The adsorption of globular proteins onto a fluorinated PDMS surface

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

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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 2008 Accepted 5 November 2008 Available online 8 November 2008 Keywords: MALDI ESR Fluorinated polymers Fluoro-tagged proteins Surfaces

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 of fluorous-tagged peptides, demonstrating that this material may be used in fluorous affinity tag technology to enrich and separate specific proteins or peptides from complex mixtures. Here, we explore the nonspecific 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 fluorinated PDMS in solutions of varying water/methanol concentration has been studied using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The interaction forces involving perfluorinated surfaces are probed using chemical force spectrometry. The denaturation of the proteins in 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 each protein. © 2008 Elsevier Inc. All rights reserved.

1. Introduction Since the term fluorous – “of, relating to, or having the characteristics of highly fluorinated saturated organic materials, molecules or molecular fragments” – was introduced by Horváth in the early 1990s, there have been extensive developments in the field of fluorous chemistry. Recently, fluorous tags have been used in synthetic applications to isolate the desired components from a reaction mixture, taking advantage of fluorophilic interactions. For example, by using solid–liquid extractions over fluorous reverse-phase silica gel, Curran and Luo [1] achieved good separation of fluorous amide products from a mixture eluted with methanol/water solvent solutions. In the field of proteomics, an approach to separating target proteins or peptides from complex mixtures using fluorous chemistry has been recently developed by Brittain and co-workers [2]. They have used fluorous affinity tag technology to enrich and separate specific proteins or peptides from complex mixtures, using mass spectrometry techniques to characterize these fluorine tagged species. They [3] have also demonstrated desorption ionization on silicon (DIOS) using fluorous-silylated materials as affinity surfaces to enrich fluorous-tagged analytes using mass spectrometric methods to test these species. We have recently reported on a similar scheme using polydimethylsiloxane (PDMS) as the substrate [4]. PDMS was modified by grafting per-

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fluorooctyltriethoxysilane via hydrolysis onto an oxidized surface and we used matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF-MS) to test the adsorption of fluorous-tagged peptides onto this fluorinated PDMS surface. Our results demonstrated that the fluorinated PDMS surface could be used for enrichment or to enhance detection of fluorous-labeled peptides, while at the same time maintaining a large zeta potential at the surface. This latter property would also allow these materials to be used in micro total analysis systems where a large and stable zeta potential is required to maintain electrophoretic mobility. One potential drawback to using any polymer substrate is that many proteins may tend to adsorb onto the surface due to its high hydrophobicity [5,6]. Our previous results [4] showed that the fluorinated PDMS surface is more hydrophobic than the unmodified PDMS surface. As untagged proteins may potentially interact strongly with the fluorinated PDMS surface, these non-specific interactions could lead to “false positives” for the presence of these species or interfere with the adsorption of fluorinated species from solution. The goal of this paper is to assess the importance and extent of non-specific protein adsorption in these systems. To do this, we report on a study of the solid–liquid extraction of some common proteins on unmodified, oxidized and fluorinated PDMS using a combination of mass spectrometric methods and chemical force spectrometry. Solid–liquid extraction from surfaces containing similar functional groups to some of the modified PDMS materials studied here have been previously carried out. For example, Mengistu et al. [7]

