The Use of Size Exclusion Chromatography to Monitor Protein Self-Assembly

crystals

Article The Use of Size Exclusion Chromatography to Monitor Protein Self-Assembly

Alaa Adawy † and Matthew R. Groves *

Department of Drug Design, Structural Biology Unit, Groningen Research Institute of Pharmacy, University of Groningen, 9713AJ Groningen, The Netherlands; [email protected] * Correspondence: [email protected]; Tel.: +31-50-36-33305 † Current address: HRTEM laboratory, Scientiﬁc and Technical Services, University of Oviedo-CINN, 33006 Oviedo, Spain.

Academic Editor: Albert Guskov Received: 6 September 2017; Accepted: 23 October 2017; Published: 31 October 2017

Abstract: High resolution size exclusion chromatography (SEC) coupled with static light scattering (SLS) analyses were conducted to study the effect of the mobile phase ionic strength and protein concentration on the output of SEC experiments. The results highlight the effect of small changes in the mobile phase composition on the estimation of molar masses estimated from retention time-based calibration curve compared with those obtained from SLS analysis. By comparing the SLS data with the SEC chromatograms, we show that SEC can provide helpful information on the protein aggregation state as macromolecules approach known precipitation points in their phase diagrams. This suggests the potential use of SEC as an easily accessible lab-based scanning methodology to monitor protein self-assembly prior to nucleation and crystallization. Implications for the use of SEC to study protein phase diagrams are discussed.

Keywords: size exclusion chromatography; static light scattering; protein aggregation; protein nucleation

1. Introduction There is no structural biology laboratory that can be functional without a chromatography system which is used for the purposes of purifying macromolecules [1–4]. One of the major purification methods used is size exclusion chromatography (SEC) which is based on the shape and size (hydrodynamic radius; Rh) of the eluted macromolecules, such that during a SEC experiment larger macromolecules (larger hydrodynamic radius) are eluted faster than the small ones which are retarded within the stationary hydrophilic resins [5]. Common wisdom is to calibrate gel filtration columns using a standard mobile phase (such as phosphate buffered saline) and use several standard proteins of known molar masses in order to create a calibration diagram (Log molar mass vs. elution time/volume) [6,7]. These diagrams are then used to retrieve information about macromolecules under investigation; including estimates of their molar masses, degree of oligomerization, and stability [8]. However, many factors other than the Rh and molar mass of macromolecules can affect the elution time. Solution pH and the ionic strength of the mobile phase can greatly affect the time at which macromolecules are eluted [9–13]. While it has long been known that solution properties can influence the elution time of a macromolecule under SEC, it unfortunately remains commonplace not to account for these solution properties before consulting a calibration curve—with many laboratories performing a single calibration curve that is applied to all subsequent buffers used. This can result in errors in estimated molar masses that may lead to the incorrect assignment of oligomeric state or other macromolecular solution properties [14,15]. This can be overcome through the use of static light scattering (SLS) measurements in-line with SEC experiments that can provide a direct measurement of

Crystals 2017, 7, 331; doi:10.3390/cryst7110331 www.mdpi.com/journal/crystals Crystals 2017, 7, 331 2 of 10 solution molecular mass, without recourse to a calibration curve [16]. In addition to the molar mass, SLS analysis also provides important information on the behavior of a macromolecule in solution. As macromolecules typically have well predictable molecular weights, deviations from quanta of these molecular weights can be interpreted as information on the aggregation state of the molecule [17]. Previous researchers have used static light scattering (SLS) measurements to assess deviations from a known molecular mass and have interpreted the results as an estimation of the second virial coefﬁcient (B2 or A22), which is a concentration independent term in the Taylor expansion approximation of the universal gas equation as applied to molecules in solution (Equation (1)) [18]

PV = nRT + c[A22] (1) where the pressure (P) and volume (V) of a solution are related to the number density of molecules (n), the universal gas constant (R), thermodynamic temperature (T), the macromolecule concentration (c), and the second virial coefficient (A22). For protein solutions, there is a correlation between the value of their osmotic second virial coefficient with solution conditions conducive to crystallization. The osmotic second virial coefficient, B22, is defined by the osmotic virial expansion (Equation 2) [19]

