR-MALDI mass spectra of type III collagen proteins from molecular sieve fractions. (a) Mass spectrum from η-fraction, collected as indicated in Figure 6a. (b) Mass spectrum from R1(III)SH-fraction, collected as indicated in Figure 6b. (c) Mass spectrum from β-fraction, collected as indicated in Figure 6b. Sum of (a) 105, (b) 30, and (c) 80 single-shot mass spectra. The mass spectra in (a) and (c) have been smoothed by 9-point FFT filtering.

reduction of disulfide bridges, are shown in Figure 8. The mass spectrum, representing the η-fraction from the nonreduced precipitate, is plotted in Figure 8a, showing a weak but clearly identifiable series of multiply charged η-ion signals. Residual γ-complexes in the fraction led to an overlap of ion distributions to a certain extent. Ions corresponding to the nonamer (ν) complex were not detected in this chromatography fraction. After reduction of disulfide bonds with 2-mercaptoethanol, the R-chain subunits form the base peaks in the molecular sieve chromatogram (Figure 6b). Moreover, two bands of lower intensity are detected, corresponding to presumably cross-linked β- and γ-complexes. The latter are likely to be of aldimine type, which are resistant to reduction and carboxymethylation. IRMALDI mass spectra of the R- and the β-fraction are displayed in Figure 8b and c, respectively. Note that in this case, molecules 3488 Analytical Chemistry, Vol. 76, No. 13, July 1, 2004

were reduced with 2-mercaptoethanol but not stabilized by carboxymethylation. Type V. Type V triple helices are of the two-chain form [R1(V)]2 [R2(V)]. Like type I, triple helices are held together essentially by noncovalent hydrogen bonds. The mass spectrum of the total NaCl protein precipitate of type V is shown in Figure 9a. Here, singly and doubly charged R-chain ions are detected next to singly and doubly charged γ-ions, which form the base peaks in the spectrum. For type V, the mass difference between R1(V) and R2(V) is large enough to allow differentiation of the two ion signals in the mass spectrum. The experimental masses of (109 100 ( 400) and (102 600 ( 400) Da are larger than the estimated theoretical values of 107 493 and 97 731 Da, respectively, for R1(V) and R2(V). However, the extent of the glycosylation was estimated from data on type V collagen from human skin.22 The mass spectrometric results suggest that, in particular, bovine R2(V) is probably more extensively modified than the human type. Assuming a complete modification of the 41 and 22.5 hydroxylysines by glucosylgalactose as the extreme case, theoretical masses of 109 951 and 102 840 were found for R1(V) and R2(V), respectively. Indeed, these values would agree well with the experimental ones. Moreover, the experimentally determined mass of the [R1(V)]2 R2(V) triple helix of (325 700 ( 1300) Da would then only be slightly higher than the thus calculated molecular weight of 322 742 Da. Thermal unfolding (heating at 43 °C for 10 min) leads to complete loss of the γ-ions by disrupture of hydrogen bonds and