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have studied the adsorption of proteins including α -casein, carbonic anhydrase, α -lactalbumin, bovine serum albumin, ubiquitin, cytochrome c, insulin and myoglobin onto methyl- and carboxylterminated porous Si surfaces. Their results showed that the proteins tend to adsorb preferentially on porous Si surfaces rather than flat surfaces, due to the increased surface area. They also found that varying the pH of the rinse solution will influence the adsorption of proteins on functionalized surfaces. However, the functional groups present on the porous Si also had a strong influence on the protein–surface interactions. Karlsson et al. [8] used three engineered variants of human carbonic anhydrase II to study the influence of protein stability on the adsorption and desorption behavior to four different surfaces (negatively charged, hydrophilic, hydrophobic, and positively charged) by using surface plasmon resonance measurements. Their results indicated that controlling the conformational stability of the protein is an important parameter in the adsorption and desorption behavior at a liquid–solid interface. Krishnan and co-workers [9] investigated nine globular blood proteins onto methyl-terminated surfaces in aqueous–buffer solution. They demonstrated that the adsorption of proteins onto hydrophobic surfaces were mainly influenced by interfacial water and were not strongly dependent on protein type. Conformational changes of proteins in organic solutions, particularly alcohols [10], have been studied by a variety of physical techniques such as fluorescence, circular dichroism and nuclear magnetic resonance [11,12]. The use of electrospray ionization mass spectrometry (ESI-MS) [13,14] to monitor the conformation changes of proteins was first described by Chowdhury and coworkers [15]. This technique has been proven to be very useful in the study of conformational changes of different proteins upon changing pH, temperature, and the presence of denaturing agents such as organic solvents [15–22]. In the work described here, we study the desorption of cytochrome c, carbonic anhydrase, insulin and ubiquitin from unmodified, oxidized and fluorinated PDMS surfaces. Here we chose methanol/water solutions of varying compositions as the liquid phase for the extraction of proteins; the original reports of fluorotagged species using DIOS techniques were eluted with such mixtures. We use the signal-to-noise ratio of the primary ion in the MALDI-MS spectrum to compare the relative adsorption of proteins on the surface after washing with different volume ratios of methanol/water solutions. These data are discussed in the context of the interaction forces observed involving perfluorinated surfaces using chemical force spectrometry, and the denaturation of the proteins in solutions of high methanol concentration, as determined by electrospray ionization mass spectrometry. In addition, we calculated the surface hydrophobicity of each protein and use this value to interpret the MALDI-MS experimental results.

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2.2. Mass spectrometric measurements Mass spectrometric measurements were carried out using a Voyager DE-STR MALDI-TOF system (Applied Biosystems, Foster City, CA). Accelerating potentials of 20 kV were used. Spectra were obtained using a nitrogen laser (337 nm) with the fluence adjusted slightly above threshold. The proteins studied here were carbonic anhydrase (Sigma-Aldrich, bovine erythrocytes, C3934), cytochrome c (Sigma-Aldrich, from horse heart, C2506), ubiquitin (Sigma-Aldrich, bovine red blood cells, U6253) and insulin (SigmaAldrich, bovine pancreas, I6634). Aqueous solutions of 1 mg/mL of each protein were prepared. The final solution pH was 4.5, 8.8, 6.5 and 6.9, respectively. The PDMS substrates were attached onto each spot of the MALDI sample plate directly. The backside (unmodified) of the PDMS samples adhere effectively to the surface of 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 was deposited onto the variously modified PDMS substrate surfaces, allowed to dry, then washed with a 1 mL aliquot of varying concentration 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 saturated in 60% acetonitrile water solution with 0.3% trifluoroacetic acid) on the washed regions. MALDI-MS was then used to detect any residual protein remaining on the PDMS surface. 2.3. Electrospray ionization mass spectrometry (ESI-MS)

2. Experimental

Electrospray ionization mass spectrometry (ESI-MS) measurements were carried out on a QSTAR XL quadrupole time-of-flight (QqTOF) mass spectrometer equipped with an electrospray ion source (Applied Biosystems, MDS-Sciex, Concorde, ON, Canada). The ESI source was operated at +4000 V and at a flow rate of 5 μL/min. All ESI-MS experiments were carried out at room temperature (21 ± 2 ◦ C). Protein stock solutions of concentration 1 mg/mL were prepared in solution of varying methanol volume ratio of 0 to 0.9. We also determined the pH values of these solutions using the method of Canals and co-workers [23]. A detailed table of the concentration factors used for the pH determinations may be found in Table S1 in the supplementary information. For carbonic anhydrase, solutions were prepared as above as well as using 0.1% acetic acid due to issues encountered with precipitation. ESI-MS experiments of carbonic anhydrase were done on a ZQ single quadrupole instrument (Waters, Milford, MA) with MassLynx processing software. All experiments carried out were at a flow rate of 10 μL/min and an ESI cone voltage of +45 V. As a protein fragment was observed for carbonic anhydrase, MS/MS experiments were carried out on the protein fragment using the QSTAR XL QqTOF MS described above.

