Journal of Colloid and Interface Science 331 (2009) 90–97

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Journal of Colloid and Interface Science

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The adsorption of globular proteins onto a fluorinated PDMS surface

Dan Wang, Michelle Douma, Brenna Swift, Richard D. Oleschuk, J. Hugh Horton ∗

Department of Chemistry, Queen’s University, Kingston, Ontario, K7L 3N6, Canada

a r t i c l e i n f o a b s t r a c t

Article history:Received 21 August 2008Accepted 5 November 2008Available online 8 November 2008

Keywords:MALDIESRFluorinated polymersFluoro-tagged proteinsSurfaces

Poly(dimethylsiloxane) (PDMS) has shown considerable promise in the fabrication of microfluidic devices.Surface modification of PDMS by the grafting of perfluorinated alkanes allows the selective adsorption offluorous-tagged peptides, demonstrating that this material may be used in fluorous affinity tag technologyto enrich and separate specific proteins or peptides from complex mixtures. Here, we explore the non-specific adsorption of proteins which may interfere with this process. The desorption of cytochrome c,carbonic anhydrase, insulin and ubiquitin onto the surfaces of unmodified, oxidized and fluorinatedPDMS in solutions of varying water/methanol concentration has been studied using matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The interaction forces involvingperfluorinated surfaces are probed using chemical force spectrometry. The denaturation of the proteinsin solutions of high methanol concentration is followed using electrospray ionization mass spectrometry(ESI-MS) and the adsorption profiles discussed in the context of the surface hydrophobicity of eachprotein.

© 2008 Elsevier Inc. All rights reserved.

1. Introduction

Since the term fluorous – “of, relating to, or having thecharacteristics of highly fluorinated saturated organic materials,molecules or molecular fragments” – was introduced by Horváthin the early 1990s, there have been extensive developments inthe field of fluorous chemistry. Recently, fluorous tags have beenused in synthetic applications to isolate the desired componentsfrom a reaction mixture, taking advantage of fluorophilic interac-tions. For example, by using solid–liquid extractions over fluorousreverse-phase silica gel, Curran and Luo [1] achieved good sep-aration of fluorous amide products from a mixture eluted withmethanol/water solvent solutions.

In the field of proteomics, an approach to separating targetproteins or peptides from complex mixtures using fluorous chem-istry has been recently developed by Brittain and co-workers[2]. They have used fluorous affinity tag technology to enrichand separate specific proteins or peptides from complex mix-tures, using mass spectrometry techniques to characterize thesefluorine tagged species. They [3] have also demonstrated desorp-tion ionization on silicon (DIOS) using fluorous-silylated materi-als as affinity surfaces to enrich fluorous-tagged analytes usingmass spectrometric methods to test these species. We have re-cently reported on a similar scheme using polydimethylsiloxane(PDMS) as the substrate [4]. PDMS was modified by grafting per-

* Corresponding author. Fax: +1 (613) 533 6669.E-mail address: hortonj@chem.queensu.ca (J.H. Horton).

0021-9797/$ – see front matter © 2008 Elsevier Inc. All rights reserved.doi:10.1016/j.jcis.2008.11.010

fluorooctyltriethoxysilane via hydrolysis onto an oxidized surfaceand we used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to test the adsorption offluorous-tagged peptides onto this fluorinated PDMS surface. Ourresults demonstrated that the fluorinated PDMS surface could beused for enrichment or to enhance detection of fluorous-labeledpeptides, while at the same time maintaining a large zeta potentialat the surface. This latter property would also allow these materi-als to be used in micro total analysis systems where a large andstable zeta potential is required to maintain electrophoretic mobil-ity.

One potential drawback to using any polymer substrate is thatmany proteins may tend to adsorb onto the surface due to itshigh hydrophobicity [5,6]. Our previous results [4] showed that thefluorinated PDMS surface is more hydrophobic than the unmodi-fied PDMS surface. As untagged proteins may potentially interactstrongly with the fluorinated PDMS surface, these non-specific in-teractions could lead to “false positives” for the presence of thesespecies or interfere with the adsorption of fluorinated species fromsolution. The goal of this paper is to assess the importance and ex-tent of non-specific protein adsorption in these systems. To do this,we report on a study of the solid–liquid extraction of some com-mon proteins on unmodified, oxidized and fluorinated PDMS usinga combination of mass spectrometric methods and chemical forcespectrometry.

