Characterization of plasma proteins adsorbed onto biomaterials by MALDI-TOFMS

*Corresponding author. Tel.:#1-204-474-7418; fax:#1-204-474-7608.E-mail address: [email protected] (H. Perreault).

Biomaterials 21 (2000) 1701}1710

Characterization of plasma proteins adsorbed onto biomaterialsby MALDI-TOFMS

R.D. Oleschuk!, M.E. McComb",#, A. Chow", W. Ens$, K.G. Standing$, H. Perreault",*,Y. Marois%, M. King%

!Department of Chemistry, University of Alberta, Edmonton, AB, Canada T6G 2G2"Departments of Chemistry and Physics, University of Manitoba, Winnipeg, MB, Canada R3 T 2N2

#Departments of Biochemistry and Biophysics, Boston University School of Medicine, Boston, MA 02118-2394, USA$Department of Physics, University of Manitoba, Winnipeg, MB, Canada R3 T 2N2%Institut des Biomate& riaux, 10, rue de l+Espinay, Que&bec, QC, Canada G1L 3L5

Received 22 October 1999; accepted 22 February 2000

Abstract

The analysis of plasma proteins adsorbed onto a polyurethane (PU) biomaterial was performed using matrix-assisted laserdesorption/ionization time-of-#ight mass spectrometry (MALDI-TOFMS). This article marks the "rst study on MALDI-TOFMSanalysis of multiple proteins adsorbed from plasma, in vitro, onto the surface of a biomaterial to easily enable their characterization.Plasma standards from three di!erent hosts were placed in contact with non-porous PU, a model biomaterial. Following the use ofwashing protocols developed in our laboratory, the biomaterial was analyzed, directly, with MALDI-TOFMS. Proteins withmolecular weights (M

3) ranging from ca. 6.5 to 150kDa were observed in the mass spectra and characterized upon comparison with

proteins of known M3. The proteins observed were tentatively identi"ed as those known to adsorb onto PU, both in vitro and in vivo.

In an attempt to model in vivo sorption, the PU biomaterial was exposed to freshly collected canine plasma, in vitro, for di!erentlengths of time. Corresponding MALDI-TOFMS spectra displayed increasing protein signal for a number of di!erent proteins withincreasing times of exposure to plasma. This method provided qualitative and semi-quantitative analysis of the proteins adsorbedonto the biomaterial surface. ( 2000 Elsevier Science Ltd. All rights reserved.

Keywords: MALDI; TOFMS; Plasma protein; Biomaterial; Biocompatible

1. Introduction

Medical devices composed of polymeric materials havebecome ubiquitous for the treatment of certain disorders,however, implantation of polymeric devices within thehuman body can lead to a number of problems. In#am-mation and thrombosis may cause the body to reject theimplant, or the polymer material may be enzymaticallydegraded, leading to the deterioration or failure of thedevice. Protein sorption onto the surface of the bio-material is a major factor contributing to the stability ofmaterials in the body, and ultimately determines thein vivo performance of the polymer [1,2]. The adsorptionof certain proteins is bene"cial to inhibit further physio-

logical response, while adsorption of others producesa detrimental e!ect, enhancing platelet adhesion andthrombus formation (shown schematically in Fig. 1)and resulting in di$culties with the implanted device.Therefore, the characterization and measurement of sur-face-adsorbed proteins is important for assessing thebiocompatibility of a material.