2.1. PDMS substrates and surface modification

2.4. Chemical force spectrometric measurements

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

Chemical force spectrometry was used here to determine the adhesive forces between chemically modified AFM tips and substrates. The data were obtained using a PicoSPM (Molecular Imaging, Tempe, AZ) and a Nanoscope IIE controller (Digital instruments, Santa Barbara, CA). Functionalized tips were prepared by immersing Au-coated contact mode silicon AFM tips (MikroMasch) in solutions of 10 mmol L−1 1-dodecanethiol, 12-thiohexadecanoic acid or perfluorodecanethiol in ethanol for 24 h to obtain methyl-, carboxylate- and perfluoro-terminated tips. The tip radius as quoted by the manufacturer was <20 nm. Substrates consisted of self-assembled monolayers (SAMs) of the same three thiol species adsorbed on flame-annealed Au(111) thermally deposited on a

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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 with methanol/water mixtures of varying concentrations: (a) cytochrome c; (b) carbonic anhydrase; (c) ubiquitin; (d) insulin. Error bars denote the standard deviation in the S/N data from ten points on the sample surface.

mica substrate. X-ray photoelectron spectra (XPS) of these samples were consistent with formation of the three alkanethiol SAMs on Au. The probe tip and substrate were immersed in a droplet of a given methanol/water solution. The adhesive force between tip and substrate was determined from the average of the well depth from the retraction portion of 200 force–distance curves. The reported values of the adhesive interaction are an average of all the force curves obtained. 3. Results MALDI mass spectra for four different proteins – cytochrome c, ubiquitin, carbonic anhydrase and insulin – adsorbed directly on the unmodified PDMS sample and without subsequent washing were obtained and may be found in the supplementary information, Figs. S.1–S.4. They showed only a primary ion peak at the expected molecular weight of each protein and a secondary peak associated with the doubly charged ion, indicating that the protein remains intact upon adsorption. In order to determine the effect of both oxidation and fluorination of the PDMS polymers on protein desorption, experiments were carried out in which the relative amounts of a specific protein remaining after washing with water/methanol solution was determined using MALDI-MS. Fig. 1a shows the S/N ratios [24] arising from the cytochrome c protein remaining on unmodified, oxidized and fluorinated PDMS surfaces following washing with methanol/water mixtures of varying concentrations. At low methanol concentrations, cytochrome c is readily desorbed from all three surfaces. However, at high methanol concentrations, cytochrome c is not strongly desorbed from the fluorinated surface. This is in contrast to the behavior of the other three proteins studied here. In Fig. 1b, which shows the results of the same experiment but using carbonic anhydrase, we can observe that this protein readily desorbs from the hydrophilic oxidized PDMS. On unmodified and fluorinated PDMS the S/N ratios are similar to one another and show the opposite trend to cytochrome c, with most protein remaining when washed using solutions of higher water concentrations. Retention of ubiquitin on the surface, the results for which are shown in Fig. 1c, shows relatively little sensitivity to either the nature of the substrate or the

Fig. 2. Adhesion forces in methanol/water solutions of varying composition between a gold-coated AFM tip terminated with a self-assembled monolayer of perfluorododecanethiol and a Au(111) surface terminated with self-assembled monolayers of perfluorododecanethiol (CF3 ), 1-dodecanethiol (CH3 ), and 16-mercaptohexadecanoic acid (CO2 H).

solution composition. Finally, the results using insulin shown in Fig. 1d demonstrate that this protein is not readily desorbed from the oxidized PDMS surface as compared to fluorinated or unmodified PDMS but, as with ubiquitin, there is not a strong dependence on the composition of the washing solution. In order to explore the interaction forces between the perfluorinated surface and both the hydrophobic and hydrophilic portions of proteins, chemical force spectrometric data – measurements of the adhesion force between and AFM tip and a substrate surface – were determined in various methanol/water solutions. Fig. 2 shows the adhesion forces observed in water/methanol solutions between an Au-coated AFM terminated with a SAM of perfluorododecanethiol and Au(111) surfaces coated with SAMs of perfluorododecanethiol, dodecanethiol, and 16-mercaptohexadecanoic acid, which form perfluoro-, methyl-, and carboxylic acid-terminated surfaces, respectively. Similar results were obtained when the terminal groups on AFM tip and substrate were reversed. In all cases,

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Fig. 3. The upper graph shows the average charge state for the proteins cytochrome c, ubiquitin and carbonic anhydrase (fragment) as determined from electrospray ionization mass 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 intact carbonic 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, in which the water/methanol solutions were prepared with 0.1 volume percent acetic acid, consequently yielding lower pH solutions as indicated. Under these conditions, intact carbonic 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 on the 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 spectrographs were 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 and intact protein may be observed.