Solid–liquid extraction from surfaces containing similar func-tional groups to some of the modified PDMS materials studied herehave been previously carried out. For example, Mengistu et al. [7]

D. Wang et al. / Journal of Colloid and Interface Science 331 (2009) 90–97 91

have studied the adsorption of proteins including α-casein, car-bonic anhydrase, α-lactalbumin, bovine serum albumin, ubiquitin,cytochrome c, insulin and myoglobin onto methyl- and carboxyl-terminated porous Si surfaces. Their results showed that the pro-teins tend to adsorb preferentially on porous Si surfaces ratherthan flat surfaces, due to the increased surface area. They alsofound that varying the pH of the rinse solution will influencethe adsorption of proteins on functionalized surfaces. However, thefunctional groups present on the porous Si also had a strong in-fluence on the protein–surface interactions. Karlsson et al. [8] usedthree engineered variants of human carbonic anhydrase II to studythe influence of protein stability on the adsorption and desorptionbehavior to four different surfaces (negatively charged, hydrophilic,hydrophobic, and positively charged) by using surface plasmon res-onance measurements. Their results indicated that controlling theconformational stability of the protein is an important parame-ter in the adsorption and desorption behavior at a liquid–solidinterface. Krishnan and co-workers [9] investigated nine globularblood proteins onto methyl-terminated surfaces in aqueous–buffersolution. They demonstrated that the adsorption of proteins ontohydrophobic surfaces were mainly influenced by interfacial waterand were not strongly dependent on protein type.

Conformational changes of proteins in organic solutions, par-ticularly alcohols [10], have been studied by a variety of phys-ical techniques such as fluorescence, circular dichroism and nu-clear magnetic resonance [11,12]. The use of electrospray ionizationmass spectrometry (ESI-MS) [13,14] to monitor the conformationchanges of proteins was first described by Chowdhury and co-workers [15]. This technique has been proven to be very usefulin the study of conformational changes of different proteins uponchanging pH, temperature, and the presence of denaturing agentssuch as organic solvents [15–22].

In the work described here, we study the desorption of cy-tochrome c, carbonic anhydrase, insulin and ubiquitin from un-modified, oxidized and fluorinated PDMS surfaces. Here we chosemethanol/water solutions of varying compositions as the liquidphase for the extraction of proteins; the original reports of fluoro-tagged species using DIOS techniques were eluted with such mix-tures. We use the signal-to-noise ratio of the primary ion in theMALDI-MS spectrum to compare the relative adsorption of pro-teins on the surface after washing with different volume ratios ofmethanol/water solutions. These data are discussed in the contextof the interaction forces observed involving perfluorinated surfacesusing chemical force spectrometry, and the denaturation of theproteins in solutions of high methanol concentration, as deter-mined by electrospray ionization mass spectrometry. In addition,we calculated the surface hydrophobicity of each protein and usethis value to interpret the MALDI-MS experimental results.

2. Experimental

2.1. PDMS substrates and surface modification

The native, oxidized and fluorinated PDMS polymers used inthis study have been previously characterized using a combinationof X-ray photoelectron spectroscopy (XPS) and AFM methods [4].Complete protocols for the surface modification have been previ-ously published [4]. Briefly, Sylgard 184 (Dow Corning Corporation)was spin-coated and cured. The resulting materials were peeled offand cut into circular samples of 0.7 cm diameter for use in theMALDI-MS experiments. Fluorinated PDMS surfaces were formedusing a two-step process: first, air plasma oxidation, followed im-mersion into a 20 mmol/L solution of perfluoro-1,1,2,2-tetrahydrooctyl-1-triethoxysilane (PFO, United Chemical Technologies, Inc.,Horsham, PA) in toluene for up to 4 h.

2.2. Mass spectrometric measurements

Mass spectrometric measurements were carried out using aVoyager DE-STR MALDI-TOF system (Applied Biosystems, FosterCity, CA). Accelerating potentials of 20 kV were used. Spectra wereobtained using a nitrogen laser (337 nm) with the fluence ad-justed slightly above threshold. The proteins studied here werecarbonic anhydrase (Sigma-Aldrich, bovine erythrocytes, C3934),cytochrome c (Sigma-Aldrich, from horse heart, C2506), ubiquitin(Sigma-Aldrich, bovine red blood cells, U6253) and insulin (Sigma-Aldrich, bovine pancreas, I6634). Aqueous solutions of 1 mg/mLof each protein were prepared. The final solution pH was 4.5, 8.8,6.5 and 6.9, respectively. The PDMS substrates were attached ontoeach spot of the MALDI sample plate directly. The backside (un-modified) of the PDMS samples adhere effectively to the surfaceof the stainless steel MALDI plate without the use of any adhesive.Similar to the DIOS experiments carried out by Brittain et al. [3],a 20 μL aliquot of 1 mg/mL aqueous solution of each protein wasdeposited onto the variously modified PDMS substrate surfaces, al-lowed to dry, then washed with a 1 mL aliquot of varying concen-tration of methanol water solutions (0–1.0 (volume fraction, v/v)of methanol/water in 0.1 increments). After washing the surface,2 μL of a sinapinic acid matrix was deposited (sinapinic acid sat-urated in 60% acetonitrile water solution with 0.3% trifluoroaceticacid) on the washed regions. MALDI-MS was then used to detectany residual protein remaining on the PDMS surface.