Currently, the characterization of adsorbed proteins iscarried out using a number of techniques [3]. Traditionalanalyses utilizing radio-labeled proteins [4,5] have beenperformed, but are limited to monitoring the adsorptionof one protein at a time. More elaborate protocolsinvolving removal of adsorbed proteins from thebiomaterial surface with a surfactant and subsequentanalysis by SDS-PAGE [6,7] have also been used, butare extremely time consuming. More recently, techniquessuch as Fourier transform infrared spectroscopy [8}10](FTIR), Raman spectroscopy, and surface plasmon reso-nance [11}13] (SPR) have been utilized to quantify the

0142-9612/00/$ - see front matter ( 2000 Elsevier Science Ltd. All rights reserved.PII: S 0 1 4 2 - 9 6 1 2 ( 0 0 ) 0 0 0 5 4 - 5

Fig. 1. Schematic representation of protein sorption onto a bio-material, believed to be responsible for the initial stages of its degrada-tion (not to scale).

amount of adsorbed protein on surfaces of materials, butare also unable to e$ciently analyze several proteinssimultaneously. Often, a physiological response to a bio-material is caused by a complementary interaction[14,15] between two or more proteins. Techniques thatcan monitor the adsorption of only a single proteinwould not detect a synergistic interaction. A techniquethat can monitor and characterize the adsorption ofseveral proteins simultaneously on the biomaterial wouldbe ideal.

Matrix-assisted laser desorption/ionization time-of-#ight mass spectrometry (MALDI-TOFMS) has becomea rapid and convenient method for the characterizationof proteins and peptides from biological samples. Thetechnique can simultaneously measure the molecularweights (M

3) of several proteins in a sample, allowing

them to be identi"ed by comparing experimentally deter-mined M

3with literature values. MALDI is tolerant of

impurities compared with other MS ionization methods.This facilitates the analysis of biological samples whichoften contain bu!er components in relatively high con-centrations.

MALDI-TOFMS has been used to analyze proteinsprepared on di!erent types of supports [16}21]. Gener-ally, these supports have been polymeric membranes andhave been used to remove polar components from pro-tein samples. Sample preparation on a membrane allowsthe removal of interfering agents such as salts and bu!ercomponents and makes the identi"cation and analysis ofproteins in complex mixtures, such as biological #uids,easier. Membranes so far used for the puri"cation ofprotein samples generally have a high binding a$nity fora wide variety of proteins. In contrast, biomaterials usu-ally adsorb certain proteins preferentially to give themenhanced in vivo stability. It has recently been suggestedthat MALDI-TOFMS would be a convenient methodfor the characterization of proteins adsorbed directlyonto surfaces, such as biomaterials [22}24]. The bio-material sample may be analyzed in an analogous man-ner to membrane MALDI-TOFMS samples and thusprovide information about the type, and amount of

protein bound to its surface following exposure to abiological #uid.

In this study polyurethane (PU) was chosen as a modelbiomaterial because of its extensive use in the construc-tion of biomedical implant devices [4]. Protein sorptiononto PU in vitro and in vivo is well characterized withinthe literature, allowing MALDI-TOFMS to be com-pared with more traditional methods of analysis. In addi-tion, our laboratory has experience with using PUmembranes as sample supports for MALDI-TOFMScharacterization of peptides and proteins derived frombiological samples [19,25,26].

Polyurethane membrane material was exposed toplasma standards from three di!erent hosts. Massspectra of the plasma standards on the PU biomaterialshowed the adsorption of several proteins ranging from6.5 to 150 kDa. Proteins observed in the mass spectradirectly correlate with those shown by other researchersto be adsorbed from plasma onto PU-based materials,thus validating MALDI-TOFMS as a technique forbiomaterial analysis. This type of application may also beused for screening among possible future biomaterials.

2. Experimental section

2.1. Reagents and materials

All chemicals used in this study were of reagent gradeunless otherwise stated. HPLC grade acetonitrile andmethanol were from Fisher Scienti"c (Fair Lawn, NJ).Deionized water, prepared with a Milli-Q plus}TOCwater puri"cation system (Millipore, Bedford, MA), wasemployed in all solutions. Plasma protein standards(human, bovine and canine) were obtained from SigmaChemicals (St. Louis, MO). Sinapinic acid, used as theMALDI matrix, was from Sigma. Protein standards wereused as MALDI calibrants. Horse heart myoglobin,16 951Da, bovine insulin, 5733Da, bovine serumalbumin, 66 430Da, were purchased from Sigma, andbovine apotransferrin, 78 030 Da, was obtained from Cal-biochem (LaJolla, CA). Solutions of these standards weremade up in water, and used without further puri"cation.

