Characterization of the folding pathway of recombinant human macrophage-colony stimulating-factor β (rhM-CSF β) by bis-cysteinyl modification and mass spectrometry

Characterization of the Folding Pathwayof Recombinant Human Macrophage-ColonyStimulating-Factor b (rhM-CSF b) by Bis-CysteinylModification and Mass SpectrometryH. Peter Happersberger,1 Janet Stapleton,2 Cynthia Cowgill,2 and Michael O. Glocker1*1Faculty of Chemistry, University of Konstanz, Konstanz, Germany2Chiron Corporation, Emeryville, California

ABSTRACT Melarsen oxide [p-(4,6-di-amino-1,3,5-triazin-2-yl)aminophenylarsonousacid (MEL)], which selectively bridges spa-tially neighboring bis-cysteinyl residues in (re-duced) proteins, was used to trap folding inter-mediates chemically during 1) time-dependentrenaturation of recombinant human macro-phage colony-stimulating factor (rhM-CSF); byredox refolding in vitro; 2) reductive unfoldingin the presence of the trapping reagent; and 3)denaturing unfolding reactions in urea andguanidinium hydrochloride. Characterizationof intermediates from folding and unfoldingreactions was performed by electrospray ion-ization mass spectometry (ESI-MS). In all fold-ing and unfolding reactions a characteristicdimeric intermediate with two attached me-larsen oxide (MEL) groups was observed, sug-gesting that these rhM-CSF b species wereimportant refolding intermediates. These inter-mediates presented a characteristic ‘‘chargestructure’’ in ESI spectra with a most abundant261 charged molecular ion whereas the ma-ture homodimeric rhM-CSF b showed a mostabundant 231 molecular ion, indicating thatthe final product was more compact. The majorlocations of the two MEL groups were identi-fied by mass spectrometric peptide mapping atcysteine residues C157 and C159 from eachmonomer. Cysteine residues C7 and C90 wereminor modification sites. The mass spectromet-ric results from the in vitro folding reactions ofrhM-CSF b are in agreement with intrinsictryptophan fluorescence measurements andare consistent with the folding pathway thatstarts with a fully reduced monomer (R), in-cludes partially folded monomeric intermedi-ates (M) and dimeric intermediates (D), andyields a final product with the native tertiarystructure (N):

2R ≠≠ 2M ≠≠ D ≠≠ N.

Our results show that selective chemical trap-ping of bis-thiol groups of proteins with MEL

permits study of folding pathways by massspectrometric structure characterization of in-termediates with otherwise transient confor-mations. Proteins Suppl. 2:50–62, 1998.r 1998 Wiley-Liss, Inc.

Key words: M-CSF; cytokine; c-fms; folding in-termediates; tryptophan fluores-cence; selective chemical modifica-tion; melarsen oxide; ESI-MS

INTRODUCTION

Macrophage colony-stimulating factor (M-CSF) isa cytokine that stimulates the survival, prolifera-tion, differentiation, and function of mononuclearphagocytes.1–3 The recombinant human M-CSF (rhM-CSF b) studied here was cloned and expressed inEscherichia coli as a truncated form (amino acids4–221) of the b-cDNA clone.4 Mass spectrometricanalyses have revealed that homodimeric rhM-CSFb contains nine disulfide bonds, three of whichparticipate in intramolecular disulfide bridges ineach monomer (C7-S-S-C90, C48-S-S-C139, C102-S-S-C146), and three of which form intermolecularbridges (C31-S-S-C31, and pairs of C157/C159-S-S-C157/C159).5 Mass spectrometric methods have been

Abbreviations: aa, amino acid; AcOH, acetic acid; D · nMEL,n-fold MEL-modified rh M-CSF b dimer; D · 4CAM, 4-foldcarboxamidomethylated rh M-CSF b dimer; D · 4SH, partiallyreduced rh M-CSF b dimer with four thiol groups; ESI-MS,electrospray ionization-mass spectrometry; GSH, L-g-glu-tamuyl-L-cysteinyl-glycine; GSSG, bis-(L-g-glutamyl)-cystinyl-bisglycine; HCCA, 4-hydroxy-a-cyano-cinnamic acid; IAA, iodo-acetamide; MALDI-MS, matrix-assisted laser desorption/ionization-mass spectrometry; M · nMEL, n-fold MEL-modifiedrh M-CSF b monomer; rhM-CSF b, recombinant human Macro-phage-Colony Stimulating Factor b; SDS, sodium dodecylsu-fate; TCEP, tris-(2-carboxyethyl)phosphine hydrochloride; TFA,trifluoroacetac acid; TFE, 2,2,2-trifluoro ethanol; VP, 4-vinylpyri-dine

Grant sponsor: Deutsche Forschungsgemeinschaft (Bonn,Germany).

*Correspondence to: Michael O. Glocker, Faculty of Chemis-try, University of Konstanz, P.O. Box M732, D-78457 Konstanz,Germany. E-mail: [email protected]

Received 8 May 1998; Accepted 10 August 1998

PROTEINS: Structure, Function, and Genetics Suppl. 2:50–62 (1998)

r 1998 WILEY-LISS, INC.

effectively employed for the molecular characteriza-tion of cystinyl disulfide structures5–7 (see Carr etal.8 and Morris and Greer9 for reviews). X-ray analy-sis (of rhM-CSF a, amino acids 4–151) revealed thatthe dimer is formed by end-to-end disulfide linkageof two four-helical bundles (helices A, B, C, and D),which run up-up-down-down.10 Selective chemicalmodification in conjunction with mass spectrometricpeptide mapping11–15 was applied for structure-function studies of rhM-CSF b16 and, like mutagen-esis studies,17 determined that histidine residues inthe A-helix play an important role in receptor-recognition and binding. The mature rhM-CSF b

homodimer is produced under renaturing conditionsin vitro, a key step in the production cycle, anddisulfide bond structures of monomeric and dimericintermediates were investigated by mass spectrom-etry.6,18,19

Protein folding is still one of the least understoodsteps in protein expression20–22 and in many caseshampers the yields of biologically active proteins inrecombinant expression systems.23 Different proteinconformations generated during protein folding canbe probed by mass spectrometric methods.24 Electro-spray ionization-mass spectrometry (ESI-MS) is es-pecially suited for monitoring conformationally differ-ent states of proteins by their characteristic ‘‘chargestructures’’11,25,26 (see Winston and Fitzgerald27, Loo28,and Przybylski and Glocker29 for reviews). For disul-fide bond-containing proteins, like rhM-CSF b, fold-ing into the ‘‘native’’ three-dimensional structure isaccompanied by the correct formation of disulfidebonds.18,22 Characterization of folding pathways bychemical trapping of intermediates with mono-thiolalkylating agents is a well-documented strat-egy.20,22,30,31 The hitherto applied trapping re-agents18,32 have been used to characterize foldingintermediates by distinguishing modified cysteinylfrom unmodified cysteinyl groups. However, charac-terization of intermediate conformations containingsulfhydryl groups that are suitably spaced but havenot yet formed disulfide bridges was not possible.

