Reaction monitorin g of succinylation of collagenwith matrix-assisted laser desorption/ionizationmass spectrometry

Sung-Ho Kim 1* , Jeoung-Hwa Lee1, Shin-Young Yun1, Jong-Shin Yoo2, Chul-Ho Jun3,Kwan-Yong Chung3 and Hwal Suh4

1Department of Chemistry, Soonchunhyang University, Asan 336-600, South Korea2Mass Spectrometry Group, Korea Basic Science Institute, Taejon 305-333, South Korea3Department of Chemistry, Yonsei University, Seoul 120-752, South Korea4Department of Medical Engineering, Yonsei University College of Medicine, Seoul 120-752, South Korea

Succinylated collagen was synthesized by the reaction of collagen with succinic anhydride under basicconditions for one hour. Using thematrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)technique, the reaction products were directly identified without multi-step separation processes. MALDI-MS monitored the reaction more accurately than the conventional method of sodium dodecyl sulfatepolyacrylamidegel electrophoresis(SDS-PAGE). From thechangein molecular massdurin g thereaction, itisobserved that about 28 succinyl groupsareattached to thecollagen strand. Copyright # 2000 John Wiley& Sons, Ltd.

Received 3 August 2000; Revised 15 September 2000; Accepted 16 September 2000

Because collagen has the ability to persist in the bodywithout developing a foreign body response that could leadto premature rejection, it has been extensively investigatedas a bioengineering material for artificial tendons, bloodvessels, carti lage, bones and skin.1,2 More than 20 types ofcollagen are already approved as an ingredient forhemostats, vascular sealants, tissue sealants, implant coat-ings, and artificial skin.3,4 However, biomaterialsoriginatedfrom collagen still have some limi tations as to their use inhuman tissue due to their local cytotoxicities, tensileproperties, and biodegradation. In order to improve itsbiocompatibility , collagen can be chemically modified bythe functionalization of its amino and/or carboxylic groupsto other functional groups.5 Succinylation of amino groupsis one of the well-known techniques to control the rate ofbiodegradation and dissociation.6–8

Although the succinylation of collagen has been fre-quently performed in tissue engineering, direct character-ization and chemical analysis are problematic due to thecomplicated structure and instability under the analyticalconditions. The hydrogen ion titration method, whichmeasures the number of carboxyl groups, requires largeamounts of sample.9 The electrophoresis method, whichdetermines the molecular weight difference betweenreactant and product, requires a long analysis time due tomulti-step separation and chemical treatment processes.10

Recently, remarkable developments have been achievedin matrix-assisted laser desorption ionization/mass spectro-metry (MALDI-MS ) for the determination of molecularweights of proteins.11–13 In this study, using the MALDI-MS technique, we monitored the succinylation reaction of

collagen as a function of reaction time to investigatemodification sites directly without laborious separation orchemical treatment processes.

EXPERIMENTAL

Materials

Aqueous calf skin collagen (Type III ) was purchased fromSigma. Succinic anhydride, sinapinic acid, acetonitrile andsodium hydroxide were purchased from Aldrich ChemicalCo. Al l chemicals were used without purification exceptsuccinic anhydride, which was recrystallized prior to use.

Succinylation of collagen

Figure 1 shows the reaction scheme of the succinylation ofcollagen. First, collagen (50 mg) was dissolved in 0.05%acetic acid in 50 mL distilled water. After completedissolution, the pH was adjusted to 10 by adding 4 NNaOH solution, and the temperature of the solution wasmaintained at 0–2°C with an ice/water bath. Then, succinicanhydride(50 mg) wasadded, and thepH wasmaintained at9–10 for 1 h by periodically adding 4 N NaOH solution.Succinylated collagen was precipitated by adjusting thepH to 4.2 with a 1 N HCl solution and collected bycentrifugation. After washing several times with distilledwater, succinylated collagen was freeze-dried and isolatedas a white fibrous material, which was characterized byMALDI-MS.