to a strong increase in the R-chain signal intensities (Figure 9b). The observed ratio between the R1(V) and R2(V) ion signal intensities reflects the 2:1 stoichiometry of the triple helix. After the heat treatment, the two R1(V)- and R2(V)-chains are detected with a shift in mass of ∼3000 and ∼6000, respectively (see discussion below). IR-MALDI-MS Performance. Generally, the best mass spectrometric results were obtained if the collagens were analyzed shortly after their preparation. Storing the dissolved collagens at 4 °C in 0.1 M acetic acid led to a decrease in analytical performance with time and, eventually, complete loss of signal after about a week. This degradation is probably caused by aggregation. The applicable A/M concentration range was tested in a dilution series with a β-fraction of type III. Optimal results were found for a molar A/M-ratio of ∼10-4. Concentrations exceeding 10-3 were found to produce distorted mass spectra with reduced peak resolution and a considerable background. The minimal concentration that still gave useful signals was ∼10-5. The maximum achievable fwhm mass resolution of the lowextraction field instrument for high-mass ions can be estimated from BSA calibrant spectra to be ∼50 to 100. The maximum observed fwhm mass resolution of the collagen signals was ∼20 to 80, depending on the sample. Therefore, on the low side, chemical inhomogeneity of the collagen molecules and adduct formation might add to the signal peak width. In comparison to BSA mass spectra acquired from samples with comparable A/M ratios, generally, a higher background was found for the collagens, indicating a partial loss of ions due to fragmentation. The ratio of signal intensities between doubly and singly charged triple helix ions, forming the base peaks in the mass spectra of nondenaturated precipitate, was found to be strongly influenced by the laser fluence (see, e.g., Figure 5a,c). For the collagen samples, this effect is much more pronounced than for standard proteins, such as BSA. For instance, for type III total protein precipitate, a reduction in laser fluence from 9000 (Figure 5a) to 5000 J m-2 (spectrum not shown) reduced the intensity ratio of γ+/γ2+ by about a factor of 8.4. At the ion detection threshold fluence of ∼3500 J m-2, singly charged ions of γ were not even detected anymore (Figure 5c). Detection of Noncovalent Complexes. At first sight, the detection of noncovalent complexes of this size may appear as a surprise. Indeed, the detection of noncovalent complexes by mass spectrometry is generally seen as a domain of electrospray ionization, and only relatively few reports demonstrate a successful adaptation of MALDI-MS to their analysis. However, due to the large number of hydrogen bonds within the triple helices, collagen complexes are exceptionally stable. Their high stability is also reflected in the relative insensitivity toward the applied laser fluence. With the exception of the hexamer and nonamer complexes of type III, a fluence range from ion detection threshold F0 to about three times this value could be applied. Moreover, measurements could be performed well in the reflector mode of the instrument. In contrast, double-stranded DNA complexes are only detected from a glycerol matrix in a rather limited fluence range, within a factor of ∼1.5 above F0.26 (25) Cheung, D. T.; Heung, D. T.; Dicesare, P.; Benya, P. D.; Libawe, E.; Nimmi, M. E. J. Biol. Chem. 1983, 258, 7774. (26) Kirpekar, F.; Berkenkamp, S.; Hillenkamp, F. Anal. Chem. 1999, 71, 2334.

Figure 10. Anion exchange chromatography of collagen V species. Chromatography was performed on a Mono Q column with 2 mg of heat-treated (50 °C, 15 min) 7% NaCl precipitate.

Shift in Mass after Thermal Unfolding. For several of the samples, thermal unfolding and dissociation was accompanied by an increase in the detected mass of the molecules. The extent of this increase apparently depends on the conformation stability of the individual collagen species. A particularly large shift was observed for type V collagen. Type V is also the only one of the tested collagen types for which the R-chains were unequivocally observed in the spectrum of the total non-heat-treated protein precipitate. Due to the large difference in mass between the two chain forms, they can directly be discriminated against a triply charged γ-ion. Type V, thus, allows a direct comparison of R-chain masses from heat-treated and non-heat-treated samples. Figure 9a, displaying the IR-MALDI-MS spectrum from the 7% NaCl precipitate, which had been stored at -20°C and for preparation was solubilized in dilute acetic acid at room temperature, demonstrates that in this case, approximately the theoretical R1(V) and R2(V) mass values are obtained. However, after thermal unfolding of the 7% NaCl precipitate, the R1(V)- and R2(V)-chains exhibit an increase in mass of ∼5000 to 6000 and ∼3000 Da, respectively (Figure 9b). To investigate this phenomenon in more detail, anion exchange chromatography was performed. After thermal unfolding of the 7% NaCl precipitate and elution with destabilizing 4 M urea, an excellent separation between both chains is achieved (Figure 10). After storage of the lyophilized chains at -20 °C and solubilization in dilute acetic acid at room temperature, heat-treated samples (in this case, heating at 50 °C for 15 min) and nonheated samples of R1(V) and R2(V) display the same difference in mass by ∼5000 and 3000 Da, respectively, as was observed from the total NaCl precipitate. Figure 11 displays the two mass spectra obtained from the heat-treated (Figure 11a) and non-heat-treated (Figure 11b) ion exchange chromatography R1(V) fraction. These results indicate that the mass-shift effect must be purely attributed to thermal unfolding of the R(V)-chains, transforming the folded polyproline into the random coil conformation. A likely explanation for the large mass shift is the formation of addition complexes between the glycerol matrix molecules (MW, 92 Da) and the galactosyl or glucosylgalactosyl saccharide units of the unfolded R(V)-chains via intermolecular hydrogen bonds. Unfolded extended structures, different from folded Analytical Chemistry, Vol. 76, No. 13, July 1, 2004

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Figure 11. IR-MALDI mass spectra of R1(V) anion exchange chromatography fractions. (a) Mass spectrum acquired from non-heattreated R1(V)-fraction. (b) Mass spectrum from heat-treated R1(V)fraction (heating at 50 °C for 10 min prior to mixing with matrix). The comparison indicates a shift in recorded mass of the R1(V)-chain ions upon thermal unfolding of ∼5000 Da. Sum of (a) 80 and (b) 105 single-shot mass spectra.