Π = R T cp (1/Mw + B22 cp + ...... ) (2) where Π is the osmotic pressure, cp the protein concentration (in mass units), R the gas constant, T the absolute temperature, and Mw the protein molecular weight. A key implication of this is that conditions that promote macromolecular self-assembly may be the conditions that also support nucleation—the initial self-assembly of macromolecules that is essential for the growth of protein crystals. This has been previously demonstrated using dynamic and static light scattering (DLS and SLS) data that show crystallization of macromolecules occurs within a narrow (slightly negative) range of A22 values corresponding to a weakly attractive solution regime [20], although it was also shown later that mildly positive values of A22 can also be conducive to crystallization [21]. Here, we report our study of the effect of the mobile phase ionic strength and sample concentration on the output of SEC analyses. We observed that the use of SEC alone can provide information on the protein aggregation state as molecules approached known precipitation points in their phase diagrams, which is conﬁrmed by the associated in-line SLS measurements. Although chromatographic analyses are nowadays implemented into different characterization methodologies [22–26] and at different beamline facilities [27–31], facilitating the determination of precise information for protein samples at the preparatory stage would certainly improve the efﬁciency of the results obtained through subsequent characterization methodologies. Our experiments serve as a proof-of-principle and theoretical background for the potential use of SEC as an easily accessible lab-based scanning methodology to monitor protein precipitation or nucleation prior to crystallization. These experiments also serve as a reminder that solution conditions can play a major role in molecular mass estimates from SEC experiments using calibration curves.

2. Results and Discussion Size exclusion chromatograms obtained were initially characterized by the peak elution point of sample and the presence of any detectable aggregation in the sample. As shown in Figure1, laser light scattering signal from the SLS measurements is more sensitive to the presence of these aggregates than the absorbance signal at 280 nm of the NGC. Since the two proteins used in this study have well-deﬁned molecular weights and the measured molecular masses are volume averaged, we have interpreted deviations in the measured molecular masses to represent the degree of assembly in the mobile phase under examination (i.e., the A22 value of Equation (1) or B22 value of Equation (2)). Such an interpretation is in line with previous DLS and SLS based studies on A22 values [19]. Crystals 2017, 7, 331 3 of 10

Crystals 2017, 7, 331 3 of 10 As shown below, the molar masses as measured by SLS correlate well with the elution position sample.of the sample. For example, For example, Figure Figure1 demonstrates1 demonstrates that under that high under salt high (2 M salt NaCl (2 M in NaCl 0.1 M in NaoAc 0.1 M NaoAcpH 4.5) thepH protein 4.5) the hen protein egg-white hen egg-white lysozyme lysozyme (HEWL) (HEWL) forms la formsrge aggregates large aggregates (eluted at (eluted ~1.8 mL) at ~1.8 and mL) a major and monomerica major monomeric fraction fraction(eluted (elutedat ~3.4 atmL). ~3.4 Under mL). Underlow salt low conditions salt conditions (0 M (0NaCl M NaCl in 0.1 in M 0.1 NaoAc M NaoAc pH 4.5),pH 4.5), no aggregation no aggregation of HEWL of HEWL can canbe se been, seen, but butthe thesample sample elutes elutes at ~3 at mL. ~3 mL. To furtherfurther examine examine this this behavior, behavior, we we screened screened the concentrationthe concentration of NaCl of NaCl in the in mobile the mobile phase duringphase duringSEC to determineSEC to determine the effects the on effects the elution on the time/volume elution time/volume as shiftsin as the shifts position in the of position the non-aggregated of the non- aggregatedHEWL absorbance HEWL peaks absorbance on the chromatogramspeaks on the chromatogr (i.e., neglectingams the(i.e., peak neglecting position the of the peak highly-aggregated position of the highly-aggregatedsample). As displayed sample). in Figure As 2displayed, these shifts in Figure were associated 2, these shifts with were changes associ inated the SLS with measured changes molarin the SLSmasses measured and sample molar polydispersity. masses and Below, sample we detailpolydispersity. our analysis, Below, demonstrating we detailfeatures our ofanalysis, protein demonstratingbehavior that have features been wellof protein documented behavior (salting-in/out), that have andbeen demonstrating well documented that SEC (salting-in/out), can be used to andidentify demonstrating changes in that protein SEC aggregation can be used stateto identify that (to changes our understanding) in protein aggregation have not state been that previously (to our understanding)demonstrated using have SEC.not Webeen could previously ﬁnd comparable demonstrated studies using using SEC. small We angle could X-ray find andcomparable neutron studiesscattering using [32 ]small and DLSangle [17 X-ray] that and support neutron our scattering results. [32] and DLS [17] that support our results.