the adhesive interactions observed were of similar magnitude, being largest in aqueous solution and becoming rapidly smaller with increasing methanol concentration. While interactions between protein and surface is one parameter that controls the protein adhesion, the folding state of the protein in solution may well have an effect on the thermodynamics or kinetics of its desorption from the surface into the solution phase. ESI data, shown in Figs. 3 and 4, were obtained on the proteins in order to determine their conformational charges in solutions of

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

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

Cytochrome c Carbonic anhydrase Insulin Ubiquitin

Hydrophobicity scale 7.1% dextran–6.8% EO30PO70

9% dextran–9% EO30PO70

Octanol/water (kcal/mol)

Cyclohexane/water (kcal/mol)

0.0043 0.0137 0.0551 0.0078

0.0126 0.0250 0.0785 0.0164

0.1048 0.1137 0.5058 −0.0088

−3.8467 −3.9304 −2.2414 −4.4037

creasing the volume ratio of methanol, the average charge state increases in a step-wise fusion, remaining relatively constant beyond a methanol volume ratio of 0.7. Carbonic anhydrase exhibited rather different behavior. In water/methanol mixtures, only a fragment ion, at molecular weight 8.567 ± 0.001 kDa was observed. The upper graph in Fig. 3 demonstrates a 7+ average charge state in methanol concentrations of 0.4 volume fraction and lower. At volume fractions higher than this, carbonic anhydrase precipitated from solution and ESI spectra were not obtained. Only on acidifying with 0.1% acetic acid, could a spectrum of the intact protein be obtained. As can be seen in the lower graph of Fig. 3, this protein was observed at an average charge state of 24+ at methanol volume fractions of 0.2 and lower. The pH conditions here were much lower—pH 3 to 5—than those for the methanol/water solutions used in the previous MALDI-MS experiments, however, indicated in the lower graph of Fig. 3. The same fragment ion also appears at all methanol concentrations, with a relative intensity of about 3% for the 0.1 volume fraction methanol solution while it dominates the spectrum at 0.2 volume fraction methanol. Fig. 4 shows selected mass spectra used to generate the data summarized in Fig. 3. For both cytochrome c and ubiquitin in water/methanol solution of 0.1 methanol volume ratio, Fig. 4a shows a narrow distribution of charge states. There is little difference in the distribution of multiply charged peaks with increasing methanol volume ratio up to 50%, with significant changes in the distribution of multiply charged peaks thereafter. A broader distribution of peaks, at higher charge states then becomes apparent in both cases with the grouping at higher charge state dominating by a methanol volume ratio of 0.8 and greater. It should be noted that ubiquitin tends to form a dispersion when first dissolved in solutions of 0.7 methanol or greater and then partially precipitates in these solutions. The carbonic anhydrase mass spectra are shown in Fig. 4c. Here, selected spectra of the acidified solutions show the presence of both the intact protein, with the largest peaks at 22+ and 23+ charge states, and the fragment species which is dominated by the 8+ ion at low methanol volume fractions and then by the 10+ ion at volume fractions above 0.5. In the non-acidified solutions (no additive), the fragment ion is still observed, but the charge distribution is quite different, with the 6+ and 7+ species dominant at all methanol concentrations studied. 4. Discussion In two-phase (liquid) organic–water mixtures, the protein surface hydrophobicity has been reported to make a significant contribution to the partitioning behavior of the protein between the organic and aqueous phases [26–31]. In our situation, the protein is partitioned between a solution phase of varying aqueous character and the solid-phase substrate which is either hydrophilic, hydrophobic or fluorophilic. Therefore the protein hydrophobicity may be one important factor in determining under what conditions the protein adsorbs preferentially on the surface. In order to interpret the results of the MALDI-MS experiments in Fig. 1, we

first consider previous attempts to quantify the hydrophobic character of proteins and apply these to the proteins studied here. Berggren et al. [32] used four different scales to calculate the hydrophobicity of proteins including bovine serum albumin (BSA), lysozyme, β -lactoglobulin A, myoglobin and cytochrome c. Two of the 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 (EO30 PO70 ) or 9% dextran–9% EO30 PO70 solution. The other two scales are based on the G for transfer of the amino acid residue from octanol or cyclohexane to water. In order to obtain the surface hydrophobicity of each protein in aqueous two-phase systems, here we use Salgado et al.’s [33] method to calculate the surface hydrophobicity (H ) for a given protein, H=

20 

ri hi .