In this study p-(4,6-diamino-1,3,5-triazin-2-yl)ami-nophenylarsonous acid [melarsen oxide (MEL)],which selectively bridges spatially neighboring bis-cysteinyl residues in (reduced) proteins33,34 was usedto trap folding intermediates chemically by 1) atime-dependent renaturation of rhM-CSF b duringredox refolding in vitro; 2) reductive unfolding in thepresence of the trapping reagent; and 3) denaturingunfolding reactions in urea and guanidinium hydro-chloride. The analytical strategy developed is de-picted schematically (Fig. 1). Folding reactions weremonitored by ESI-MS and in the case of denaturingunfolding compared with intrinsic tryptophan fluo-rescence studies. Chemical bridging of topologicallyneighboring bis-cysteinyl residues with nonlocal in-teractions35 permitted characterization of otherwise

transient intermediates present in the disulfide-mediated folding pathway of rhM-CSF b.

MATERIALS AND METHODSRedox Refolding of rhM-CSF b and Trappingof Intermediates

Highly purified mature rhM-CSF b dimer (cour-tesy of Chiron, Emeryville, CA) was stored frozen aslyophilized powder. A thawed aliquot was redis-solved in 260 µl of a 8 M urea solution containing 12mM EDTA, 5 mM tris-(2-carboxyethyl)phosphine(TCEP), 50 mM NaH2PO4, pH 8.5. Protein concentra-tion (cM-CSF) was 15 µg/µl. The sample was incubatedat room temperature for 3 hr to ensure completereduction and denaturation. Monomer was diluted1:30 into precooled (4°C) 50 mM NH4HCO3, 1 mMEDTA, 0.4 mM L-g-glutamyl-L-cysteinyl-glycine(GSH), 0.4 mM bis-(L-g-glutamyl)-cysteinyl-bisgly-cine (GSSG), pH 8.5. Final protein concentration was0.5 µg/µl. Aliquots (200 µg, 4 nmol) were removed atgiven time points (0 h, 15 min, and 1, 3, 6, 18, and 42hr) and blocked by the addition of MEL (600 µl, 0.1mM), which was dissolved in 10 mM NH4HCO3, 3 Murea, pH 8.5. After incubation for 10 min at 25°C,samples were acidified (pH 3) by the addition of 0.1%trifluoroacetic acid (TFA), and folding intermediateswere desalted using a microconcentration device(Microcon, Amicon, Beverly, MA; cutoff: 3,000 Da).Retentates were washed three times with 200 µl of0.1% TFA dissolved in CH3CN / H2O (4:6, v/v), pH 2.The final protein concentration was approx. 1 µg/µl.Aliquots were used directly for sodium dodecyl sul-fate-polyacrylamide gel electrophoresis (SDS-PAGE;5 µl) and mass spectrometric analyses (1 µl) withoutfurther purification.

Reductive Unfolding of rhM-CSF band Trapping of Intermediates

A solution of rhM-CSF b-homodimer (80 µl, 2µg/µl) in 10 mM NH4HCO3, pH 8.2, was added to asolution of MEL (920 µl, 0.2 mM), dissolved in 10 mMNH4HCO3, pH 8.2. After incubation for 75 min, aTCEP solution (Pierce, Rockford, IL; 2.4 µl, 250 mMin H2O) was added. Final pH was set to 7.5. Aliquotswere acidified by addition of 200 µl of 0.1% TFA after24 hr of incubation and desalted by ultrafiltration asdescribed above.

Denaturing Unfolding of rhM-CSF band Trapping of Intermediates

rhM-CSF b aliquots (200 µg), dissolved in 25 mMNaH2PO4, 1 mM EDTA, pH 8, containing differentconcentrations of chaotrops (urea, 1–8 M; guanidin-ium hydrochloride, 1–5 M) were incubated with 44µM MEL, dissolved in the respective buffers, to yielda molar ratio of 1:11 (rhM-CSF b/MEL) and a finalvolume of 1 ml. After incubation for 2 min at roomtemperature, a solution of 125 mM TCEP (pH wasadjusted to 8 with solid NH4HCO3) was added (result-

51ESI-MS CHARACTERIZATION OF FOLDING INTERMEDIATES

ing molar ratios of rhM-CSF b/TCEP were 1:180).After 60 min of reaction at room temperature, asolution of 125 mM 4-vinylpyridine (VP) was added,resulting in molar ratios of 1:400 (rhM-CSF b/VP),and the reaction mixtures were incubated for 30 minat room temperature. After acidification by additionof 1.5 ml of a solution consisting of CH3CN / 4.2%TFA (2:1, v/v) to result in a final CH3CN concentra-tion of 40% (v/v), excess of reagents was removed bygel permeation chromatography (PD-10 colums,Pharmacia and Upjohn, Freiburg, FRG). Sampleswere eluted with 0.1% TFA dissolved in CH3CN /H2O (4:6, v/v), pH 2. Protein-containing fractionswere collected, lyophilized, and analyzed by massspectrometry.

For fluorescence emission spectroscopy, rhM-CSFb (0.1 µg/µl), dissolved in 10 mM sodium citratebuffer, pH 6.5, containing different concentrations ofchaotropes (urea, 1–8 M; guanidinium hydrochlo-ride, 1–6 M) was incubated for several time periods(0, 1, and 24 hr) at 25°C. Fluorescence emission wasrecorded at 350 nm on a Hitachi F-2000 fluorimeter(Hitachi Europe, Berks, UK) after excitation at 295

nm and gave comparable readings for all incubationperiods. The values obtained after 1 hr incubationare displayed (cf. Fig. 6). The 100% fluorescencesignal was obtained from a mixture with 5:2 molarratio of N-acetyl-L-tyrosine ethyl ester and N-acetyl-L-tryptophanamide, dissolved in 6 M guanidiniumhydrochloride, and 8 M urea, respectively.