Figure 1. LightLight scattering scattering curves curves with with the the corresponding corresponding calculated molar masses obtained for HEWL eluted inin 0.10.1 M M NaOAc NaOAc buffer buffer containing containing 0 M 0 (red) M (red) and 2 and M (blue) 2 M NaCl.(blue)At NaCl. 2 M NaCl,At 2 HEWLM NaCl, displayed HEWL displayeda signiﬁcant a significant delay in its delay elution in its from elution the from column the andcolumn aggregates and aggregates were ﬁrst were eluted first and eluted were and shown were shownby the lightby the scattering light scattering signal (solidsignal line)(solid as line) a small as a peaksmall (eluted peak (eluted around around 1.8 mL) 1.8 preceding mL) preceding the main the mainpeak (elutedpeak (eluted around around 3.4 mL). 3.4 The mL). absorbance The absorbance curves (dashed)curves (dashed) demonstrate demonstrate the same the elution same volumeelution volumebehavior behavior and are and very are much very aligned much aligned with the with LS curvethe LS of curve HEWL of HEWL eluted eluted with 0 with M NaCl. 0 M NaCl. This is This not isthe not case the for case HEWL for HEWL eluted witheluted 2 Mwith NaCl, 2 M which NaCl, does which not does show not any show information any information about higher about molecular higher molecularweight protein weight aggregation. protein aggregation