2.3. Electrospray ionization mass spectrometry (ESI-MS)

Electrospray ionization mass spectrometry (ESI-MS) measure-ments were carried out on a QSTAR XL quadrupole time-of-flight(QqTOF) mass spectrometer equipped with an electrospray ionsource (Applied Biosystems, MDS-Sciex, Concorde, ON, Canada).The ESI source was operated at +4000 V and at a flow rateof 5 μL/min. All ESI-MS experiments were carried out at roomtemperature (21 ± 2 ◦C). Protein stock solutions of concentration1 mg/mL were prepared in solution of varying methanol volumeratio of 0 to 0.9. We also determined the pH values of these solu-tions using the method of Canals and co-workers [23]. A detailedtable of the concentration factors used for the pH determinationsmay be found in Table S1 in the supplementary information. Forcarbonic anhydrase, solutions were prepared as above as well asusing 0.1% acetic acid due to issues encountered with precipita-tion. ESI-MS experiments of carbonic anhydrase were done on aZQ single quadrupole instrument (Waters, Milford, MA) with Mass-Lynx processing software. All experiments carried out were at aflow rate of 10 μL/min and an ESI cone voltage of +45 V. Asa protein fragment was observed for carbonic anhydrase, MS/MSexperiments were carried out on the protein fragment using theQSTAR XL QqTOF MS described above.

2.4. Chemical force spectrometric measurements

Chemical force spectrometry was used here to determine theadhesive forces between chemically modified AFM tips and sub-strates. The data were obtained using a PicoSPM (Molecular Imag-ing, Tempe, AZ) and a Nanoscope IIE controller (Digital instru-ments, Santa Barbara, CA). Functionalized tips were prepared byimmersing Au-coated contact mode silicon AFM tips (MikroMasch)in solutions of 10 mmol L−1 1-dodecanethiol, 12-thiohexadecanoicacid or perfluorodecanethiol in ethanol for 24 h to obtain methyl-,carboxylate- and perfluoro-terminated tips. The tip radius asquoted by the manufacturer was <20 nm. Substrates consisted ofself-assembled monolayers (SAMs) of the same three thiol speciesadsorbed on flame-annealed Au(111) thermally deposited on a

92 D. Wang et al. / Journal of Colloid and Interface Science 331 (2009) 90–97

Fig. 1. Signal-to-noise (S/N) ratios from MALDI-TOF MS arising from four proteins remaining on unmodified, oxidized and fluorinated PDMS surfaces following rinsing withmethanol/water mixtures of varying concentrations: (a) cytochrome c; (b) carbonic anhydrase; (c) ubiquitin; (d) insulin. Error bars denote the standard deviation in the S/Ndata from ten points on the sample surface.

mica substrate. X-ray photoelectron spectra (XPS) of these sam-ples were consistent with formation of the three alkanethiol SAMson Au. The probe tip and substrate were immersed in a droplet ofa given methanol/water solution. The adhesive force between tipand substrate was determined from the average of the well depthfrom the retraction portion of 200 force–distance curves. The re-ported values of the adhesive interaction are an average of all theforce curves obtained.

3. Results

MALDI mass spectra for four different proteins – cytochrome c,ubiquitin, carbonic anhydrase and insulin – adsorbed directly onthe unmodified PDMS sample and without subsequent washingwere obtained and may be found in the supplementary informa-tion, Figs. S.1–S.4. They showed only a primary ion peak at theexpected molecular weight of each protein and a secondary peakassociated with the doubly charged ion, indicating that the pro-tein remains intact upon adsorption. In order to determine theeffect of both oxidation and fluorination of the PDMS polymers onprotein desorption, experiments were carried out in which the rel-ative amounts of a specific protein remaining after washing withwater/methanol solution was determined using MALDI-MS. Fig. 1ashows the S/N ratios [24] arising from the cytochrome c proteinremaining on unmodified, oxidized and fluorinated PDMS surfacesfollowing washing with methanol/water mixtures of varying con-centrations. At low methanol concentrations, cytochrome c is read-ily desorbed from all three surfaces. However, at high methanolconcentrations, cytochrome c is not strongly desorbed from thefluorinated surface. This is in contrast to the behavior of the otherthree proteins studied here. In Fig. 1b, which shows the resultsof the same experiment but using carbonic anhydrase, we canobserve that this protein readily desorbs from the hydrophilic ox-idized PDMS. On unmodified and fluorinated PDMS the S/N ratiosare similar to one another and show the opposite trend to cy-tochrome c, with most protein remaining when washed using so-lutions of higher water concentrations. Retention of ubiquitin onthe surface, the results for which are shown in Fig. 1c, shows rel-atively little sensitivity to either the nature of the substrate or the