compact ones, should facilitate binding of glycerol to the chains. It can be speculated that the hydroxyl groups of one glycerol molecule associate with one monosaccharide unit. For the unfolded R1(V)-chain, a maximum possible increase in mass of 7544 Da (assuming complete diglycosylation of all 41 hydroxylysines) and for R2(V) a maximum shift of 4140 Da (assuming complete diglycosylation of all 22.5 hydroxylysines) might then be expected. Qualitatively, the model, therefore, explains the different increases in mass for both chains in the unfolded state. In contrast, for the R1(I)/R2(I) system with only two known glycolysation sites for R1(I) (and presumably also for R2(I)), the possible mass shift fell into the experimental error in mass determination. Even after gel filtration (Figure 4a), no significant shift was observed. On the other hand, cross-linked species of type I, detected from molecular sieve chromatography fractions, are detected with a notably higher mass than predicted by simple multiplication of the R-chain masses. For instance, cross-linked γ-complexes (Figure 4c) are detected at 291 000 Da and, hence, with a shift in mass of ∼8000 Da if compared with the theoretical mass value of 282741 for the [R1(I)]2 R2(I) complex. It is at present not clear whether this shift is due to a change in molecular conformation as a result of the gel filtration, which then again would lead to extensive adduct formation with glycerol, or whether it rather reflects a systematic error in the measurement of these cross-linked species. An increase in the applied laser fluence was found to reverse the observed shifts by up to ∼1 kDa for the crosslinked y species, which can be interpreted as a partial loss of 3490

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previously associated glycerol molecules. More extensive attachment of alkali metals to the collagens, which has been observed in some other IR-MALDI studies with glycerol and proteins, would also add to the mass shift. These effects were not further investigated in the present study. In the case of type III collagen, the extent of available free binding sites can be altered by chemical reduction. R1(III)SH molecules, obtained by reduction of the 1.5 M precipitate and marked by free sulfhydryl functions (no stabilization by carboxymethylation), exhibit a distinct increase in mass by ∼8000 Da (Figure 8b). The conformational stability of the R1(III)SHpolyproline structure is believed to be decreased and to be associated with a lowered thermal unfolding transition temperature. Melting curves of R1(III)SH obtained by scanning calorimetric measurements at pH 6.0 indicate a transition start temperature of 17 °C and a melting temperature, T1/2, of 27.7 °C.27 Both parameters are shifted to lower values by decreasing the pH of the R1(III)SH solution. At pH 3.0, values of 14 °C and 25 °C, respectively, are found, providing evidence that under the conditions of mass spectrometric analysis (20 °C, pH 2.7 of a dilute acetic acid solution), a portion of R1(III)SH is present in the unfolded state. Obviously, the equilibrium between the folded and unfolded states of R1(III)SH may be shifted slightly toward the destabilized conformation already at room temperature. When R1(III)SH unfolds, glycerol molecules can bind to the newly exposed polar regions of the chain, in this case, preferentially to the hydroxyl groups of the hydroxyproline residues (135 residues per chain). This model is supported by UV-MALDI measurements on the R1(III)SH fraction which result in the expected molecular weight of the R-chain (see Figure 12b, below). An increase in the applied laser fluence was again found to reverse the observed shift in mass in the IR-MALDI mass spectrum. For instance, an increase in laser fluence by a factor of 2, as compared to the one used in the acquisition of the spectrum in Figure 8b of 6000 J m-2, was found to reduce the detected m/z of the R1(III)SH ions by ∼2 kDa. UV-MALDI-MS. UV-MALDI mass spectra were acquired in linear-TOF mode of the Reflex III instrument and with 2,5-DHBs as matrix. Molecular ions corresponding to intact triple helices were not detected in the UV-MALDI mass spectra of any of the tested samples. Tests with low-field ion extraction conditions, similar to those applied for the IR-MALDI experiments, showed no improvement. Two representative UV-MALDI mass spectra are plotted in Figure 12. Figure 12a displays a mass spectrum acquired from a non-heat-treated 7% NaCl precipitate containing native collagen of type V. Within the error margins, the determined masses for the R-chains of ∼103 [R2(V)] and ∼110 kDa [R1(V)] coincide with those from the IR-MALDI measurements on non-heat-treated samples (Figure 9a), pointing again to an extensive modification of hydroxylysine residues by glucosylgalactose. Notable for the UV mode is an increase in the R1(V)/R2(V) signal intensity ratio with increasing charge state, as well as a significantly increased mass resolution for the higher charged ion species. Figure 12b displays a mass spectrum acquired from a molecular sieve fraction containing R1(III)SH, which was obtained from (27) Beermann, B.; Hinz, H.-J. Institute of Physical Chemistry, University of Mu ¨ nster, personal communication, 03/24/2004.