Crystals 2017, 7, 331 4 of 10 Crystals 2017, 7, 331 4 of 10

Figure 2. The change in the retention volume of 1 mg/mL HEWL at increasing concentrations of NaCl Figure 2. The change in the retention volume of 1 mg/mL HEWL at increasing concentrations of NaCl in the mobile phase. The retention volume of identical injection volumes at identical concentrations in the mobile phase. The retention volume of identical injection volumes at identical concentrations shows a marked dependence upon the mobile phase in SEC. This stacked area plot focuses on the showsnon-aggregated a marked dependence region of the upon chromatogram the mobile (rig phaseht peak in SEC.in Figure This 1), stacked with retention area plot volumes focuses on the non-aggregatedassociated with aggregated region of fraction the chromatogram not shown. (right peak in Figure1), with retention volumes associated with aggregated fraction not shown. Effects of Ionic Strength on Elution (Salting in) Effects of IonicAs shown Strength in Figure on Elution 2, the addition (Salting of in) low amounts of NaCl to the mobile phase triggered HEWL Asto elute shown faster in than Figure it did2, when the addition the mobile of phas lowe was amounts NaCL-free. of NaCl The measured to the mobile molar mass phase was triggered also lower, as displayed in Figure 3 at NaCl ≤ 0.2 M. This clearly demonstrates the known salting in effect HEWL to elute faster than it did when the mobile phase was NaCL-free. The measured molar mass by which proteins become more soluble (less prone to aggregation/self-assembly) and thus are eluted was also lower, as displayed in Figure3 at NaCl ≤ 0.2 M. This clearly demonstrates the known faster from the size exclusion column [33,34]. The volume-averaged masses measured by SLS confirm saltingthat in this effect is accompanied by which proteins by a lowering become of the more tendency soluble of (lessthe molecules prone to to aggregation/self-assembly) self-assemble (i.e., large and thusnegative are values eluted of faster A22). A from NaCl the concentration size exclusion of 0.4 columnM provides [33 a,34 mobile]. The phase volume-averaged in which 1 mg/mL masses measuredHEWL by shows SLS conﬁrmthe lowest that degree this isof accompaniedself-association by (i.e., a loweringhigh negative of the values tendency of A22 of), whereas the molecules 0 M to self-assembleNaCl shows (i.e., a significantly large negative higher values degree of of A self-association22). A NaCl concentration (i.e., large positive of 0.4values M providesof A22). Asa can mobile phasebe in observed which in 1 mg/mLFigure 2, at HEWL increasing shows NaCl the concentration lowest degree (>0.4 ofM for self-association 1 mg/mL HEWL (i.e., and high 0.2 M negative for 2 mg/mL HEWL), the monomer peaks again begin to shift further to the right of the chromatograms, values of A22), whereas 0 M NaCl shows a signiﬁcantly higher degree of self-association (i.e., large displaying increasing retention times. This observation has been earlier reported to be due to the positive values of A22). As can be observed in Figure2, at increasing NaCl concentration (>0.4 M relatively high content of the charged ions in the aqueous media that are expected to compete with for 1 mg/mL HEWL and 0.2 M for 2 mg/mL HEWL), the monomer peaks again begin to shift the bound proteins for the charged resin [35]. This should decrease the proteins’ electrostatic further to the right of the chromatograms, displaying increasing retention times. This observation interactions with the resin and promotes their intrinsic hydrophobic interactions. Therefore, the has beenprotein earlier becomes reported structurally to be duedisconnected to the relatively from the highaqueous content mobile of phase the charged and is retained ions in longer the aqueous in mediathe that column. are expected Here, we tohave compete neglected with the thechange bound in th proteinse viscosity for of thethe chargedmobile phase resin in [ 35this]. analysis This should decreaseas, within the proteins’ the range electrostatic of concentrations interactions used, it withshould the beresin very minor and promotes compared their to the intrinsic solubility hydrophobic effect. interactions.[36]. Therefore, the protein becomes structurally disconnected from the aqueous mobile phase and is retainedDespite longer the delaying in the column.retention Here,volumes we at have increasing neglected NaCl the concentrations, change in the the viscosity results correlate of the mobile phasewell in thiswith analysis the ‘salting as, withinout’ effect the that range is ofshown concentrations in Figure 3 used,as a general it should tendency be very for minor an increase compared in to the solubilitymeasured effect.molecular [36]. mass accompanied by an increase in the sample polydispersity, indicative of a broadening of the population assembly distribution. We also observed that at certain NaCl Despite the delaying retention volumes at increasing NaCl concentrations, the results correlate well concentration ranges of the mobile phase (0.6–1.1 M NaCl for 1 mg/mL HEWL and 0.4–0.7 M NaCl withfor the 2 ‘salting mg/mL out’HEWL, effect shown that in is Figures shown 2 in and Figure 3, respective3 as a generally), there tendency were peak for shifts an increase to the left in (faster measured molecular mass accompanied by an increase in the sample polydispersity, indicative of a broadening of the population assembly distribution. We also observed that at certain NaCl concentration ranges of the mobile phase (0.6–1.1 M NaCl for 1 mg/mL HEWL and 0.4–0.7 M NaCl for 2 mg/mL HEWL, shown in Figures2 and3, respectively), there were peak shifts to the left (faster elution) that also correlated Crystals 2017, 7, 331 5 of 10 Crystals 2017, 7, 331 5 of 10 withelution) an increase that also in thecorrelated molar with masses an increase as determined in the molar using masses SLS. Theseas determined reversed using shifts SLS. (i.e., These against thereversed ionic strength shifts (i.e., effect against of the the mobile ionic strength phase) implyeffect of chances the mobile of nucleation, phase) imply because chances the of nucleation, mobile phase usedbecause in this the case mobile is known phase to easilyused in crystallize this case theis known protein to under easily test. crystallize However, the theprotein amount under of injectedtest. proteinHowever, per trial the (100–200 amountµ ofg HEWL)injected that protein should per have trial also (100–200 encountered µg HEWL) a sort that of dilution should inhave the columnalso is notencountered expected toa sort really of dilution lead to thein the growth column of evenis not aexpected critical nucleus.to really lead While to onlythe growth subtle of increases even a in thecritical molar massesnucleus. were While recorded, only subtle the increases change inin elutionthe molar position masses correlateswere recorded, well the with change the SLS in elution measured massesposition and correlates the changes well in with the measuredthe SLS measured masses aremasses significantly and the changes above thein the errors measured of the measurement,masses are as displayedsignificantly in above Figure the3. Beyond errors of 0.7 the and measurement, 1.2 M NaCl as in displayed the mobile in phaseFigure of3. 1Beyond and 2 mg/mL0.7 and 1.2 HEWL, M NaCl in the mobile phase of 1 and 2 mg/mL HEWL, respectively, the samples were prone to the respectively, the samples were prone to the formation of aggregates, shown as light scattering peak formation of aggregates, shown as light scattering peak preceding the main analyzed peak as shown preceding the main analyzed peak as shown in Figures1 and3. in Figures 1 and 3.