Fig. 2. Adhesion forces in methanol/water solutions of varying composition betweena gold-coated AFM tip terminated with a self-assembled monolayer of perfluorodo-decanethiol and a Au(111) surface terminated with self-assembled monolayers ofperfluorododecanethiol (CF3), 1-dodecanethiol (CH3), and 16-mercaptohexadecanoicacid (CO2H).

solution composition. Finally, the results using insulin shown inFig. 1d demonstrate that this protein is not readily desorbed fromthe oxidized PDMS surface as compared to fluorinated or unmodi-fied PDMS but, as with ubiquitin, there is not a strong dependenceon the composition of the washing solution.

In order to explore the interaction forces between the perfluo-rinated surface and both the hydrophobic and hydrophilic portionsof proteins, chemical force spectrometric data – measurementsof the adhesion force between and AFM tip and a substrate sur-face – were determined in various methanol/water solutions. Fig. 2shows the adhesion forces observed in water/methanol solutionsbetween an Au-coated AFM terminated with a SAM of perfluorodo-decanethiol and Au(111) surfaces coated with SAMs of perfluorodo-decanethiol, dodecanethiol, and 16-mercaptohexadecanoic acid,which form perfluoro-, methyl-, and carboxylic acid-terminatedsurfaces, respectively. Similar results were obtained when the ter-minal groups on AFM tip and substrate were reversed. In all cases,

D. Wang et al. / Journal of Colloid and Interface Science 331 (2009) 90–97 93

Fig. 3. The upper graph shows the average charge state for the proteins cytochrome c, ubiquitin and carbonic anhydrase (fragment) as determined from electrospray ionizationmass spectrometry in methanol/water solutions of varying composition, but with no other additive. The pH of each solution is also indicated. Under these conditions, no intactcarbonic anhydrase was observed, only a fragment of molecular weight 8.567 kDa. The lower graph shows results from a similar experiment for carbonic anhydrase only, inwhich the water/methanol solutions were prepared with 0.1 volume percent acetic acid, consequently yielding lower pH solutions as indicated. Under these conditions, intactcarbonic anhydrase was observed over a limited methanol concentration range.

Fig. 4. Electrospray ionization spectrographs for (a) cytochrome c, (b) ubiquitin and (c) carbonic anhydrase in methanol/water solutions of varying volume ratios indicated onthe figure. The spectra in (a) and (b) were both obtained with no additive to the methanol/water mixtures, and the proteins were observed intact. For (c), mass spectrographswere obtained in solutions obtained with both no additive (uppermost) in which case only ion signals arising from a fragment of molecular weight 8.567 kDa were observed.The lower spectrographs in (c) were acquired with solutions containing 0.1% volume percent acetic acid and in these cases ion signals arising from both the fragmented andintact protein may be observed.

the adhesive interactions observed were of similar magnitude, be-ing largest in aqueous solution and becoming rapidly smaller withincreasing methanol concentration.

While interactions between protein and surface is one parame-ter that controls the protein adhesion, the folding state of the pro-tein in solution may well have an effect on the thermodynamics orkinetics of its desorption from the surface into the solution phase.ESI data, shown in Figs. 3 and 4, were obtained on the proteinsin order to determine their conformational charges in solutions of

varying methanol concentration. As the solution pH is also a func-tion of methanol concentration, these data are also presented inFig. 3; the pH ranged from 7.0 to 8.8 for most experiments con-ducted here. ESI spectra were not obtained for bovine insulin, asthis protein tends to precipitate and is known to exist as a mixtureof monomer, dimer and hexamer under neutral pH conditions [25].As shown in Fig. 3, the calculated average charge states of the ESImass spectrum of cytochrome c or ubiquitin remains constant insolutions of methanol volume ratio less than 0.5–0.6. Upon in-

94 D. Wang et al. / Journal of Colloid and Interface Science 331 (2009) 90–97

Table 1Calculated hydrophobicity values for the four proteins studies here according to the hydrophobicity scales of Berggren et al. [31,32]. Smaller numbers indicate less hydrophobicspecies.