2.2. PU membrane model biomaterial

The non-porous ether-type PU membrane, 50lm inthickness (XPR625-FS) was supplied by Stevens Elas-tomerics (Northampton, MA). The PU membrane usedconsists of a co-polymer of polytetramethylene etherglycol and diphenylmethane diisocyanate (MDI) [27].The membrane was washed with water and methanolprior to use in order to remove polar and non-polarsurface contaminants.

1702 R.D. Oleschuk et al. / Biomaterials 21 (2000) 1701}1710

Fig. 2. (a) Two-phase bulk structure of PU membrane consisting ofhard (moderately polar) and soft (non-polar) segments. (b) Chemicalstructure of PU used in these experiments.

2.3. MALDI-TOFMS

MALDI-TOFMS was performed in the linear modeon a re#ecting time-of-#ight mass spectrometer, an in-strument built in-house in the Time-of-Flight Lab, Uni-versity of Manitoba [28]. Accelerating potentials of 25and 30 kV were used. Spectra were obtained using a ni-trogen laser (337 nm) (VSL 337 ND, Laser Science Inc.,Cambridge, MA, USA) with the #uence adjusted slightlyabove threshold. In order to avoid saturation of thedetector by low mass matrix ions, the detector was pulsedon ca. 19 000 ns after each laser shot. External calib-rations for measurements using the PU membrane wereperformed with standards (bovine insulin, equine myo-globin, human transferrin) prepared on similar mem-brane targets. Spectra presented here are summations ofca. 50}100 consecutive shots.

2.4. Preparation of plasma standard samples

Samples were prepared on PU based on protocolsdeveloped in our laboratory [19]. Samples were preparedon the membrane by "rst depositing 2lL of the plasmasolution onto the surface of the membrane and allowingit to dry. A mark was placed on the underside of themembrane to allow the sample spot to be located afterwashing. The membrane was then washed with 50lL ofwater for 1min to remove the non-adsorbed plasmacomponents. The washing step was repeated. After dry-ing, immediately before analysis, matrix (2lL, saturatedsinapinic acid in 1 : 1 H

2O/acetonitrile) was placed on the

membrane and allowed to crystallize. The membrane wasthen trimmed to size, a$xed to the MALDI target usingan adhesive spray (Spraymount, 3M) and subsequentlyanalyzed by MALDI-TOFMS.

2.5. In vitro experiments

In vitro experiments were performed at the Institut desBiomateH riaux du QueH bec, UniversiteH Laval. Samples ofPU membrane material were cut into 2.0 cm]2.0 cmpieces and exposed to pooled samples of freshly collectedcanine plasma in neutral glass vials (Anchor Ltd, Lon-don, Ontario). Membrane samples were exposed (in trip-licate) for periods of time from 0 to 4h at 373C. Followingexposure, the samples were immediately washed withwater to remove non-adsorbed plasma components,dried under vacuum and later shipped to our laboratoryat the University of Manitoba for MALDI-TOFMSanalysis.

2.6. Safety considerations

Suitable precautions should be taken when handlingbiological #uids. Gloves were worn during collection andmanipulation of samples.

3. Results and discussion

3.1. Analysis of a human plasma standard

The nature of protein sorption ultimately determinesthe in vivo stability of a material. PU possesses a two-phase bulk structure consisting of hard (moderately po-lar) and soft (non-polar) segments as shown in Fig. 2a[29,30]. The chemical structure of each of these segmentsgreatly in#uences the ability of the polymer to adsorbproteins [31]. Protein binding is believed to occurthrough a combination of hydrogen bonding and hydro-phobic interactions between the protein and the surfaceof the polymer [32]. Several di!erent types of PU havebeen used and tested for the construction of biomedicaldevices [33]. For our study the polytetramethylene oxide(PTMO) methylene bis(p-phenyl diisocyanate (MDI)-type PU (Fig. 2b) was chosen because it can adsorb largeramounts of protein than PU prepared with more hy-drophilic soft segments can [29]. Previously, we haveshown that MALDI-TOFMS resolution and mass accu-racy for proteins directly deposited onto the PTMO/MDI-type PU membrane used here were equivalent tothose obtained using a metallic target [19,26].