1 mg/ml HEWL in 0.1M NaOAc, pH 4.5 1 mg/ml HEWL in 0.1M NaOAc, pH 4.5 8.0 8.0 7.8 Salting 7.8 Salting 7.6 In Out 7.6 In Out 7.4 7.4 7.2 7.2 7.0 7.0 Retention time (min) time Retention 6.8 Retention time6.8 (min) 6.6 6.6 1.5 1.5 1.4 Nucleation 1.4 Nucleation 1.3 1.3 1.2 1.2 Polydispersity 1.1 1.1Polydispersity 1.0 1.0 100 100 80 80 60 60 40 40 20 20 Mass Recovery (%) Mass Recovery (%) Recovery Mass 0 0 15 15 12 12 9 9 6 6 Molar mass (KDa) mass Molar Molar mass (KDa) mass Molar 3 3 0 0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 0.00.20.40.60.81.01.21.41.61.82.02.2 NaCl (M) NaCl (M) (a) (b)

Figure 3. Graphical representation for the change in retention time obtained from SEC. Also given are Figure 3. Graphical representation for the change in retention time obtained from SEC. Also given are the polydispersity, mass recovery, and molar mass obtained from in line SLS data analysis (horizontal the polydispersity, mass recovery, and molar mass obtained from in line SLS data analysis (horizontal red line on which are the error bars). In the molar mass panel, the molar masses calculated from the red line on which are the error bars). In the molar mass panel, the molar masses calculated from standard calibration curve of the gel filtration column are shown in white columns. Figure 3a,b the standard calibration curve of the gel ﬁltration column are shown in white columns. Figure3a,b indicate the data for HEWL eluted using a mobile phase containing a wide range of NaCl indicateconcentrations the data for at HEWLa protein eluted concentration using a mobile of 1 and phase 2 mg/mL, containing respectively. a wide The range retention of NaCl time concentrations increases at awith protein increasing concentration NaCl concentration of 1 and 2 mg/mL, in the mobile respectively. phase. At The two retention supersaturation time increases levels, the with retention increasing NaCltime concentration showed a slight in the drop mobile before phase. it continued At two again supersaturation its increase. levels, One drop the retentionwas detected time at showed the a slightinterface drop between before the it continued salting in (when again the its protein increase. beca Oneme more drop soluble was detected by the first at theaddition interface of sodium between thechloride) salting in and (when salting the out protein (when became the protein more became soluble less by soluble the ﬁrst at increasing additionof NaCl sodium concentrations). chloride) and saltingOn the out graphs, (when the dashed protein blue became line shows less soluble the limit at between increasing the NaCl saltin concentrations).g in and the salting On out the as graphs,per theour dashed observations. blue line showsThe second the limit drop between (indicated the by salting the black in and arrow) the saltingoccurred out at as the per supersaturation our observations. Thelevel second that dropis most (indicated probably coinciding by the black with arrow) nucleati occurredon (the area at thebetween supersaturation the two dashed level red thatlines), is as most probablywas shown coinciding by the with drop nucleation of mass (therecovery area betweenand subtle the increase two dashed in the red molar lines), mass. as was At shownhigher by thesupersaturation drop of mass recovery levels, additional and subtle bands increase for aggregates in the molar preceding mass. the At bands higher under supersaturation study appeared levels, additionalon LS graphs bands (shown for aggregates on the graph preceding as blue the circles), bands which under coincided study appeared with a drop on LSin molar graphs masses (shown of on thethe graph main as protein blue circles), peaks and which incr coincidedease in their with polydispersity. a drop in molar masses of the main protein peaks and increase in their polydispersity. Crystals 2017, 7, 331 6 of 10 Crystals 2017, 7, 331 6 of 10