(1)

i =1

In Eq. (1), the index i is over all 20 naturally occurring amino acids. h is an experimentally determined hydrophobicity value for each amino acid residue, based on one of four different aqueous/organic systems as noted above. The values of h used here are those previously published by Berggren [31], and found in Table S2, in the supplementary information. The term r i in Eq. (1) is the relative superficial surface area of amino acid residue i, given as r i = S i / S where S i is the total accessible superficial area of the amino acid residue i in the protein and S is the sum of the accessible superficial area (ASA) for all the amino acids of type i [34]. We calculated the ASA value using the software STRIDE [29] by inputting the protein data base (PDB) file (1HRC for cytochrome c [35], 1V9E for carbonic anhydrase [36], 2BN3 for insulin [37] and 1V81 for ubiquitin [38]) for each protein [39] studied here. The resulting H values for the four proteins using each of the four hydrophobicity scales studied by Berggren [31] are listed in Table 1. These workers have indicated that the best fit for the correlation between H and log P (a quantitative descriptor of lipophilicity) was obtained using the hydrophobicity scales measured using the dextran/EO30 PO70 system, suggesting that this set of calculated H values are the most reliable. In any case, insulin appears to be the most hydrophobic of all four proteins studied here, regardless of scale used, while cytochrome c is generally much more hydrophilic. It should be noted that the hydrophobicity values are calculated based on ASA values for the proteins in aqueous solution. As we shall see below, the solution of higher methanol concentration used in some experiments here may have the effect of denaturing these proteins, affecting the hydrophobicity values. Likewise, adsorption on the surface may also affect this parameter. We may first then consider what insight the chemical force spectrometric results shown in Fig. 2 might have on the partitioning behavior of the protein between solid and liquid phases. Many groups, including our own [40,41], have found that the adhesion forces between the same pair of functional groups can be strongly affected by media of differing polarity, such as the methanol–water mixtures used here. Previous chemical force spectrometric mea-

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surements on methyl- and perfluoro-terminated SAMs [42] have shown results similar to those that we see here – the adhesive interactions become smaller by orders of magnitude when the polarity of the solution is decreased. This has been ascribed to the interaction forces being dominated by intrasolvent polar interactions. That is, the tip–sample adhesive interaction is mainly driven by the fact that to separate the tip and sample, an SAM–solvent interface must be formed and solvent–solvent bonds disrupted. In the particular case of the perfluoro- and methyl-terminated tip– sample pairs involving perfluoro or methyl-functionated groups in aqueous solutions this means forming an unfavorable interface between the hydrophobic tip and/or sample and a hydrophilic solvent. At the same time, the strong H-bonding aqueous network is disrupted. Hence, in all three interaction pairs studied here, the adhesive force is much stronger in more aqueous solutions. This result would suggest that, all else being equal, the adhesive interaction between protein and the two more hydrophilic surfaces would be strongest in solutions of higher water content. The MALDI-MS data in Fig. 1 – which essentially indicate how much protein is left adhered the surface after washing – are not all consistent with this. The most consistent set of data is that for one of the more hydrophobic proteins, carbonic anhydrase which shows, at least on the perfluorinated and unmodified PDMS, a pattern of retention at high water content and removal at higher methanol concentration. The minimal amount of carbonic anhydrase found remaining on the hydrophilic oxidized PDMS, regardless of wash solution concentration, is also consistent with this relatively hydrophobic protein failing to retained strongly on a hydrophilic surface. The remaining data in Fig. 1 are not so consistent with this model. While the retention of ubiquitin and insulin is relatively independent of wash solution composition, cytochrome c shows a trend opposite to that of carbonic anhydrase, with the strongest retention occurring at high methanol concentrations. Cytochrome c is the most hydrophilic of the four proteins studied here, so this suggests that its solubility within the liquid phase, and unfolding of this protein in solutions of high methanol concentration, may also have an important impact on its partitioning between the surface and solution. The ESI data provide some insight into this aspect of the MALDI results. Douglas and Konermann [22] found the ESI spectra of cytochrome c in solutions of 0.03 and 0.5 methanol are strongly dependent on the pH conditions. The average charge state is 8+ at neutral pH and 17+ at acidic pH at 0.03 methanol concentration. By contrast, for 0.5 methanol solutions the average charge state changes from 8+ to 10+ when going from neutral to acidic conditions. These results thus show that cytochrome c changed its folding conformation only at low pH values for methanol concentrations of less than 0.6. Cytochrome c retains its folding conformation at low methanol concentration, of less than 0.6 volume ratios. At methanol/water volume ratios above 0.6, however, we can see an increasing number of multiple charged peaks showing up in the MS spectra, indicating that cytochrome c is denatured under these conditions. Even in highest methanol concentrations the average charge state was 10+ , lower than that observed by Douglas et al. [22], presumably because we remained at the more neutral pH 7 to 8 range. It is notable that the methanol concentration at which, according to the ESI spectra, cytochrome c begins to denature are exactly those at which, according to the MALDI-MS measurement in Fig. 1, the protein begins to adhere more strongly to the PDMS surface. This suggests, that in the case of cytochrome c at least, partitioning between the surface and solution may be strongly controlled by the conformational state of the protein in solution. Bychkova et al. [20] suggested there are two different denatured forms of cytochrome c upon the addition of methanol: the intermediate form which is relatively stable under moderate con-