MALD I sample preparation

Sinapinic acid was selected as matrix, and dissolved tosaturation in a1:1 mixtureof distilled water and acetonitrile.The samples of collagen and succinylated collagen weredissolved in distilled water. After thecollagen solutionshad

*Correspondence to: S.-H. Kim, Department of Chemistry, Soon-chunhyang University, Asan 336-660, South Korea.E-mail: shkim71@sch.ac.krContract/grant sponsor: Korea Ministry of Health and Welfare;Contract/grant number: HMP-99-E-05-0001.

Copyright # 2000 John Wiley & Sons, Ltd.

RAPID COMMUNICATIONS IN MASS SPECTROMETRYRapid Commun. Mass Spectrom. 14, 2125–2128 (2000)

beenincubatedfor 1 h at varioustemperaturesbetween25and70°C, they weremixed with matrix solutionanddriedin air for 3 min.

Instr umentation

MALDI-MS analysis wasconducted using a Voyager DE-STRinstrument from PEBiosystems.Thelaserdesorption/

ionization wasaccomplishedat 337nm nitrogen laser.Theintensity was attenuated to about 70% full power foroptimumpeakintensity. Theionswereacceleratedat25kVwith delayedextraction.Theinstrument in linearmodewascalibrated against bovine serum albumin as externalstandard. From the mass measurements of the BSAmolecularion [M � H]� at m/z66,431,andthecorrespond-ing doubly charged ion [M � 2H]2� at m/z 33,216, theinstrumental error was within 0.1% for the 60 kDa massregion.

RESULTS AND DISCUSSION

Identificat ion of collagen

It is well knownthat tropocollagen,thebasic structuralunitof collagen, hasa massof about285 kDa, andconsistsofthree polypeptide chains of the same size. This triply

Figure 1. Reactionschemeof thesuccinylationof collagen.

Figure 2. MALDI spectraof collagenobtainedat severalelevatedtemperatureup to 70°C.

Figure 3.Temperaturedependencyfor (a) intensitychangeof collagenpeaksand(b) helix contentsof calf skin collagen.

Rapid Commun.MassSpectrom.14, 2125–2128(2000) Copyright# 2000JohnWiley & Sons,Ltd.

2126 MALDI-MS MONITORINE OF SUCCINYLATION OF COLLAGEN

stranded collagen is believed to form a helix, andstructuraltransition occurswithin a narrowrangeof temperatures.Inthecaseof calf skincollagen,this transitionoccursbetween30 and40°C. Figure 2 showsthe MALDI massspectraofcollagen obtained at various incubation temperatures ofsamplesolution between 25 and 70°C. Below 30°C, themolecular massof collagenwasnot detected,while above35°C, themasspeakof collagen wasobservedbetweenm/z93,456and 93,600.Considering the instrumental error of0.3%in themassmeasurement,thedetectedspeciesof 93.5kDa can be assignedas one-third of the triply strandedcollagen of molecular mass285 kDa. The intensity of thispeak increases rapidly near 40°C, and remainsconstantabovethattemperature.Theintensity changeof thecollagenpeak,asa function of the incubationtemperaturebetweenroom temperature and 70°C, is plotted in Fig. 3(a). Thiscorrespondswell to thehelix contents of calf skin collagenat different temperaturesreported in the literature14 (Fig.3(b)). From this result,we concludethat, at low tempera-ture, the molecular mass of collagen was not determinedeasily due to the complicated triply strandedstructure.However, above the transition temperature, at which thetriple strandof collagen changesinto a single strand,themolecular massappearedat m/z93.5kDa.