Protein Hydrophobicity scale

7.1% dextran–6.8% EO30PO70 9% dextran–9% EO30PO70 Octanol/water(kcal/mol)

Cyclohexane/water(kcal/mol)

Cytochrome c 0.0043 0.0126 0.1048 −3.8467Carbonic anhydrase 0.0137 0.0250 0.1137 −3.9304Insulin 0.0551 0.0785 0.5058 −2.2414Ubiquitin 0.0078 0.0164 −0.0088 −4.4037

creasing the volume ratio of methanol, the average charge stateincreases in a step-wise fusion, remaining relatively constant be-yond a methanol volume ratio of 0.7.

Carbonic anhydrase exhibited rather different behavior. In wa-ter/methanol mixtures, only a fragment ion, at molecular weight8.567±0.001 kDa was observed. The upper graph in Fig. 3 demon-strates a 7+ average charge state in methanol concentrations of0.4 volume fraction and lower. At volume fractions higher thanthis, carbonic anhydrase precipitated from solution and ESI spectrawere not obtained. Only on acidifying with 0.1% acetic acid, coulda spectrum of the intact protein be obtained. As can be seen inthe lower graph of Fig. 3, this protein was observed at an averagecharge state of 24+ at methanol volume fractions of 0.2 and lower.The pH conditions here were much lower—pH 3 to 5—than thosefor the methanol/water solutions used in the previous MALDI-MSexperiments, however, indicated in the lower graph of Fig. 3. Thesame fragment ion also appears at all methanol concentrations,with a relative intensity of about 3% for the 0.1 volume fractionmethanol solution while it dominates the spectrum at 0.2 volumefraction methanol.

Fig. 4 shows selected mass spectra used to generate the datasummarized in Fig. 3. For both cytochrome c and ubiquitin inwater/methanol solution of 0.1 methanol volume ratio, Fig. 4ashows a narrow distribution of charge states. There is little differ-ence in the distribution of multiply charged peaks with increasingmethanol volume ratio up to 50%, with significant changes in thedistribution of multiply charged peaks thereafter. A broader distri-bution of peaks, at higher charge states then becomes apparent inboth cases with the grouping at higher charge state dominating bya methanol volume ratio of 0.8 and greater. It should be noted thatubiquitin tends to form a dispersion when first dissolved in solu-tions of 0.7 methanol or greater and then partially precipitates inthese solutions.

The carbonic anhydrase mass spectra are shown in Fig. 4c. Here,selected spectra of the acidified solutions show the presence ofboth the intact protein, with the largest peaks at 22+ and 23+charge states, and the fragment species which is dominated by the8+ ion at low methanol volume fractions and then by the 10+ ionat volume fractions above 0.5. In the non-acidified solutions (noadditive), the fragment ion is still observed, but the charge distri-bution is quite different, with the 6+ and 7+ species dominant atall methanol concentrations studied.

4. Discussion

In two-phase (liquid) organic–water mixtures, the protein sur-face hydrophobicity has been reported to make a significant con-tribution to the partitioning behavior of the protein between theorganic and aqueous phases [26–31]. In our situation, the proteinis partitioned between a solution phase of varying aqueous char-acter and the solid-phase substrate which is either hydrophilic,hydrophobic or fluorophilic. Therefore the protein hydrophobicitymay be one important factor in determining under what condi-tions the protein adsorbs preferentially on the surface. In order tointerpret the results of the MALDI-MS experiments in Fig. 1, we

first consider previous attempts to quantify the hydrophobic char-acter of proteins and apply these to the proteins studied here.

Berggren et al. [32] used four different scales to calculate thehydrophobicity of proteins including bovine serum albumin (BSA),lysozyme, β-lactoglobulin A, myoglobin and cytochrome c. Two ofthe scales are based on the logarithm of the partition constant(log K ) of amino acid residues between an aqueous 7.1% dextran–organic 6.8% ethylene oxide/propylene oxide polymer (EO30PO70)or 9% dextran–9% EO30PO70 solution. The other two scales arebased on the ��G for transfer of the amino acid residue fromoctanol or cyclohexane to water. In order to obtain the surface hy-drophobicity of each protein in aqueous two-phase systems, herewe use Salgado et al.’s [33] method to calculate the surface hy-drophobicity (H) for a given protein,

H =20∑

i=1

rihi . (1)

In Eq. (1), the index i is over all 20 naturally occurring amino acids.h is an experimentally determined hydrophobicity value for eachamino acid residue, based on one of four different aqueous/organicsystems as noted above. The values of h used here are those pre-viously published by Berggren [31], and found in Table S2, in thesupplementary information. The term ri in Eq. (1) is the relativesuperficial surface area of amino acid residue i, given as ri = Si/Swhere Si is the total accessible superficial area of the amino acidresidue i in the protein and S is the sum of the accessible superfi-cial area (ASA) for all the amino acids of type i [34]. We calculatedthe ASA value using the software STRIDE [29] by inputting the pro-tein data base (PDB) file (1HRC for cytochrome c [35], 1V9E forcarbonic anhydrase [36], 2BN3 for insulin [37] and 1V81 for ubiq-uitin [38]) for each protein [39] studied here.