SDS-PAGE Analyses of Folding Intermediates

SDS-polyacrylamide gels36 were used directly af-ter polymerization. Protein samples (5 µg) weredissolved in 20 µl sample buffer consisting of 4%SDS, 12% glycerol, 50 mM Tris-(hydroxymethyl)-aminomethane (pH 6.8) and 0.02% Coomassie Bril-liant Blue G-250, and deposited in one gel-slot. Afterelectrophoresis the gels were fixed in methanol/AcOH/water (5:1:4) for 0.5–1 hr, stained with 0.05%Coomassie Brilliant Blue G-250 in 10% AcOH, anddestained in 10% AcOH. The dimeric rhM-CSF bfolding intermediates (D) showed different mobilitiesthan the mature homodimer (N). Besides the strongbands for monomer and dimer, respectively, minorbands were visible in the gel that resulted as arti-

Fig. 1. Analytical concept of bis-thiol selective chemicaltrapping in protein folding reactions. The bis-thiol selective me-larsen oxide (MEL) can be applied as a trapping reagent in bothdenaturing unfolding (left ) and redox refolding reactions (right ) as

it is compatible with a broad range of folding reaction conditions.The covalently trapped intermediates permit mass spectrometricstructure characterization of distinct but otherwise transient pro-tein conformations.

52 H.P. HAPPERSBERGER ET AL.

facts from SDS-PAGE sample preparation proce-dures. For example, cleavage of the C-terminal partof rhM-CSF b at Asp-Pro bonds has been described.5

Selective Removal of the MEL Modificationand Carboxamidomethylation of the D · 2MELFolding Intermediate

A solution of reductively unfolded rhM-CSF b(D · 2MEL), was prepared by dissolving 100 µg ofD · 2MEL in 100 µl of a freshly prepared solutionthat consisted of 10% AcOH / TFE (7:3, v/v), pH 2.The solution was incubated with 0.5 µl of 1 M2,3-dimercaptopropanol, dissolved in DMSO, result-ing in a molar ratio of 1:260 (rhM-CSF b/2,3-dimercaptopropanol). After 30 min at room tempera-ture, excess of reagents was removed byultrafiltration using 10% AcOH/TFE (7:3, v/v), pH 2,as described above. Aliquots were taken for massspectrometric molecular weight determinations andshowed that MEL was completely removed by thisprocedure. To a 50 µg aliquot, dissolved in 10%AcOH/TFE (7:3, v/v), pH 2, a solution of 250 mMiodoacetamide e (IAA) was added to result in a finalmolar ratio of 1:1800 (rhM-CSF b/IAA). The pH wasadjusted to 8 by addition of approx. 50 µl of 1 MNH4HCO3. After incubation for 30 min at roomtemperature in the dark, the reaction mixture wasacidified by addition of glacial AcOH, and excess ofreagents was removed by ultrafiltration using 0.1%TFA in CH3CN/H2O (4:6, v/v), pH 2, as solvent.Aliquots were taken for mass spectrometric molecu-lar weight determinations and showed the additionof 4 carboxamidomethyl groups to the dimeric fold-ing intermediate of rhM-CSF b (D · 4CAM). Lyophi-lized retentates were used for proteolytic digestions.

Proteolytic Digestion of D · 4CAM

The D · 4CAM derivative of rhM-CSF b (50 µg)was dissolved in 50 µl 50 mM NaH2PO4, pH 8, andincubated for 5 min at 95°C. This procedure causeddisulfide bond scrambling but rendered rhM-CSF bsusceptible to proteolytic degradation.6 Tryptic diges-tion was carried out after cooling by addition of 2 µlof a trypsin solution (1 µg/µl), dissolved in the samebuffer. E/S was 1:25. For AspN digestions an E/S of1:15 was used. Digestions were performed for 5 hr at37°C. The reactions were stopped by freezing and themixtures were stored at -20°C before high-perfor-mance liquid chromatography (HPLC) purificationor mass spectrometric analyses.

HPLC Purification of Tryptic Peptides

Purification and separation of tryptic peptides wasperformed by HPLC on a 250 3 4.6 mm Vydacreversed phase C-18 column (300 A, 10 µm) equippedwith a Vydac precolumn. A Waters Millipore HPLCsystem, consisting of two HPLC pumps (WatersM510 and Waters M45) was employed with solventA, 0.1% TFA in H2O, and solvent B, 0.1% TFA in

CH3CN. The flow rate was 0.8 ml/min. The solventmixture was kept constant at 10% B for 5 min andwas then raised to 60% B over 50 min. Fractionswere collected, lyophilized, and redissolved in 10 µlof 0.1% TFA dissolved in CH3CN/H2O (4:6, v/v), pH 2,for MALDI-MS analyses.

On Target Reduction of Disulfide Bonds

On target reduction in the presence of HCCA-matrix7 was carried out with the tryptic peptides ofthe rhM-CSF b derivative after matrix-assistedlaser desorption/ionization-mass spectrometry(MALDI-MS) peptide mapping (see below). The ma-trix/peptide mixture on the target was dissolved byadding 1.5 µl of a 12.5 mM TCEP solution in 100 mMNH4HCO3, pH 8, and 0.5 µl CH3CN. The reactionmixture adopted pH 8 (yellow color). After approx. 15min at room temperature the solvent was evapo-rated. To improve the signal intensities of the MALDImass spectra, 0.7 µl 4-hydroxy-a-cyano-cinnamicacid (HCCA) matrix was added. The matrix/peptidemixture was recrystallized and washed by additionof 2 µl of CH3CN/0.1% TFA (2:1, v/v), pH 2, and 2 µl of0.1% TFA, respectively, prior to MALDI-MS analy-ses.