Although MALDI-TOFMS is relatively tolerant ofimpurities such as salts and bu!er components, the re-moval of these species yields greater resolution and massaccuracy. In addition, proteins with no a$nity for themembrane material must be removed prior to analysis.To accomplish this a rinsing step may be performed. Allof the components with a high a$nity for the biomaterialremain adsorbed while polar components and non-ad-sorbed proteins are washed o! the material. The washing

R.D. Oleschuk et al. / Biomaterials 21 (2000) 1701}1710 1703

Fig. 3. MALDI-TOF mass spectra of human plasma obtained using PU as a sample support. (a) Low M3

region. (b) High M3

region. Tentativeassignments are found in Table 1.

Table 1Tentative assignments for human plasma proteins adsorbed on polyurethane and detected by MALDI-TOFMS

Protein Molecular weight (Da) Relative peak area! Concentration"

Literature# Experimental RSD$ % RSD (%) RSD$ % RSD (g/L)

Immunoglobulin G 150000 148361 1900 1.3 20 5 23 8}18Transferrin 79 754 78 513 980 1.3 17 3 20 2.0}4.0Comp C3-b 75 000 73 953 590 0.80 31 9 27 (1Albumin 66 472 66 463% * * 100 * * 35}55a2-HS Glycoprotein 49 000 49 575 400 0.80 20 3 16 0.4}0.85

Apo A-4 43 400 44 175 170 0.40 16 12 77 (1Apo E 34236 33 109 200 0.60 56 28 49 (1Apo A-1 28 000 27 502 14 0.05 135 107 79 (1Apo D 22500 22 174 30 0.15 17 8 47 (1Apo H-III (HDL) 17 000 16 327 460 3 25 17 68 3Transthyretin 13 761 13 555 170 1.3 171 159 93 (1Apo C-II 8914 8946 16 0.20 * * * (1Apo C-III 8764 8783 10 0.20 * * * (1Apo C-I 6630 6631 10 0.20 * * * (1

!Peak areas of high M3

proteins normalized to albumin."Concentration in whole blood plasma from Ref. [34].#Literature molecular weights from Refs. [34,35].$RSD and % RSD based on 4 replicate measurements.%Albumin was used as the internal calibrant.

process is analogous to membrane puri"cation of pro-teins prior to MALDI-MS analysis [18,19].

Aliquots of human plasma standard (2lL) were placedonto the surface of the membrane and allowed to dry.Following rinsing and application of matrix, the sampleswere placed onto a MALDI probe and inserted in themass spectrometer for analysis. Fig. 3 shows MALDI-TOF mass spectra of human plasma proteins adsorbedonto the PU biomaterial (also see Table 1). In order toobtain reasonable signals in the high M

3range

('20 kDa) it was necessary to suppress the backgroundfrom the matrix by turning on the microchannel platedetector on, only after the matrix ions had time to reachthe detector. Good-quality MALDI mass spectra werethus obtained after removal of excess of salts and bu!ercomponents present in plasma (Table 2). Ions from at least

14 di!erent proteins were visible. The proteins ranged inM

3from ca. 6.5 to 150 kDa. Mass resolution and accuracy

were su$cient for identi"cation upon comparison withM

3values from the literature (Table 1) [34,35].