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centrations (0.25–0.4) and the “final” form which is stable at high concentrations (>0.4). They also suggested that the increase of the protein helicity (such as the helical transition of β -structural or irregular chain regions) results in the changes of the conformation of protein in alcohol solutions. Our results show two relatively stable forms, one below 0.5 methanol concentration and the other above 0.7 methanol. The significantly changed charge distribution of cytochrome c at higher methanol concentration presumably would be consistent with the formation of this highly helical state in methanol. Turning to the interpretation of the ESI results for ubiquitin, we note that Daggett and co-workers [43] used NMR to study the conformation of ubiquitin in 0.6 methanol and found that it shows a partially unfolded state at this stage. This partially unfolded protein can refold when then placed in pure water. Our results show us that ubiquitin showed a higher average charge state when the concentration 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 for ubiquitin from 5+ to 7+ . In this regard, the ubiquitin ESI data strongly resemble that for cytochrome c, although with lower average charge state. The solution phase conformation does not have a strong affect on the adhesion of ubiquitin to the PDMS surface, although there is an indication in Fig. 1c for a modest increase in retention at higher methanol concentration, at least on unmodified PDMS. The adsorption of ubiquitin on the surface may be influenced by the dispersion of this protein and its poor solubility in high methanol concentration solutions. Chait and co-workers [16] suggested that in folded ubiquitin, the formation of multiply charged peaks is attributed to the protonation of the basic residues Arg-74, Arg-72, Arg-42, Lys-63, Lys-33 and Lys-6 of ubiquitin which are exposed to the solvent. His-68 and Arg-54 are partially exposed to the solvent, while Lys-48, Lys29, Lys-27 and Lys-11 are not accessible to the solvents. Once the methanol was added to the aqueous solution, the protein tends to unfold and exposes parts of these residues to the solvent. Our results were consistent with their conclusions. For the folded ubiquitin, we can only observe protonation of the six exposed sites, while upon unfolding, the partially exposed sites and even the inaccessible sites were exposed to the solvent, increasing the charge state. The MALDI-MS results for carbonic anhydrase showed the greatest contrast with those for the other proteins, with the greatest adsorption taking place at low methanol concentrations. We have already noted that this may be due to the relatively hydrophobic nature of this protein. The MALDI-MS spectra (supplementary information) showed only the presence of a primary ion peak, so there is no evidence that the protein is in any way hydrolyzed or otherwise fragmented when adsorbed on the polymer surface. However, the ESI results do demonstrate that fragmentation of the protein takes place in solution under the conditions used here. A previous ESI study of carbonic anhydrase [44] reported no evidence of fragmentation, but was carried out in aqueous solutions acidified with 0.2% acetic acid, similar to those conditions in which we also observed intact protein. The fragment observed here, at 8.567 kDa, was one of many previously observed in a tandem mass spectrometry experiment [45], but was not assigned to a peptide fragment in that work. As the goal was to study the protein’s conformation in solution conditions equivalent to the wash step prior to MALDI analysis, acidification was undesirable. H/D exchange studies performed by Babu et al. [46] found that when the pH was decreased for protein solutions with varying methanol concentrations, an increased alpha helix structure was observed for several proteins. Furthermore, the increase in alpha helices was more pronounced at high methanol concentrations of 0.9. The formation of these non-native