Mass Spectrometry

MALDI-MS analyses were carried out with aBruker Biflex linear time-of-flight spectrometer(Bruker Franzen, Bremen, FRG), equipped with aUV-nitrogen laser (337 nm) and a dual microchannelplate detector. The acceleration voltage was set to 20kV, and spectra were calibrated with insulin. Ali-quots of 1.0 µl of the HPLC-purified tryptic peptideswere mixed with 0.7 µl of matrix solution (10 µg/µl;HCCA) dissolved in CH3CN/0.1% TFA (2:1, v/v), pH2, directly on the target. Spectra were recorded afterevaporation of the solvent and processed using theX-MASS data system.

ESI-MS was performed on a Perkin Elmer-SciexAPI-III triple quadrupole mass spectrometer (Thorn-hill, Ontario, Canada) in the single quadrupolescanning mode. The solvent delivery system con-sisted of an Applied Biosystems (Foster City, CA)140B syringe pump. A mixture of 0.1% TFA in H2Oand 0.07% TFA in acetonitrile (6:4 v/v) was used assolvent. The flow rate was adjusted to 40 µl/min.Samples (5 µl, 0.1 µg/µl) were dissolved in the samesolvent and injected using a Rheodyne (Cotati, CA)injection port (model 8125) equipped with a 5 µlsample loop.

Nanospray-electrospray ionization mass spectrom-etry (nano-ESI-MS) was performed with a Vestec-201A quadrupole mass spectrometer (Vestec, Hous-ton, TX). The ion-spray interface temperature wasapproximately 40–50°C for all measurements. Themass analyzer with a nominal m/z range of 2,000was operated at 1/8 unit resolution. A self-developednano-ESI source37 was used for nano-ESI-MS analy-


ses. Borosilicate glass capillaries (GC120F-10, ClarkElectromedical Instruments, Pangbourne, UK) wereused for manufacturing the micro-capillaries using acapillary puller (P-97, Sutter, Novato, CA). Thecapillaries were coated with a thin layer of gold by asputter process (International Scientific Instru-ments). Capillaries were filled by dipping the capil-lary tip into the sample solution (0.5–2.5 µl). Anelectrospray voltage at the capillary tip of 1.2–1.5 kVand a repeller voltage of 3–40 V were employed.Spectra were recorded with a scan rate of 7 sec/scanwith a mass window of m/z 200–2,000. Mass calibra-tion was performed with the 81 to 121 charged ionsof hen egg white lysozyme (Mr 14305.1), and rawdata were analyzed using a Vector-2 data system(Teknivent, Houston, TX). Samples were diluted to afinal concentration of 0.1 µg/µl with 10% (v/v) AcOH/TFE (7:3, v/v), pH 2.

Computer-Assisted Data Analysisand Curve Fitting

The average degree of MEL modification wasdetermined from relative intensities of individualcharge states in the ESI spectra for monomer anddimer ions, respectively. Monomer/dimer ratios weredetermined from relative intensities of characteris-tic ion signals, the [M126H]261 molecular ion of thedimer and the [M116H]161 molecular ion of themonomer, respectively. These ions were selected asthey did not show overlap of the monomer and dimersignals, respectively. Observed m/z values of theindividual ion signals were: 1889 (D), 1899(D · 1MEL), 1909 (D · 2MEL), 1920 (D · 3MEL), 1534(M), 1551 (M · 1MEL), 1569 (M · 2MEL), 1586(M · 3MEL), and 1602 (M · 4MEL). Relative fluores-cence was determined as described elsewhere.38 Thedata were analyzed using the ‘‘Peakfit’’ non-linearcurve-fitting software (Jandel Scientific, Erkrath,Germany) applying a sigmoidal curve fit.

Molecular Modeling and Visualization

The chemical structure of melarsen oxide was semi-empirically minimized applying the HYPERCHEM(Hypercube, Gainesville, GA) program package us-ing the PM3 parameter set.39 The schematic represen-tation of M-CSF a was created with the programMolMol 2.5.40 using the coordinates of M-CSF a.10

RESULTSESI-MS Monitoring of In Vitro RedoxRefolding of rhM-CSF b

The redox refolding reaction was started by dilu-tion of denatured and reduced monomer into a bufferof high reducing potential (TCEP and reduced gluta-thione/oxidized glutathione 5 2.8:1) that graduallybecame more oxidizing by air (oxygen) oxidation,giving a half-time for dimer formation of approxi-mately 28 hr. Aliquots were taken at various timepoints, blocked with MEL, purified, and subjected to

SDS-PAGE analysis and ESI-MS molecular weightdetermination, respectively. SDS-PAGE analysis in-dicated that during the early time points of refolding(0–3 hr), there was no dimer formation, at least nonethat could be detected (data not shown). ESI-MSmolecular weight determinations of the initial (0 hr)time point of refolding showed abundant signals forthe completely MEL-modified rhM-CSF b monomer(M · 4MEL) forming an evenly distributed ion seriescentering around the 151 charged molecular ion(Fig. 2A). Additional tiny signals in the spectrumindicated the presence of small amounts of M · 1MEL.MEL forms bis-thioesters bridging two closely spacedsulfhydryl groups in proteins.33,34 Hence, the ninecysteine residues per rhM-CSF b monomer added atotal of four MEL groups. The number of incorpo-rated MEL groups was instantly determined bymass spectrometry from the characteristic gain inmass of the folding intermediates (Dm for one MELgroup is 276 Da; experimental and calculated massesof the observed intermediates are given in Table I).This result indicated that even at t 5 0 hr, therhM-CSF b monomer was not a linear extension, butrather had its cysteinyl groups suitably spaced sothat MEL would react. Determinations of modifica-tion sites in these intermediates are currently beingperformed.

A dimer became apparent by SDS-PAGE analysisafter 6 hr of exposure of the monomer to renatur-ation conditions (Fig. 3, lane 1). ESI-MS analysis ofthis sample (Fig. 2B) showed odd-charged and even-charged ions of the doubly MEL-modified dimer(D · 2MEL) in the gaps between the monomer ions,due to the different degrees of modification formonomer and dimer intermediates, respectively (cf.Materials and Methods). The ion series of the dimershows an intensity maximum around the 261charged molecular ion. However, the still dominantion series in this spectrum corresponds to thepredominantly singly MEL-modified monomer(M · 1MEL). Further modification degrees were de-tected for the monomer (Fig. 2B; blow-up of the 151charged molecular ion). The charge states of thesedifferently modified monomer ions were again foundevenly distributed around the 151 charged molecu-lar ion. The appearance of the doubly MEL-modifieddimer intermediate (D · 2MEL) suggested that thisrhM-CSF b species was an important intermediatein the refolding pathway, as it also appeared in thereductive unfolding and denaturing unfolding reac-tions (see below).