MALDI-TOFMS has been used for both the qualita-tive, semi-quantitative and quantitative analysis of pro-tein and peptide mixtures [36,37] and recently for thequantitative measurement of single protein sorption ontopolymer surfaces [24]. This investigation reports on thesemi-quantitative and qualitative determination of sev-eral plasma proteins simultaneously adsorbed onto a PUmembrane using relative MALDI-TOFMS peak inten-sities. Tentative mass assignments for each of the proteins[38] are shown in Table 1 along with integrated peakareas measured relative to albumin. Reproducibility inM

3measurements was good, however the % RSD


Table 2Concentration of bu!er components in plasma

Component Concentration (mM)!

Bicarbonate 42}54Calcium 4.2}5.2Chloride 95}103Cholesterol 0.038}0.063Glucose 0.036}0.055Iron 1.0}2.68]10~4

Magnesium 3.0}5.2Phosphate 1.8}2.6Potassium 8}9.6Sodium 136}142Sulfate 0.4}2.6Urea 0.013}0.033Uric acid 1.2}3.1]10~3

!Values adapted from Ref. [38].

measurements of relative peak areas ranged considerablyfrom 16 to 93%. The principle error in peak integrationwas due to slight di!erences in mass spectra obtained ondi!erent samples. Slight variations in the matrix prepara-tion steps led to non-uniform co-crystallization of theplasma proteins with the matrix and the production ofirregular crystals on the surface of the PU membrane.This resulted in variable signal intensity in the massspectra dependent on the location of the target sampledwith the laser.

Additional care must be taken in the interpretation ofthe spectra. The MALDI signal intensity observed is notnecessarily indicative of the amount of protein on thesurface of the membrane, neither is the amount of a par-ticular protein adsorbed onto the membrane indicative ofits concentration in solution. The a$nity of the proteinfor the membrane plays a more signi"cant role than itsconcentration. This is demonstrated by the absence ofions of several major proteins, and by the presence ofseveral low concentration proteins ions (Fig. 3b, Table 1).Only proteins with an a$nity for the membrane materialare adsorbed and later detected, while those with lowa$nity are washed o! [21,22]. Additionally, it is knownthat MALDI signal intensity is dependent on the amountof the protein and the ease with which it will co-crystalizewith the matrix and ionize in the MALDI plume [39].We have shown such discrimination phenomena for mix-tures of medium and high M

3wheat proteins prepared on

PU supports vs. steel targets [26].Plasma proteins which are known to adsorb onto PU

materials in vivo include lipoproteins [40,41], albumin[42], immunoglobulin G (IgG) [43], C3, "brinogen aswell as others [4]. With the exception of "brinogen, theseproteins were observed in the mass spectra obtained onPU (Fig. 3). This indicates a direct correlation betweenthe proteins detected in the mass spectrum of humanplasma on PU and proteins known to adsorb onto PUin vivo, determined using other techniques.

Biomaterials development has focused on the adsorp-tion of three proteins in particular, as indicators of bio-compatibility [3]. The adsorption levels of albumin,IgG and "brinogen are used to assess the in vivoperformance of a material. For biomaterials, albuminadsorption is bene"cial owing to this protein'srelative lack of glycosylation, which prevents plateletadhesion. On the other hand, adsorption of IgG and"brinogen may cause a host defense reaction which in-creases platelet adhesion and promotes a physiologicalresponse. Since the adsorption of both albumin and IgGwas observed in this study it follows that MALDI-TOFMS could be used to assess biocompatibility ofdi!erent materials by analyzing the amount of albuminor IgG adsorbed by a particular material. The thirdmajor protein, "brinogen, was not detected because of itshigh M

3. In addition, "brinogen is normally observed

after longer periods of exposure than were used in thisinvestigation.