proteins, on the other, might be directly related to the distinct linear structure of the fibrillar collagen species with lengths of the single R-chains of ∼300 nm. These dimensions are large compared to the shallow depth of material removal in UV-MALDI of only a few nanometers per laser pulse.28 On the other hand, IR-MALDI with glycerol goes along with ejection depths on the order of 0.5-1 µm29 and even the expulsion of small micrometersized droplets.30 In these, the collagen chains may be favorably incorporated before they are finally released into the gas phase. Future experiments have to show whether this limitation also holds for other UV-MALDI matrixes than the 2,5-DHBs employed here. In fact, in their conference abstract, Zaia et al. report the detection of triple helices also by UV-MALDI-MS with sinapinic acid as matrix.2 Sinapinic acid is known to form hydrophobic faces upon crystallization,31 which could, indeed, facilitate the incorporation of the complexes.

Figure 12. (a) UV-MALDI mass spectrum of type V collagen proteins from total NaCl precipitate. (b) UV-MALDI mass spectrum of R1(III)SH molecular sieve chromatography fraction. Matrix: 2,5DHBs. Sum of (a) 500 and (b) 650 single-shot mass spectra.

heat-treated and mercaptoethanol-reduced 1.5 M NaCl precipitate (cf. Figure 8b for the corresponding IR-MALDI mass spectrum). In contrast to the IR-MALDI measurement, in which extensive adduct formation was observed, the R1(III)-chains are here detected with about their theoretical mass value of 95 373 Da. Compared to the IR-MALDI mode, the analytical sensitivity is clearly reduced for UV-MALDI-MS, even for the detection of the single R-chains. The signal-to-noise ratios of the R-chain ion signals are also strongly diminished if compared to UV-MALDI mass spectra of “globular” proteins of comparable size, for example, BSA (MW, 66.4 kDa) or monoclonal antibodies (MW, ∼150 kDa; data not shown). An advantage of the UV-MALDI-MS mode is that (extensive) adduct formation with matrix molecules does apparently not occur; and mass accuracy is, hence, increased. The distinct differences between the IR- and the UV-MALDI modes, on one hand, and those between globular and the collagen (28) Dreisewerd, K. Chem. Rev. 2003, 103, 395. (29) Rohlfing, A.; Menzel, C.; Kukreja, L. M.; Hillenkamp, F.; Dreisewerd, K. J. Phys. Chem. B 2003, 107, 12275. (30) Leisner, A.; Rohlfing, A.; Berkenkamp, S.; Hillenkamp, F.; Dreisewerd, K. J. Am. Soc. Mass Spectrom. 2004, 15, 934. (31) Beavis, R. C.; Bridson, J. N. J. Phys. D, Appl. Phys. 1993, 26, 442. (32) Lowell-Baronas, D.; Lauer-Fields, J. L.; Fields, G. B. J. Liq. Chromatogr. R. T. 2003, 26, 2225.

CONCLUSIONS IR-MALDI-MS with glycerol as matrix allows the sensitive detection of fibril-forming collagen proteins in their triple helical form and, after dissociation, in the form of their R-chain subunits. Cross-linked substructures can be identified by combined chromatography/mass spectrometry studies. Compared to electrophoresis, the IR-MALDI-MS method needs a much more complex instrumentation, but it is faster and more precise, although improvements in accuracy and mass resolution are clearly desirable. Moreover, only small amounts of sample in the microgram range for a total protein precipitate are required. Potential fields of application are, therefore, in those areas where speed, accuracy, and sensitivity are of primary importance. One example would be monitoring of reaction products in the production of collagenbased biomaterials. Another important one might be the molecular analysis of the collagen expression on the cellular level. In the context of proteome research,32 those fibrillar collagens, which for medical reasons have been recognized as interesting, could be identified by the masses of their molecules as well as their subunits. The studies might be supplemented by collagen-derived peptide mass fingerprints by means of chemical fragmentation reagents, such as the highly specific cyanogen bromide, providing only a few but very characteristic peptides. ACKNOWLEDGMENT The authors thank F. Hillenkamp and J. Rauterberg for helpful discussions and support of this project and B. Beermann and H.J. Hinz for providing us with the calorimetric data on R1(III)SH. A.R. is grateful to the University of Mu¨nster for a Ph.D. grant. Received for review January 12, 2004. Accepted March 29, 2004. AC049928Q

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