Dimer formation increased substantially after 42hr of refolding as judged from the broad rhM-CSF bdimer band by SDS-PAGE analysis (Fig. 3, lane 2).ESI-MS analysis of the protein intermediates fromthis refolding time point (Fig. 2C) showed again anion series for the singly MEL-modified monomer(M · 1MEL) with an intensity maximum at the 151charged molecular ion together with an ion series for


the unmodified dimer (D) that now revealed twointensity maxima. The first was centered around the261 and the second around the 231 charged molecu-lar ion.

After the denatured rhM-CSF b had been exposedfor 72 hr to the refolding conditions, SDS-PAGEanalysis detected only dimeric protein that migratedlike final product (Fig. 3, lane 3). The ESI-MSanalysis of this sample showed an evenly distributedion series for unmodified dimer (N) with an intensitymaximum at the 231 charged molecular ion (Fig.2D). This ‘‘charge structure’’ was indicative for themature homodimeric rhM-CSF b (see below) andindicated the termination of the refolding reaction.

The existence of more than one protein formgenerated in the course of refolding was apparent inESI spectra of the trapped refolding mixtures by 1)the multiple ion series of intermediates differenti-ated by mass due to different modification states and2) by the clearly distinguished ‘‘charge structures’’ ofthe intermediates. The less modified intermediatesshowed individual multiply charged ion signals thatshifted to lower m/z values (vertical arrows in Fig. 2),indicating that the numbers of closely spaced thiolgroups diminished with increasing refolding time.This could be due to an increase in disulfide bondformation or to keeping the thiol groups far apartfrom each other in the intermediate structures.

Fig. 2. ESI-MS monitoring of redox invitro refolding of rhM-CSF b. ESI-spectra ofMEL-trapped folding intermediates at 0, 6,and 42 hr of refolding (A–C, respectively)and of mature homodimer (D). M · 4MEL,monomeric intermediate with four MELgroups; D · 2MEL, dimeric intermediate withtwo MEL groups; N, mature homodimer.Charge numbers denote [M1nH]n1 ions.The different charge states of the intermedi-ates are indicated by the dashed (dimer)and dotted (monomer) lines around themultiply charged ion series, respectively. Ionsignals for the dimeric derivatives are drawnin gray. Spectra were obtained from 0.1%TFA in H2O/0.07% TFA in acetonitrile (6:4,v/v) solutions.


The ESI spectra were recorded after rebufferingthe trapped folding intermediates into acidic solu-tion. The ‘‘charge structures’’ of the monomer inter-mediates with different degrees of modification werenot distinguishable, suggesting no drastic differ-ences in the conformations of the solution structuresin the solvents used for ESI-MS. By contrast, themaxima of the ion series of the dimeric intermedi-ates D · 2MEL and D (dashed lines in Fig. 2B,C)were observed at lower m/z values than that of themature homodimer (N), indicating an increase incompactness of final product, at least in the solventsused for ESI-MS. This shows that dimeric foldingintermediates could be isolated in a characteristicstructure and a ‘‘consolidation step’’ during redox

refolding of rhM-CSF b was necessary to yield thenative homodimeric protein structure. The dimericrhM-CSF b folding intermediates (D) producedbroader bands in SDS-PAGE than the mature ho-modimer (N) (cf. Fig. 3) and ‘‘consolidation’’ could becorrelated with maximum activity in the M-NSF 60proliferation assay; the intermediate showed no bio-logical activity (data not shown).

The mass spectrometric results from the redoxrefolding reaction studied here are in agreementwith the suggested in vitro folding pathway forrhM-CSF b:

2R ≠ 2M ≠ D ≠ N.

Completely reduced monomers (R) form mono-meric intermediates (M) in early refolding eventswith different degrees of MEL modification (alreadyat t 5 0 hr). The subsequently formed dimericintermediates (D) (some of which contained no freesulfhydryls), seemed to exhibit a more open struc-ture than the mature homodimer (N). These resultsare consistent with observations from a previousstudy.18

ESI-MS Monitoring of ReductiveUnfolding of rhM-CSF b

Reductive unfolding reactions present yet anothersource of intermediates possessing distinct conforma-tions and are particularly useful for studying ‘‘late’’folding steps.32 To study unfolding derivatives, rhM-CSF b was incubated in aqueous buffer in thepresence of MEL and reduction was started by theaddition of TCEP. SDS-PAGE analysis of the reduc-tive unfolding reaction (Fig. 3, lane 4) revealed thatreduction of rhM-CSF b in the presence of thetrapping reagent (MEL) produced only bands migrat-ing as dimer under conditions that usually led tocomplete reduction into monomers (data not shown).ESI-MS investigations of the obtained product (Fig.4) revealed a predominantly twofold MEL-modifiedrhM-CSF b derivative (D · 2MEL). In contrast to thenative rhM-CSF b homodimer (N) that exhibitedmultiply charged molecular ions with an intensitymaximum around the 231 charged molecular ion(Fig. 4A), the D · 2MEL derivative showed an ionseries centering around the 261 charged molecularion (Fig. 4B) comparable to the D · 2MEL derivative(cf. Fig. 2B) obtained by redox refolding. Only afterprolonged exposure of rhM-CSF b to the reductiveunfolding conditions (.4 weeks) was a D · 3MELderivative formed in higher abundance (data notshown). These results showed that initial reductionof rhM-CSF b by TCEP in the presence of thetrapping reagent rapidly led to modified homodimerswith two attached MEL groups, preventing furtherreduction almost completely.

TABLE I. Molecular Masses of rhM-CSF b,MEL-Trapped Folding Intermediates, Partially

Reduced, andAlkylated Derivatives

rhM-CSFb/intermediatea Mr Mobsd.