3.2. Sample-to-sample reproducibility

Initially, the mass spectra of human plasma proteinson PU exhibited relatively poor reproducibility in peakintegration. Sample-to-sample reproducibility was ex-plored with care taken to ensure that the preparation/washing and subsequent MALDI-TOFMS measure-ments were obtained under nearly identical conditions.The results are shown in Fig. 4, for four replicate samplesof bovine plasma standard prepared on PU. Each spec-trum shown is the summation of 50 consecutive shotssampled over the entire surface of the target. Good shot-to-shot reproducibility was obtained when samples weresubjected to the same sample preparation and MALDIconditions. Relative peak areas and heights are given inTable 3. As with the human plasma proteins integrationof peak areas resulted in relatively high % RSD values(mean 42%). Improved results were obtained by measur-ing the peak heights which had a mean % RSD of ca.28%. Peak height variations overall were less than thoseobtained with integration and ranged from 9 to 54%.The larger #uctuation in peak areas arose mainly fromvariation in peak widths possibly due to inconsistentformation of adducts (i.e. with the matrix) from onesample to another. Di!erences in baseline assignmentassociated with manual integration may have also con-tributed to the larger error. Comparison of the twosemi-quantitative sets of measurements and the errorsassociated with them are better visualized in Fig. 5. Hereit can bee seen that, even though there exists some vari-ance in the measurements, semi-quantitative results ofpeak area or height may still be obtained and are respect-ive of the amount of protein adsorbed onto the surface ofthe membrane.

Relative MALDI peak areas or heights can thus be usedas a semi-quantitative assessment of the concentration


Fig. 4. Reproducibility of MALDI-TOFMS measurements obtainedfor four replicate samples of bovine plasma standards. Masses corres-pond to values in Table 3.

Table 3Reproducibility in MALDI-TOFMS measurements of bovine plasma proteins adsorbed on polyurethane, High M

3

Molecular weight! Peak area" Peak height#

(Da) RSD$ % RSD (%) RSD % RSD (%) RSD % RSD

12571 161 1.27 75 38 51 288 48 1613 657 126 0.92 341 104 30 551 87 1514 881 147 0.98 93 24 26 216 21 915 946 192 1.20 28 7 24 96 20 2122 034 690 3.10 26 21 82 47 12 2522 831 510 2.21 22 23 103 48 12 2427 470 193 0.70 157 35 22 190 58 3033 153 179 0.54 57 5 8 78 10 1238 796 292 0.75 12 7 61 28 12 4448 942 397 0.80 9 3 35 27 13 5053 007 517 0.97 16 8 52 28 15 5466 433! * * 100 * * 100 * *

73 324 279 0.38 31 13 42 40 16 4077 606 341 0.44 17 8 49 36 19 54

Average % RSD 42 Average % RSD 28

!Albumin was used as the internal calibrant."Peak areas of high M

3proteins normalized to albumin.

#Peak heights of high M3

proteins normalized to albumin.$RSD and % RSD based on four replicate measurements.

Fig. 5. Graphical representation of statistical values from Table 3.Mean and standard deviation in (a) peak area, (b) peak height.

of di!erent proteins adsorbed onto the biomaterial.In addition, small mass shifts in the protein adsorptionpro"le of a material can be determined and allow betterbiomaterial characterization.


Fig. 6. MALDI-TOF mass spectra of three di!erent plasma standards:bovine, canine and human on PU membrane. (a) Low M

3region.

(b) High M3

region.

Fig. 7. MALDI-TOF mass spectra of bovine plasma proteins on PUmembrane using di!erent sample preparation methods. (a) Plasma,dried, washed, dried, matrix. (b) Plasma, dried, methanol, dried, washed,dried, matrix. (c) Plasma#methanol, dried, washed, dried, matrix.(d) Plasma#matrix solution, dried, washed, dried, matrix.

3.3. Plasma standards of diwerent hosts

Studies suggest that there are slight di!erences in pro-tein adsorption patterns between plasma samples fromdi!erent hosts and that these are due to variations inhematological pro"les [44]. Plasma samples from threehosts (i.e. human, canine and bovine) were used to deter-mine the ability of MALDI-TOFMS to distinguish be-tween protein adsorption patterns for di!erent plasmastandards.