The complementary experiment, monitoring the protein assembly by injecting increasing concentrations of of the the protein protein under under test test when when the the mobile mobile phase phase concentration concentration was waskept kept constant, constant, was wasmore more straightforward. straightforward. In this In this case, case, the the mobile mobile phase phase ionic ionic strength strength will will not not interfere interfere with with the the peak position–molecular size/mass relationship relationship as as the ionic strength isis keptkept constant.constant. Increasing the concentration ofof injected injected HEWL HEWL to theto the mobile mobile phase phase that containsthat contains a crystallizing a crystallizing agent (NaCl) agent resulted(NaCl) inresulted peak shiftsin peak to shifts the left to the as shownleft as shown in Figure in Figure4. These 4. These shifts shifts again again coincided coincided with with an increasean increase in thein the molar molar mass mass as measured as measured by SLS. by ThisSLS. againThis again clearly clearly indicates indicates the possibility the possibility of monitoring of monitoring protein inter-molecularprotein inter-molecular interactions interactions that might that be might indicative be indicative for assembly for assembly by observing by observing peak shifts peak on shifts SEC chromatograms.on SEC chromatograms. At low salt At concentrationlow salt concentration in the mobile in the phase mobile (0.5 phase and 1.0 (0.5 M NaCl),and 1.0 one M populationNaCl), one waspopulation detected was as displayeddetected as in displayed Figure4a,b. in However, Figure 4a,b. at high However, salt concentration at high salt (1.5concentration M NaCl), protein (1.5 M assemblyNaCl), protein was alsoassembly associated was also with associated protein aggregation with protein as canaggregation be seen in as Figure can be4 c.seen in Figure 4c. ) ) n n

0.5M NaCl in 0.1M NaOAc i 1.5M NaCl in 0.1M NaOAc 6.6 i 1.0M NaCl in 0.1M NaOAc 6.6 m m 6.6 ( ( 6.4 6.4 me me 6.4 me ti ti 6.2 on 6.2 on 6.2 ti ti en en t 6.0 6.0 t 6.0 e e R Retention time (min) time Retention R

100 100 100 % % 80 80 80 60 60 60 ecovery ecovery ecovery ecovery

40 R R 40 40 20 20 ass ass 20 Mass % Recovery M 0 M 0 0 30 30 ) 30 a 25 25 (kD 25 20 20 20 15

15 massar

l 15 o M Molar massMolar (kDa) Molar massMolar (kDa) 10 10 10

0.5 1.0 1.5 2.0 0.5 1.0 1.5 2.0 0.5 1.0 1.5 2.0 HEWL (mg/mL) HEWL (mg/mL) HEWL (mg/mL) (a) (b)(c)

Figure 4. 4. GraphicalGraphical representation representation for the for change the change in retention in retention time obtained time obtained from SEC, from mass SEC, recovery mass recoveryand molar and mass molar obtained mass obtainedfrom SLS from data SLSanalysis data analysisfor HEWL for prepared HEWL prepared at four concentrations at four concentrations (0.5–2.0 (0.5–2.0mg/mL) mg/mL)eluted through eluted NaCl through buffered NaCl with buffered 0.1 M with NaOAc 0.1 MpH NaOAc 4.5 at three pH 4.5 concentrations at three concentrations (0.5 (a), 1.0 (0.5(b) and (a), 1.01.5M (b ()c and)). In 1.5M the molar (c)). In mass the molarpanels, mass the molar panels, masses the molar calculated masses from calculated the standard from the calibration standard calibrationcurve of the curve gel filtration of the gel column ﬁltration are columnshown in are wh shownite columns. in white Blue columns. circles Bluein the circles molar in mass the molarpanel massindicate panel where indicate small where aggregat smalles appeared aggregates on appeared LS graphs. on LS graphs.