M 24523.7 24520.1 6 5.0M · 1MEL 24791.8b 24789.0 6 6.0M · 2MEL 25067.9b 25065.1 6 4.2M · 3MEL 25344.0b 25341.2 6 4.7M · 4MEL 25620.1b 25622.5 6 4.6N 49029.4 49028.2 6 8.1D · 1MEL 49305.5c 49304.6 6 11.8D · 2MEL 49581.6c 49582.0 6 8.1D · 3MEL 49857.7c 49896.9 6 26.1d

D · 4SH 49033.4c 49032.2 6 10.8D · 4CAM 49261.4c 49263.1 6 5.1aM, completely reduced monomer; M · nMEL, MEL-trappedmonomeric intermediates; D · nMEL, MEL-trapped dimericintermediates; N, native homodimer, final refolding product;D · 4SH, partially reduced dimeric derivative; D · 4CAM, di-meric carboxamidomethylated derivative.bCalculated value for MEL-modified monomeric derivativeswith one free cysteine residue. Further unmodified cysteineresidues are regarded as being involved in disulfide bonds.cCalculated values for MEL-modified dimeric derivatives inwhich unmodified cysteine residues are regarded as beinginvolved in disulfide bonds.dSignals with low intensities.

Fig. 3. SDS-PAGE analysis of rhM-CSF b folding reactions.Lane 1 , 6 hr redox refolding; lane 2 , 42 hr redox refolding; lane 3 ,dimeric rhM-CSF b (final refolding product); lane 4 , reductiveunfolding; lane 5 , molecular weight standards. The location of themonomer and dimer are indicated. Molecular weights from theprotein standard mixture are given in kDa.


Identification of MEL Modification Sitesin the D · 2MEL Folding IntermediateObtained by Reductive Unfolding

Tryptic and AspN digestions of rhM-CSF b andderivatives led to peptide fragments that were iden-tified unambiguously by MALDI-MS peptide map-ping. The observed fragments were in conformitywith results from previous studies5,16 and selectedpeptides were confirmed by gas-phase Edman se-quencing (data not shown). In addition, disulfidebond-containing peptides were analyzed byMALDI-MS peptide mapping prior to and afterreduction; hence, assignments of peptides were madein consistence with the observed chemical proper-ties. However, as the digests had to be carried outat pH 8, MEL-modification sites could not bedetermined directly. Although MEL is a potent trap-ping reagent that is applicable in a broad pH range,its products with bis-cysteinyl groups were onlyfound to be stable under digestion conditions when

(macro)cyclic structures remained.33,34 Peptide bondcleavage may open up the cyclic structures and leadto dissociation of MEL. This process may afford freethiol groups that could cause substantial disulfidebond scrambling at pH 8, preventing the assignmentof modification sites.31 Hence, we selectively ex-changed the MEL modifications in the D · 2MELfolding intermediate by carboxamidomethyl groups,a modification that survives proteolytic digestions.

To achieve selective exchange of MEL by carbox-amidomethylations, D · 2MEL was incubated withhigh excess of 2,3-dimercaptopropanol 41 at pH 2.These conditions allowed complete removal of MEL33

but prevented reduction of disulfide bonds, as indi-cated by mass spectrometric molecular weight deter-minations of low molecular weight model compounds(data not shown); this is in agreement with knownthiol/disulfide exchange reactions.42,43 Removal ofMEL from the D · 2MEL intermediate was complete,as shown by multiply charged ion signals for aprotein with a molecular mass of 49,032.2 in ESI-MSanalyses (Table I). After removal of excess reagents,IAA was added in high excess and pH was shifted to8. After incubation in the dark and subsequentacidification to stop the reaction, excess of reagentswas again removed and mass spectrometric molecu-lar weight determinations showed the addition ofpredominantly four carboxamidomethyl groups (CAMgroups) to the dimeric folding intermediate of rhM-CSF b (cf. Table I). The dimeric D · 4CAM derivativewas formed in the above-described procedure onlywhen D · 2MEL was the starting material, as wasconfirmed by control reactions with unmodified rhM-CSF b. The D · 4CAM derivative was subjected totryptic digestion after heat denaturation6 that per-mitted identification of cysteine-modified peptidesby MALDI-MS (Table II). MALDI spectra were ac-quired before and after on-target reduction andshowed predominant modification of cysteine resi-dues C157 and C159.

These two residues are involved in intermoleculardisulfide bonds that hold the native rhM-CSF bdimer together.5 The peptide at amino acids 138–163, which contained four cysteine residues (C139,C146, C157, and C159), was found to carry two CAMgroups, as shown by the ion observed at m/z 2,993(calcd. 2,993.2). Asp-N digestion of the D · 4CAMderivative yielded a peptide at amino acids 156–168that contained cysteine residues C157 and C159 andwas found modified with two CAM groups resultingin an ion at m/z 1,526 (calcd. 1,525.6). A minor signalfor the unmodified AspN-derived peptide at aminoacids 156–168 at m/z 1,410 (calcd. 1,409.6) was alsoobserved (Table II). In addition, ion signals of lowabundance were observed at m/z 2,092 for a peptideat amino acids 4–21 that carried CAM-modifiedcysteine residue C7 (calcd. 2,091.3), indicating minorMEL modification of this residue. Cysteine residueC7 is linked intramolecularly to C90 via a disulfide

Fig. 4. ESI-MS monitoring of reductive unfolding of rhM-CSFb. ESI-spectra before (A) and after addition of reducing agent (B).D · 2MEL, dimeric MEL-trapped unfolding product with two at-tached MEL groups; N, native homodimer. Charge numbersdenote [M1nH]n1 ions; the determined molecular weights aregiven. Spectra were obtained from 0.1% TFA in H2O/0.07% TFA inacetonitrile (6:4, v/v) solutions.


bond in the native rhM-CSF b structure. The C7-S-S-C90 disulfide-linked peptide amino acids 4–21-S-S-amino acids 89–93 produced an intense ion signal atm/z 2618 (Table II), showing that cysteine residuesC7 and C90 were mostly unmodified in this foldingintermediate.All other cysteine- and cystine-contain-ing peptides were found unmodified by MALDI-MSmolecular weight determination of the peptide mix-tures (Table II) and of HPLC-separated fractions,respectively. Whether the D · 2MEL derivative ob-tained by redox refolding had the same disulfidebond structure as the D · 2MEL intermediate stud-ied here remains to be investigated. However,D · 2MEL obtained by redox refolding was alwaysaccompanied by other intermediates, which maycomplicate unambiguous disulfide bond assignment.