PU samples were exposed to each plasma standardand then analyzed to compare adsorption patterns. Thespectra of three di!erent plasma standards adsorbed onPU are shown in Fig. 6. These results indicate thatproteins of similar high M

3were adsorbed (Fig. 6b) for

each plasma standard. There were di!erences in the lowM

3pro"les (Fig. 6a). The human plasma standard result-

ed in the highest number of adsorbed proteins in the5.5}10kDa mass range, possibly lipoproteins. In com-parison, the spectra obtained for bovine and canineplasma showed adsorption of fewer proteins in this re-gion. The adsorption of lipids onto PU surfaces has beenassociated with the biodegradation of PU implants [45].It has also been suggested that the interaction betweenlipoproteins and di!erent domains of the PU facilitateslipid adsorption [4].

The variations between protein adsorption patternscould lead to di!erences in biocompatibility, questioningthe validity of testing biomaterials with other hosts [40].MALDI-TOFMS is capable of distinguishing betweendi!erent protein adsorption patterns and therefore maybe seen as a valid method for biomaterial analysis.

3.4. Inyuence of sample preparation conditions

Di!erent sample preparation methods were tested fortheir e!ect on ion signal intensities and to further deter-mine conditions which may be used for in vitro testing. InFig. 7a, bovine plasma was applied onto the PU mem-brane followed by washing and addition of matrix. InFig. 7b, methanol was applied following the applicationand drying of the plasma sample. The addition of meth-anol resulted in an increase in the overall MALDI signal.A similar phenomenon was reported in an earlier publi-cation [19]. We associated this to enhanced protein bind-ing on the membrane, an e!ect commonly exploited inSDS-PAGE when electroblotting onto porous mem-branes. Methanol enhanced protein adsorption was alsodescribed for MALDI-TOFMS on porous membranes[17]. In Fig. 7c, methanol was added to the sample beforethe plasma sample had a chance to dry on the membrane.This resulted in the precipitation of the proteinsfrom the plasma. Alcohol precipitation is often used toremove proteins from plasma/serum in order to analyze


Fig. 8. MALDI-TOF mass spectra obtained on PU membranes ex-posed to canine plasma for (a) 0 (b) 0.5 (c) 1 and (d) 4 h.

other components. The result was an increase in signalintensity for some of the proteins, notably, albumin.

Fig. 7d shows the e!ect of adding matrix solution tothe sample before the plasma had a chance to dry on themembrane. In this case, the spectrum showed substantialdi!erences relative to features observed in Fig. 7a}c.Even though the matrix material is dissolved in 50% (v/v)acetonitrile, it is acidic in nature. Protonation in solutionwill result in a net charge on the plasma proteins and willdisfavor their sorption onto the predominantly hydro-phobic membrane. The acidity of the matrix facilitatesthe removal of some bound proteins from the surface sothey may co-crystallize with the matrix for analysis byMALDI [19].

The di!erent sample preparation protocols led tochanges in the amount and nature of the proteinsadsorbed and detected by MALDI. These discrepanciesshow that a consistent sample preparation method mustbe employed to directly compare protein adsorptionpatterns on di!erent biomaterials. With the additionof methanol to the samples (Fig. 7b and c) it waspossible to improve spectral quality compared withthe neat sample. However, these approaches do notre#ect solution conditions which would be observedin vivo. Therefore, the conditions chosen for thein vitro experiments where based on the "rst protocolused (Fig. 7a).

3.5. In vitro analysis

In vitro testing of biomaterials is commonly performedbecause it is relatively inexpensive and faster than in vivomethods. In vitro experiments involve the isolation of#uid or tissue from a biological source, followed byexperimental characterization of this material. In thisstudy, the MALDI analysis of proteins adsorbed in vitroonto PU over di!erent times of exposure to canineplasma was performed. Canine plasma is commonly usedby biomaterials researchers and represents the worst casescenario for protein adsorption since it produces exten-sive platelet adhesion and thrombus formation. Freshlycollected canine plasma was placed in contacted with thePU material under speci"ed conditions to facilitate pro-tein sorption. Triplicate samples were exposed to canineplasma for di!erent lengths of time (0, 0.5, 1 and 4 h) at370C.