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helices resulted in a decrease in H/D exchange to a rate that is similar to the native protein confirmation, indicative of the protective nature of acid against protein unfolding. The increased fragmentation of carbonic anhydrase observed in solutions with higher organic content could be a result of the disruption of hydrogen bonds between amino acid residues in the given protein by the organic solvent which disrupts the secondary structure of the protein and may increase the probability of fragmentation since the protein is now in a more exposed state. Observations of a higher charge state of the fragment at higher organic concentrations, with an average charge of 7.4+ at 0.2 methanol compared to 9.7+ at 0.8 methanol, confirm that the protein is unfolding. The presence of a higher charge state protein envelope for an unfolded protein in comparison to the native protein structure has been studied by Konermann [47] who performed molecular dynamic simulations to study protein conformation and charge state during the ESI process. It was concluded that both electrostatic and steric effects have an impact on protein conformation and charge state distribution leaving an unfolded protein to have more sites accessible for protonation. Although protein unfolding is one possible reason for why fragmentation of carbonic anhydrase is observed, the exact reason or mechanism for this observation is unclear at this time. As the observed protein fragment of carbonic anhydrase has a large molecular weight of approximately 8566 Da there are several amino acid sequences that could correspond to the given fragment. We performed an ESI-MS/MS analysis of the given fragment with an m/ z of [M + 9H]9+ = 952.3. Although the fragment cannot be conclusively identified, the amino acid sequence of 158–234 and a molecular weight of 8567.4 Da was a possibility since this sequence provides several peak matches including potentially one y ion. At higher molecular weights identification becomes more difficult as common amino acid residues are observed among most potential fragments. Further analysis would be required to provide confirmatory evidence of the identity of the fragment by ruling out other possibilities, however the results of the MS/MS provide some initial insight into the sequence of the fragment of carbonic anhydrase.

cytochrome c was that ubiquitin partially precipitated at higher methanol concentrations. The relatively stable adsorption profile may then be due to partially precipitated protein prefers being dispersed by the wash solution. Carbonic anhydrase MALDI-MS results show that this compound adheres most effectively to fluorinated and unmodified PDMS 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, it appears that hydrophobic interactions between substrate and protein dominate the adsorption profile. In solution, ESI experiments suggest that carbonic anhydrase denatures in methanolic solutions to a greater extent than the other proteins under study. Ultimately, whether a globular protein is retained on the surface following elution is due to a complex balance between the strength of the interaction of the protein with the surface and its solubility conformation in the wash solution. The proteins studied here show a wide range of adsorption behavior and demonstrate that in any separation of a fluoro-tagged protein or peptide, care must be taken to account for non-specific adsorption.

5. Conclusions

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We have examined the elution of the proteins ubiquitin, carbonic anhydrase, cytochrome c and insulin on the surface of perfluorinated and oxidized PDMS using MADLI-TOF methods. As perfluorinated PDMS is a robust material which can be used as the substrate in a microfluidic device and is selective to peptides carrying fluorous affinity tags, it is a good model to study the nonselective retention of proteins onto fluorinated surfaces, and how such untagged proteins might interfere with a fluoro-tag-based assay. MALDI-MS shows that changing the concentration of the eluting methanol water solution has the most pronounced trend on the adsorption of cytochrome c on the fluorinated PDMS surface, and indeed unmodified and oxidized PDMS substrates. Retention of cytochrome c increased with increasing concentration of methanol. ESI-MS results demonstrate that cytochrome c denatures at methanol concentrations >60% due to the increases in the protein helicity. This change appears to influence the adsorption of protein on the fluorinated PDMS surface since more hydrophobic residue sites become available to interact with the surface and it prefers to adhere rather than dissolve in the solvent. For ubiquitin, the MALDI- MS results show a relatively stable adsorption profile of this protein on fluorinated PDMS as a function of methanol concentration. ESI-MS results show that the ubiquitin denatured in the water/methanol solutions only at concentrations >70%. A pronounced difference between ubiquitin and

Acknowledgments Financial support from the Natural Sciences and Engineering Research Council (NSERC) of Canada, and the Canadian Foundation for Innovation (CFI) is gratefully acknowledged. We thank Dr. Yimin She for help with some experiments and Dr. Berndt Keller for helpful discussions. Supplementary information Supporting information available: MALDI-TOF mass spectra, tables of hydrophobicity constants for various amino acids and correction factors for pH in solutions of varying methanol concentration. Please visit DOI: 10.1016/j.jcis.2008.11.010. References

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