Nano-ESI-MS and Tryptophan FluorescenceMonitoring of Denaturing Unfoldingof rhM-CSF b

The differences in MEL modification of the rhM-CSF b (un)folding intermediates provided directinformation on changes in protein structures. Dur-ing both redox refolding and reductive unfoldingreactions, the D · 2MEL derivatives were observed,indicating that these trapped intermediates wereimportant for rhM-CSF b folding. In additionalstudies we investigated the products obtained bydenaturing unfolding reactions. For this purposerhM-CSF b was incubated in urea (1–8 M) andguanidinium hydrochloride (1–5 M), respectively, inthe presence of MEL. Reduction was started by theaddition of TCEP as described above and sampleswere analyzed by SDS-PAGE (data not shown) andnanospray ESI-MS molecular weight determination.

We observed no differences in the spectra obtainedwith nanospray ESI-MS compared to those obtainedwith conventional ESI-sources. In 3 M urea, forexample, D · 2MEL was again the dominant rhM-CSF b derivative (Fig. 5A) and no monomer ionswere detected. In 8 M urea, however, a substantialportion of rhM-CSF b was completely reduced andsubsequently modified to form the M · 4MEL deriva-tive producing a mixture with the D · 2MEL deriva-tive (Fig. 5B). The MEL-modified folding intermedi-ates in this sample were further subjected toalkylation of thiol groups with 4-vinylpyridine. TheD · 2MEL derivative showed unchanged ion series inthe ESI spectra, indicating that no free thiol groupswere present. By contrast, the M · 4MEL derivativeadded one 4-vinylpyridine molecule, showing thepresence of a free thiol group in the monomericfolding intermediate (Fig. 5B). Whether theM · 4MEL derivative observed here had the samemodification pattern as the M · 4MEL derivativeobtained during redox refolding is presently investi-gated. The rhM-CSF b dimer showed the dominantion series in all ESI spectra obtained from thevarious chaotrope concentrations. The average de-grees of MEL modifications and the monomer/dimerratios obtained by denaturing unfolding with increas-ing concentrations of chaotropes were calculatedfrom relative abundances of the ion signals in theESI spectra (cf. Materials and Methods). A steadyincrease in average MEL modification up to a maxi-mum of 47% was observed in urea (Fig. 6A, dashedline). By contrast, denaturation of rhM-CSF b inguanidinium hydrochloride and subsequent ESI-MSmolecular weight determinations revealed a sigmoi-dal shaped course of unfolding typical of a ‘‘two-

TABLE II. MassAssignments of Proteolytically Derived Peptides From FoldingIntermediate D · 2MEL†

Cys-residue/disulfide bondaAmino acid sequence positions/

number of modified residues [M 1 H]calc.1 [M 1 H]obsd.

1

C7 (4–21)/— 2,034.3 2,035C7 (4–21)/1 2,091.3 2,092b

C31, C48 (22–51)/— 3,551.1c 3,551C102 (94–104)/— 1,365.5 1,366C139, C146, C157, C159 (138–163)/2 2,993.2c 2,993C157, C159 (156–168)/—d 1,409.6c 1,410b

C157, C159 (156–168)/2d 1,525.6 1,526C7-S-S-C90 (4–21)-S-S-(89–93) 2,617.0 2,618C31-S-S-C48 (22–44)-S-S-(45–51) 3,569.1 3,570C48-S-S-C102 (45–52)-S-S-(101–116) 2,875.4 2,878C48-S-S-C102 (45–66)-S-S-(101–104) 3,075.7 3,078C90-S-S-C102 (67–93)-S-S-(101–125) or 6,077.1 6,077

(89–104)-S-S-(69–104) 6,077.8†Tryptic digestion after incubation with 2,3-dimercaptopropanol and carboxamidomethylation. Heat-induced denaturation was required to render rhM-CSf b susceptible to proteolytic digestion.aVerified by on-target reduction and subsequent MALDI-MS peptide mapping analyses.bMALDI-MS signal with very low intensity.cPeptide mass with one disulfide bond.dAspN digestion.


state’’ unfolding reaction. Average MEL modificationof the intermediates reached up to 95% (Fig. 6B,dashed line) and the midpoint of the denaturationcurve was approx. at 2.5 M guanidinium hydrochlo-

ride. In 3 M guanidinium hydrochloride and above,the completely modified monomeric rhM-CSF b de-rivative (M · 4MEL) presented the dominant ionseries in the ESI spectra.

Denaturing unfolding of rhM-CSF b was alsostudied by tryptophan fluorescence measurementswith increasing chaotrope concentrations. In urea,tryptophan fluorescence of rhM-CSF b increased upto approx. 66% of the relative fluorescence (Fig. 6A,solid line) of the amino acid standards, representing100% denaturation. Note that the course of denatur-ation in urea monitored by ESI-MS of MEL-trappedrhM-CSF b intermediates showed a steady increasesimilar to the denaturation curve obtained by trypto-phan fluorescence measurements from the nonre-duced homodimer. By contrast, tryptophan fluores-cence of the rhM-CSF b denaturation in guanidiniumhydrochloride showed again a ‘‘two-state’’ unfoldingprocess and was complete (100% relative fluores-cence) at high concentrations, as indicated by thesigmoidal shaped curve of tryptophan fluorescence(Fig. 6B). The midpoint of the denaturation curvewas approx. 3.5 M for the nonreduced homodimericrhM-CSF b, whereas an unfolding midpoint at 2.5 Mguanidinium hydrochloride concentration was ob-tained with reduced monomeric rhM-CSF b (Fig. 6B,solid lines). The guanidinium hydrochloride denatur-ation from the reduced rhM-CSF b monomer moni-tored by tryptophan fluorescence measurementsnearly superimposed on the unfolding curve ob-tained by ESI-MS of trapped intermediates. Theabove-described results consistently showed partialdenaturation of the rhM-CSF b dimer even with highurea concentrations, whereas guanidinium hydro-chloride had much stronger denaturing effects onrhM-CSF b and led to complete denaturation. Theresults from both methods are consistent and sug-gest increase of accessibility of all disulfide bonds/cysteinyl groups with denaturation, finally leadingto completely reduced and MEL-modified rhM-CSFb monomers.