The resulting H values for the four proteins using each ofthe four hydrophobicity scales studied by Berggren [31] are listedin Table 1. These workers have indicated that the best fit forthe correlation between H and log P (a quantitative descriptor oflipophilicity) was obtained using the hydrophobicity scales mea-sured using the dextran/EO30PO70 system, suggesting that this setof calculated H values are the most reliable. In any case, insulinappears to be the most hydrophobic of all four proteins studiedhere, regardless of scale used, while cytochrome c is generallymuch more hydrophilic. It should be noted that the hydrophobic-ity values are calculated based on ASA values for the proteins inaqueous solution. As we shall see below, the solution of highermethanol concentration used in some experiments here may havethe effect of denaturing these proteins, affecting the hydrophobic-ity values. Likewise, adsorption on the surface may also affect thisparameter.

We may first then consider what insight the chemical forcespectrometric results shown in Fig. 2 might have on the partition-ing behavior of the protein between solid and liquid phases. Manygroups, including our own [40,41], have found that the adhesionforces between the same pair of functional groups can be stronglyaffected by media of differing polarity, such as the methanol–watermixtures used here. Previous chemical force spectrometric mea-

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surements on methyl- and perfluoro-terminated SAMs [42] haveshown results similar to those that we see here – the adhesiveinteractions become smaller by orders of magnitude when the po-larity of the solution is decreased. This has been ascribed to theinteraction forces being dominated by intrasolvent polar interac-tions. That is, the tip–sample adhesive interaction is mainly drivenby the fact that to separate the tip and sample, an SAM–solventinterface must be formed and solvent–solvent bonds disrupted. Inthe particular case of the perfluoro- and methyl-terminated tip–sample pairs involving perfluoro or methyl-functionated groups inaqueous solutions this means forming an unfavorable interface be-tween the hydrophobic tip and/or sample and a hydrophilic sol-vent. At the same time, the strong H-bonding aqueous network isdisrupted. Hence, in all three interaction pairs studied here, theadhesive force is much stronger in more aqueous solutions.

This result would suggest that, all else being equal, the adhesiveinteraction between protein and the two more hydrophilic sur-faces would be strongest in solutions of higher water content. TheMALDI-MS data in Fig. 1 – which essentially indicate how muchprotein is left adhered the surface after washing – are not all con-sistent with this. The most consistent set of data is that for one ofthe more hydrophobic proteins, carbonic anhydrase which shows,at least on the perfluorinated and unmodified PDMS, a pattern ofretention at high water content and removal at higher methanolconcentration. The minimal amount of carbonic anhydrase foundremaining on the hydrophilic oxidized PDMS, regardless of washsolution concentration, is also consistent with this relatively hy-drophobic protein failing to retained strongly on a hydrophilic sur-face.

The remaining data in Fig. 1 are not so consistent with thismodel. While the retention of ubiquitin and insulin is relativelyindependent of wash solution composition, cytochrome c shows atrend opposite to that of carbonic anhydrase, with the strongest re-tention occurring at high methanol concentrations. Cytochrome c isthe most hydrophilic of the four proteins studied here, so this sug-gests that its solubility within the liquid phase, and unfolding ofthis protein in solutions of high methanol concentration, may alsohave an important impact on its partitioning between the surfaceand solution. The ESI data provide some insight into this aspect ofthe MALDI results.

Douglas and Konermann [22] found the ESI spectra of cy-tochrome c in solutions of 0.03 and 0.5 methanol are stronglydependent on the pH conditions. The average charge state is 8+at neutral pH and 17+ at acidic pH at 0.03 methanol concentra-tion. By contrast, for 0.5 methanol solutions the average chargestate changes from 8+ to 10+ when going from neutral to acidicconditions. These results thus show that cytochrome c changed itsfolding conformation only at low pH values for methanol concen-trations of less than 0.6. Cytochrome c retains its folding conforma-tion at low methanol concentration, of less than 0.6 volume ratios.At methanol/water volume ratios above 0.6, however, we can seean increasing number of multiple charged peaks showing up in theMS spectra, indicating that cytochrome c is denatured under theseconditions. Even in highest methanol concentrations the averagecharge state was 10+, lower than that observed by Douglas et al.[22], presumably because we remained at the more neutral pH 7to 8 range. It is notable that the methanol concentration at which,according to the ESI spectra, cytochrome c begins to denature areexactly those at which, according to the MALDI-MS measurementin Fig. 1, the protein begins to adhere more strongly to the PDMSsurface. This suggests, that in the case of cytochrome c at least,partitioning between the surface and solution may be stronglycontrolled by the conformational state of the protein in solution.