Representative mass spectra showing the e!ect of thetime of exposure of canine plasma on the amount ofproteins adsorbed are shown in Fig. 8. In the controlsample spectrum, (Fig. 8a) no peaks are observed. Analy-sis of the sample exposed to plasma for 0.5 h showsseveral peaks due to adsorption of proteins onto thesurface of the PU biomaterial. Continued exposure, up to4h, yielded more intense protein signals. This may beinterpreted as increasing amounts of protein being adsor-bed on the surface of the polymer.

These data supports the notion that protein adsorp-tion occurs quite rapidly upon exposure of the polymericmaterial [1]. It has been suggested that for relativelyshort periods of exposure, protein adsorption "ts aLangmuir isotherm [46]. The distribution of proteinsadsorbed at the surface of a material is, however, timedependent. Proteins are constantly deposited, and dis-placed and thus the surface composition changes [47].The rates of deposition and displacement can be deter-mined by the relative concentrations of proteins withinthe plasma and their a$nity for the material. Althoughproteins with a high plasma concentration may be adsor-bed "rst, those with low concentrations but with a highera$nity for a particular surface will eventually displacethem. The displacement of proteins occurs after the sur-face of the polymer has become saturated. This phenom-enon was not observed in this study, even over the fourhour exposure. Instead, analysis of PU samples exposedto canine plasma in vitro yield spectra suggesting that thelonger the exposure time, the larger the amount of pro-tein present on the surface of the material.

The mass spectral pro"le observed, although weaker inabsolute intensity, was similar to the pro"le shown forthe canine standard (Fig. 6b). This resemblance indicatesa strong similarity between the proteins observed fromthe in vitro experiments and those observed for thecanine plasma standard. The use of plasma standardsfor biomaterials testing may be su$cient as an initial


screening process for the biomaterial. Here, the use ofplasma standards is preferred because it is faster, simplerand less expensive than performing either in vivo orin vitro experiments.

4. Conclusions

Among the various kinds of interfacial phenomenaoccurring in the "eld of biomaterials, the adsorption ofproteins at the interface between a liquid and a solid isone of the most signi"cant areas that has been investi-gated. The analysis of proteins adsorbed onto bio-materials is a new application of MALDI-TOFMS. Thetechnique is able to analyze several proteins simul-taneously with su$cient resolution to allow the easycharacterization of adsorbed proteins based on literatureM

3. Experiments performed in this study showed direct

correlations between the proteins known to adsorb onPU from plasma and those seen in the mass spectra ofplasma proteins on PU.

The analysis of proteins adsorbed onto PU fromplasma standards yielded good-quality mass spectra, dis-playing a variety of proteins from 6.5 to 150 kDa. TheMALDI spectra exhibited good shot-to-shot reproduci-bility with only minor shifts in peak height. These fea-tures enabled the semi-quantitative analysis of adsorbedproteins. Comparison of plasma standards from di!erenthosts showed only small di!erences in the high M

3pro-

tein distribution, and more pronounced di!erences forlow M

3proteins. Di!erences in sample manipulation

were shown to have a profound e!ect on spectra, there-fore care should be taken when developing experimentalprocedures for biomaterial characterization.

This study clearly demonstrates that MALDI-TOFMS may be used to monitor the adsorption ofdi!erent proteins onto biomaterials. Therefore, this tech-nique is helpful for determining the biocompatibility ofthe biomaterial in vivo. The method may be readilyexpanded to include testing implant devices removedfrom patients following implantation.

Acknowledgements

This work was supported by the National Science andEngineering Research Council of Canada (NSERC). Wethank the members of the Time-of-Flight Laboratory,University of Manitoba, for helpful discussion. Finally,we wish to thank the University of Manitoba for a Grad-uate Fellowship awarded to M. E. McComb.

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Characterization of plasma proteins adsorbed onto biomaterials by MALDI-TOFMS

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