Fig. 5. Nano-ESI-MS and trypto-phan fluorescence monitoring of denatur-ing unfolding of rhM-CSF b. ESI-spectraof MEL-trapped intermediates in 3 Murea (A) and in 8 M urea (B) afteraddition of reducing agent. M · 4MEL,monomeric intermediate with four MELgroups; D · 2MEL, dimeric unfoldingproduct with two attached MEL groups.Charge numbers denote [M1nH]n1 ions.The different charge states of the inter-mediates are indicated by the dashed(dimer) and dotted (monomer) linesaround the multiply charged ion series,respectively. Spectra were obtained from10% (v/v) AcOH/TFE (7:3 v/v), pH 2,solutions.

Fig. 6. Correlations of relative tryptophan fluorescence and %MEL modification of rhM-CSF b. Course of fluorescence and MELmodification are shown as functions of urea concentration (A) andguanidinium hydrochloride (gua(HCl)) concentration (B), respec-tively. The tryptophan fluorescence denaturation curves (solidlines) are depicted for the nonreduced dimer (squares) and thereduced monomer (diamonds). Courses of relative MEL-modifica-tion degrees (dashed lines) are averaged from monomeric anddimeric derivatives (circles).


DISCUSSION

Our results show that selective chemical trappingof bis-thiol groups of proteins with MEL permitsstudy of folding pathways by mass spectrometricstructure characterization of intermediates with oth-erwise transient conformations. The results from theredox folding reactions are consistent with earlierinvestigations in which the overall sequence of redoxrefolding of rhM-CSF b was determined by fast atombombardment-mass spectrometry using dithiothrei-tol as a reducing agent and IAA as a trappingreagent.18 Bis-thiol-selective derivatization of inter-mediates during redox refolding in glutathione-based redox buffers, however, is different in principlefrom the mono-thiol trapping strategy.44,45 Bis-thiolmodification is a way to cross-link closely spacedcysteine residues,33,34 thereby providing tertiarystructure information on folding intermediates thathave not yet formed disulfide bonds but have adoptedthe appropriate conformation to undergo specificnonlocal interactions.35 This information is com-pletely lost in the case of mono-thiol derivatization.Additionally, the hitherto applied mono-thiol deriva-tizing agents such as iodoacetic acid simultaneouslymodify protein thiols and thiols from the redox buffersystem (e.g., GSH/GSSG; cystine/cysteine), therebyshifting the redox potential during trapping.22,46 Bycontrast, MEL does not form stable products withmono-thiols and selectively traps bis-thiols of the

protein folding intermediates without interferingwith the redox potential.34

Reductive unfolding is particularly useful for thestudy of ‘‘late’’ folding steps.32 Unfolding is thought tobe initiated by local events such as reduction of thehighest accessible disulfide bond by which the com-pact structure of the disulfide linked protein isopened up. Subsequent reduction of further, nowaccessible, disulfide bonds results in complete dena-turation of the protein. Alkylation of the resultantthiol groups renders reductive unfolding irrevers-ible. As shown, MEL is readily applicable for thisapproach. The chemical specificity of MEL, however,distinguishes this trapping reagent from mono-thiol-selective reagents. The initial reduction of accessibledisulfide bonds in the presence of MEL leads tomodification of the bis-thiol groups in statu nascendi.Hence, reduction will not continue to completionsince the MEL-bridged protein is maintained as acompact structure,34 which is reflected in the ESI-MS‘‘charge structure’’ of the intermediate.11,29,47 Withthis approach, crucial folding intermediates may bespecifically trapped; whether such intermediatesdetermine the rate-limiting step during folding32

depends on the individual folding pathways of agiven protein.

Intrinsic tryptophan fluorescence has been widelyused as a means of monitoring unfolding/refoldingtransitions.48 Recorded changes in tryptophan fluo-

Fig. 7. rhM-CSF a is represented as ribbon diagram includingthe structurally resolved amino acid residues (4–151). The mono-mers (circled) contain the four-helix bundle structure. The mono-mer-monomer interface of the covalent dimer is indicated (open

oval). Cysteine residues are shown as wireframe models, markingthe disulfide bonds. The location of a tryptophan residue (W132) isshown in one monomer using the X-ray coordinates of the M-CSFa homodimeric protein.10


rescence revealed a two-state unfolding process ofrhM-CSF b in guanidinium hydrochloride, reflectingalterations of the local environment of W132, whichis located at the outer edges of the molecule (Fig. 7).By contrast, chemical trapping of bis-thiol groupswith MEL monitors conformational changes occur-ring in two different areas of the molecule simulta-neously. Changes in the compact four-helix bundlestructure of each rhM-CSF b monomer are measuredby monitoring the degree of modification of thecysteinyl residues participating in intramoleculardisulfide bonds. These intramolecular disufide bondsare located at the outer edges of the molecule (Fig. 7).In addition, alterations at the monomer-monomerinterface are reflected by modifications of cysteinylresidues involved in intermolecular disulfide bonds.Mass spectrometric peptide mapping of the D · 2MELderivative obtained from reductive unfolding re-vealed that cysteine residues C157 and C159 (in-volved in dimer formation) were predominantly MEL-modified. This is in agreement with an initialunfolding reaction of rhM-CSF b at the monomer-monomer interface. Unfolding of the monomer units,as reflected by minor modification of cysteine resi-dues (C7) involved in intramolecular disulfidebridges, e.g., C7-S-S-C90, may occur later duringunfolding. Alternatively, unfolding of rhM-CSF bmay follow two independent pathways. The majorunfolding pathway involved initial structure changesin the monomer-monomer interface and the minorroute required local unfolding at the outer edges ofthe dimer around the disulfide bond C7-S-S-C90.The trapping strategy and analytical concept appliedhere permit characterization of disulfide bond-mediated protein folding pathways by 1) monitoringthe average degree of modification reflecting thegeneral unfolding behavior of rhM-CSF b; and 2)pinpointing the modification sites in folding interme-diates.

ACKNOWLEDGMENTS

We are grateful to Dr. Michael Przybylski, inwhose laboratory parts of this work was carried out.We extend our thanks to Dr. Max L. Deinzer formaking his ESI-MS equipment available. We thankDr. S.-H. Kim and Dr. K. Koths for providing theX-ray coordinates of rhM-CSF a.

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Documents

Characterization of the folding pathway of recombinant human macrophage-colony stimulating-factor β (rhM-CSF β) by bis-cysteinyl modification and mass spectrometry