Bychkova et al. [20] suggested there are two different dena-tured forms of cytochrome c upon the addition of methanol: theintermediate form which is relatively stable under moderate con-

centrations (0.25–0.4) and the “final” form which is stable at highconcentrations (>0.4). They also suggested that the increase of theprotein helicity (such as the helical transition of β-structural or ir-regular chain regions) results in the changes of the conformation ofprotein in alcohol solutions. Our results show two relatively stableforms, one below 0.5 methanol concentration and the other above0.7 methanol. The significantly changed charge distribution of cy-tochrome c at higher methanol concentration presumably wouldbe consistent with the formation of this highly helical state inmethanol.

Turning to the interpretation of the ESI results for ubiquitin, wenote that Daggett and co-workers [43] used NMR to study the con-formation of ubiquitin in 0.6 methanol and found that it shows apartially unfolded state at this stage. This partially unfolded pro-tein can refold when then placed in pure water. Our results showus that ubiquitin showed a higher average charge state when theconcentration of methanol reached 0.6, consistent with unfolding.Upon increasing the concentration of methanol from 0.6 up to 0.9,the ESI spectra show a transition of the average charge state forubiquitin from 5+ to 7+. In this regard, the ubiquitin ESI datastrongly resemble that for cytochrome c, although with lower av-erage charge state. The solution phase conformation does not havea strong affect on the adhesion of ubiquitin to the PDMS surface,although there is an indication in Fig. 1c for a modest increase inretention at higher methanol concentration, at least on unmodifiedPDMS. The adsorption of ubiquitin on the surface may be influ-enced by the dispersion of this protein and its poor solubility inhigh methanol concentration solutions.

Chait and co-workers [16] suggested that in folded ubiquitin,the formation of multiply charged peaks is attributed to the proto-nation of the basic residues Arg-74, Arg-72, Arg-42, Lys-63, Lys-33and Lys-6 of ubiquitin which are exposed to the solvent. His-68and Arg-54 are partially exposed to the solvent, while Lys-48, Lys-29, Lys-27 and Lys-11 are not accessible to the solvents. Once themethanol was added to the aqueous solution, the protein tendsto unfold and exposes parts of these residues to the solvent. Ourresults were consistent with their conclusions. For the folded ubiq-uitin, we can only observe protonation of the six exposed sites,while upon unfolding, the partially exposed sites and even the in-accessible sites were exposed to the solvent, increasing the chargestate.

The MALDI-MS results for carbonic anhydrase showed thegreatest contrast with those for the other proteins, with the great-est adsorption taking place at low methanol concentrations. Wehave already noted that this may be due to the relatively hy-drophobic nature of this protein. The MALDI-MS spectra (supple-mentary information) showed only the presence of a primary ionpeak, so there is no evidence that the protein is in any way hy-drolyzed or otherwise fragmented when adsorbed on the polymersurface. However, the ESI results do demonstrate that fragmenta-tion of the protein takes place in solution under the conditions usedhere. A previous ESI study of carbonic anhydrase [44] reported noevidence of fragmentation, but was carried out in aqueous solu-tions acidified with 0.2% acetic acid, similar to those conditionsin which we also observed intact protein. The fragment observedhere, at 8.567 kDa, was one of many previously observed in a tan-dem mass spectrometry experiment [45], but was not assigned toa peptide fragment in that work.

As the goal was to study the protein’s conformation in solutionconditions equivalent to the wash step prior to MALDI analysis,acidification was undesirable. H/D exchange studies performed byBabu et al. [46] found that when the pH was decreased for pro-tein solutions with varying methanol concentrations, an increasedalpha helix structure was observed for several proteins. Further-more, the increase in alpha helices was more pronounced at highmethanol concentrations of 0.9. The formation of these non-native

96 D. Wang et al. / Journal of Colloid and Interface Science 331 (2009) 90–97

helices resulted in a decrease in H/D exchange to a rate that is sim-ilar to the native protein confirmation, indicative of the protectivenature of acid against protein unfolding.