In summary, the SEC elution point of HEWL correlated with SLS determined molecular masses In summary, the SEC elution point of HEWL correlated with SLS determined molecular masses of of the samples, demonstrating that SEC alone can provide data on the self-assembly of the samples, demonstrating that SEC alone can provide data on the self-assembly of macromolecules. macromolecules. It should be stressed that at no point were masses indicative of dimers or higher It should be stressed that at no point were masses indicative of dimers or higher order oligomers. order oligomers. SLS measures a volume averaged molar mass, meaning that changes in the recorded SLS measures a volume averaged molar mass, meaning that changes in the recorded mass indicate mass indicate a small subset of the sample being present as higher order assemblies, allowing an a small subset of the sample being present as higher order assemblies, allowing an estimate of A22 estimate of A22 to be made. Our data suggest that the same A22 estimate may also be made directly to be made. Our data suggest that the same A estimate may also be made directly from SEC from SEC chromatograms in the absence of SLS data.22 chromatograms in the absence of SLS data. Since the effect of NaCl on most proteins is an increased solubility, we selected another Since the effect of NaCl on most proteins is an increased solubility, we selected another precipitant, precipitant, (NH4)2SO4, that is known to have the same salting out effect on a different model protein: (NH ) SO , that is known to have the same salting out effect on a different model protein: bovine bovine4 2 trypsin4 [37]. We followed the same two systematic studies we conducted for HEWL; with trypsin [37]. We followed the same two systematic studies we conducted for HEWL; with increasing increasing (NH4)2SO4 concentration at constant trypsin concentration and vice versa. It was difficult (NH ) SO concentration at constant trypsin concentration and vice versa. It was difﬁcult to follow to follow4 2 4the variations with increasing trypsin concentrations, due to the early appearance of the variations with increasing trypsin concentrations, due to the early appearance of aggregates. aggregates. However, increasing the (NH4)2SO4 concentrations led to similar variations as shown in However, increasing the (NH4)2SO4 concentrations led to similar variations as shown in Table1. Table 1. Trypsin eluted earlier when (NH4)2SO4 was added to the mobile phase and this coincided Trypsin eluted earlier when (NH ) SO was added to the mobile phase and this coincided with with an increase in molar mass and4 2 polydispersity4 . The protein showed higher solubility (lower molar mass and polydispersity) at (NH4)2SO4 concentrations higher than 1 M and eluted later from the column (salting in). However, trypsin showed an increasing degree of assembly at higher

Crystals 2017, 7, 331 7 of 10 an increase in molar mass and polydispersity. The protein showed higher solubility (lower molar mass and polydispersity) at (NH4)2SO4 concentrations higher than 1 M and eluted later from the column (salting in). However, trypsin showed an increasing degree of assembly at higher (NH4)2SO4 concentrations (salting out). Using a mobile phase of 1.5 M (NH4)2SO4, trypsin displayed two peaks on the chromatogram—one corresponding to a trimeric assembly and a second monomeric peak. It is notable that two distinctly different degrees of assemblies in cases of using 0 and 1.5 M (NH4)2SO4 in the mobile phase eluted at similar volumes (Table1). In addition, the larger trimeric assembly of trypsin eluted in 1.5 M (NH4)2SO4 eluted later than the monomeric assembly eluted in 0.5 M (NH4)2SO4. These observations are consistent with our observations above for HEWL in increasing NaCl concentrations.

Table 1. The variations of bovine trypsin retention volume, molar mass, and polydispersity with

increasing concentrations of (NH4)2SO4 in 0.1 M TRIS buffer pH 8.5.

(NH4)2SO4 (M) Retention Volume (mL) Measured Molar Mass (kDa) Polydispersity 0.0 2.73 28.688 (±0.846) 1.002 (0.042) 0.5 2.55 37.571 (±6.305) 1.042 (0.248) 1.0 2.91 25.673 (±2.765) 1.033 (0.143) 1.5 2.73 85.202 (±7.919) 1.061 (0.177) 2.96 26.516 (±7.972) 1.014 (0.387) 2.0 1.21 1232.341 (±88.439) 1.019 (0.099) 1.46 1119.304 (±28.018) 1.018 (0.035)