Succinylation of collagen

Succinylationof collagen wasperformedby thereactionofsuccinic anhydride with collagen for one hour. Figure 4shows the MALDI mass spectra of (a) collagen, (b)succinylated collagen, and (c) a 2:1 mixture of collagen

andsuccinylatedcollagen,eachobtainedafter 1 h incuba-tion at 50°C. The native collagen was observed at m/z93,497,andthesuccinylatedproduct of thesingly strandedcollagen at m/z96,321.During the reaction, the succinyla-tion resulted in a massincreaseof about2800Da. Becausethe molecular mass of the succinyl group is 100Da, it isdeduced that about28 succinyl groupsare attachedto thecollagen strand. This corresponds to an increment of290mmol of succinyl groupsper gram of collagen. Thereare 38 different lysine residues in the collagen studied(theoreticalMW of 93,650Da), and5 of theselysinesarehydroxylated.The 28 succinylated sites, therefore,corre-spond to 85% derivatization of available lysine residuesbasedon the collagen molecular structure.15 Further studywill be needed to rationalize the specific sites forsuccinylation of collagen.

Until now, the modification of proteinswasconvention-ally identified by molecularweight determination by SDS-PAGE (sodiumdodecyl sulfate polyacrylamide gel electro-phoresis). Figure 5 shows the experimental results fromSDS-PAGE for the succinylation of collagen. Figure 5(a)indicates the collagen and Fig. 5(b) the succinylatedcollagen. Even though this technique requires laboriousmulti-step separation processes, it fails to show anymolecular massdifferencebetweenreactant and product.However, using MALDI-MS, the reaction was monitoreddirectly without any pre-separation processes.

Figure 4. MALDI massspectrumof succinylatedcollagenobtainedafter 1 h incubationat 50°C. (a) Collagen,(b) succinylatedcollagen,and(c) 2:1 mixture of collagenandsuccinylatedcollagen.

Figure 5. Molecular weight determinationwith SDS-PAGEof (a)collagenand(b) succinylatedcollagen.

Figure 6. Molecular massesof succinylatedcollagendependingonvariousreactiontime from 1 to 24 h.

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MALDI-M S MONITORINE OF SUCCINYLATION OF COLLAGEN 2127

Reaction-time dependenceof the succinylation reaction

In order to examine the stability of succinylated collagen,the molecular masschanges of succinylated products, asafunctionof reaction time,wereinvestigatedby MALDI-MS.As shownin Fig. 6, thesuccinylatedcollagenover1 to 24hreaction time showsmolecular massesbetween96,138and96,555 Da (Table 1). Consideringthe instrumentalerror of0.3% (300Da for 100 kDa), theseproducts havethe sameaveragemolecular massof 96.3 kDa. Neither increasenordecreaseof the molecularmassof product was observedwithin experimental uncertainty. This means that thereaction was completewithin 1 h, resulting in about 28succinyl groupsattachedto a singlecollagen strand.Eventhough the reactiontime was extendedup to 24h, furtherattachmentof succinylgroupsdid not occur.

CONCLUSIONS

Succinylated collagen was synthesizedby the reactionofcollagen with succinic anhydride underbasicconditionsforone hour. Using the MALDI-TOF technique, the reactionproducts weredirectly identified without multi-stepsepara-tion processes.MALDI-MS wasableto monitor thereaction

more accurately than the conventional method of SDS-PAGE,which failed totally. From the changein molecularmass during the reaction, it is observedthat about 28succinylgroupsareattachedto thecollagen strand.Fromtheanalysesof the succinylation product as a function ofreaction time, the succinyl groups seem to be attachedcompletelyto the reactionsite of Lysyl e-aminogroupsofcollagen within one hour. Further succinylation did notoccur. For further study, the MALDI-MS methodwill beapplied to the identification of the availablemodificationsitesof biocompatible proteins.

Acknowledgement

This work wassupportedby researchgrantsfrom the KoreaMinistryof HealthandWelfare(HMP-99-E-05-0001).

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Table 1. Molecular massesof succinylatedcollagendependingonvarious reaction time

Reactiontime (hour) MW (Da)

0 934971 965554 964218 96384

12 9639816 9634320 9613824 96204

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2128 MALDI-MS MONITORINE OF SUCCINYLATION OF COLLAGEN