The increased fragmentation of carbonic anhydrase observed insolutions with higher organic content could be a result of the dis-ruption of hydrogen bonds between amino acid residues in thegiven protein by the organic solvent which disrupts the secondarystructure of the protein and may increase the probability of frag-mentation since the protein is now in a more exposed state. Obser-vations of a higher charge state of the fragment at higher organicconcentrations, with an average charge of 7.4+ at 0.2 methanolcompared to 9.7+ at 0.8 methanol, confirm that the protein is un-folding. The presence of a higher charge state protein envelope foran unfolded protein in comparison to the native protein structurehas been studied by Konermann [47] who performed moleculardynamic simulations to study protein conformation and chargestate during the ESI process. It was concluded that both electro-static and steric effects have an impact on protein conformationand charge state distribution leaving an unfolded protein to havemore sites accessible for protonation. Although protein unfolding isone possible reason for why fragmentation of carbonic anhydraseis observed, the exact reason or mechanism for this observation isunclear at this time.

As the observed protein fragment of carbonic anhydrase has alarge molecular weight of approximately 8566 Da there are severalamino acid sequences that could correspond to the given fragment.We performed an ESI-MS/MS analysis of the given fragment withan m/z of [M + 9H]9+ = 952.3. Although the fragment cannot beconclusively identified, the amino acid sequence of 158–234 anda molecular weight of 8567.4 Da was a possibility since this se-quence provides several peak matches including potentially one yion. At higher molecular weights identification becomes more dif-ficult as common amino acid residues are observed among mostpotential fragments. Further analysis would be required to provideconfirmatory evidence of the identity of the fragment by ruling outother possibilities, however the results of the MS/MS provide someinitial insight into the sequence of the fragment of carbonic anhy-drase.

5. Conclusions

We have examined the elution of the proteins ubiquitin, car-bonic anhydrase, cytochrome c and insulin on the surface of per-fluorinated and oxidized PDMS using MADLI-TOF methods. As per-fluorinated PDMS is a robust material which can be used as thesubstrate in a microfluidic device and is selective to peptides car-rying fluorous affinity tags, it is a good model to study the non-selective retention of proteins onto fluorinated surfaces, and howsuch untagged proteins might interfere with a fluoro-tag-based as-say.

MALDI-MS shows that changing the concentration of the elut-ing methanol water solution has the most pronounced trend onthe adsorption of cytochrome c on the fluorinated PDMS sur-face, and indeed unmodified and oxidized PDMS substrates. Re-tention of cytochrome c increased with increasing concentrationof methanol. ESI-MS results demonstrate that cytochrome c dena-tures at methanol concentrations >60% due to the increases in theprotein helicity. This change appears to influence the adsorption ofprotein on the fluorinated PDMS surface since more hydrophobicresidue sites become available to interact with the surface and itprefers to adhere rather than dissolve in the solvent.

For ubiquitin, the MALDI- MS results show a relatively sta-ble adsorption profile of this protein on fluorinated PDMS as afunction of methanol concentration. ESI-MS results show that theubiquitin denatured in the water/methanol solutions only at con-centrations >70%. A pronounced difference between ubiquitin and

cytochrome c was that ubiquitin partially precipitated at highermethanol concentrations. The relatively stable adsorption profilemay then be due to partially precipitated protein prefers being dis-persed by the wash solution.

Carbonic anhydrase MALDI-MS results show that this com-pound adheres most effectively to fluorinated and unmodifiedPDMS when washed with solutions of high aqueous concentration.It does not adhere strongly to oxidized PDMS under any conditions.As one of the most hydrophobic of the four proteins under study, itappears that hydrophobic interactions between substrate and pro-tein dominate the adsorption profile. In solution, ESI experimentssuggest that carbonic anhydrase denatures in methanolic solutionsto a greater extent than the other proteins under study.

Ultimately, whether a globular protein is retained on the sur-face following elution is due to a complex balance between thestrength of the interaction of the protein with the surface and itssolubility conformation in the wash solution. The proteins studiedhere show a wide range of adsorption behavior and demonstratethat in any separation of a fluoro-tagged protein or peptide, caremust be taken to account for non-specific adsorption.

Acknowledgments

Financial support from the Natural Sciences and EngineeringResearch Council (NSERC) of Canada, and the Canadian Founda-tion for Innovation (CFI) is gratefully acknowledged. We thank Dr.Yimin She for help with some experiments and Dr. Berndt Kellerfor helpful discussions.

Supplementary information

Supporting information available: MALDI-TOF mass spectra, ta-bles of hydrophobicity constants for various amino acids and cor-rection factors for pH in solutions of varying methanol concentra-tion.

Please visit DOI: 10.1016/j.jcis.2008.11.010.

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