3. Discussion The conventional application of SEC in structural biology laboratories is to fractionate a given biological sample, for instance by separating monomers from aggregates and perform a molecular weight distribution analysis as well as facilitating protein storage by changing the mobile phase composition. In the general knowledge regarding SEC, it is assumed that, while the sample concentration can interfere with the resolution of sample fractionation, elution points on chromatograms are largely independent of the components of the mobile phase. In contrast, we show here that variations in the composition of the mobile phase can have a large impact on the retention time/volume. Not only can these variations affect the subsequent evaluation of the eluted macromolecules, but also can result in detectable changes in their state (e.g., self-assembly, aggregation or precipitation). This variation on the mobile phase components could be highly problematic for experiments performed with the intention of fractionating or purifying an injected macromolecular sample. Therefore, precise calibration curves specific for every mobile phase should be plotted. In addition, careful choice of the mobile phase may provide a mechanism to further enhance separation of proteins of interest from contaminants using SEC approaches. Furthermore, we believe this to be the ﬁrst demonstration that SEC can be used to study changes in molecular mass that we propose are due to variations of A22 values across a phase diagram. Our observations are conﬁrmed by measurable changes in the molecular masses retrieved from our inline SLS measurements. Similar variations in measured solution molecular mass have been reported by both DLS and X-ray/neutron scattering and have been accepted to represent the effects of changes in A22 with solution composition. SEC experiments using recently available microcolumns hold the potential for a similar scanning of solution behavior and may yet provide an alternative route to the prediction of crystallization conditions. We are currently developing an experimental setting that will make it possible to test mobile phase conditions for proteins that have not been previously crystallized.

4. Materials and Methods

4.1. Solution Conditions Hen egg-white lysozyme (HEWL) and bovine trypsin were purchased from Sigma-Aldrich Darmstadt, Germany (Cat. No. L4919) and Amresco LLC, Ohio, USA (Lot 2555C052), respectively and Crystals 2017, 7, 331 8 of 10 used without further purification. Per the product information the material was ~98% pure monomer. All other chemicals were reagent grade. HEWL was dissolved in 0.1M sodium acetate (NaOAc) that was adjusted to pH 4.5 using acetic acid. The mobile phases were prepared from a range of sodium chloride (NaCl) concentrations buffered with 0.1 M NaOAc, pH 4.5, because this combination is known to facilitate the crystallization of HEWL in two polymorphic forms: tetragonal and orthorhombic crystals [38–40]. Similarly, trypsin was dissolved in water with adequate amount of calcium chloride and benzamidine HCl to prevent self-cleavage. The mobile phases for trypsin were prepared from a range of ammonium sulfate (NH4)2SO4 that was adjusted to pH 8.5 using 0.1 M Tris buffer. This combination is known to lead to the growth of orthorhombic trypsin crystals [41]. Before every SEC experiment a freshly prepared mobile phase was degassed and filtered through a 0.22 µm membrane. Relying on the known phase diagram and solubility values of HEWL, we performed our scans in two ways to study both the effect of mobile phase ionic strength and sample concentration [42,43]. First, we scanned a defined HEWL concentration at increasing precipitant concentrations in the mobile phase. For this, we selected two HEWL concentrations (1 & 2 mg/mL) against a wide range of NaCl concentrations (0.1–2 M) in the mobile phase. The second approach was to scan increasing HEWL concentrations (0.5–2 mg/mL) at discrete constant mobile phase concentrations (0.5, 1.0 and 1.5 M NaCl). Scanning increasing Trypsin concentrations against relatively low (NH4)2SO4 concentrations (0.5M) lead to aggregation on the column. Therefore, we focused on performing SEC at low trypsin concentration (2.0 mg/mL) with increasing (NH4)2SO4 concentrations (0–2 M).

4.2. Experimental Setups A brand new microcolumn (Superdex 75 5/150 GL) mounted on an NGC (Next Generation Chromatography) chromatography system, (BioRad, Berkeley, CA, USA) operated at a ﬂow rate of 0.35 mL/min was used for the whole set of SEC experiments. The system was equilibrated using the selected NaCl concentration using the buffer blending module, ensuring a minimization of random errors in the preparation of the mobile phase. For every chromatographic run, equal volumes of the required HEWL or trypsin concentration were dissolved in their respective buffer (water in case of trypsin) and manually injected into the NGC system. The selected injected volume to total column volume ratio facilitated a high-resolution fractionation regime. The sample molar mass, mass recovery, and polydispersity were monitored by using static light scattering (SLS) measurements provided by an in-line miniDAWN TREOS, (Wyatt, Santa Barbara, CA, USA) and its associated Astra 6.1 software (Wyatt, Santa Barbara, CA, USA).

Acknowledgments: We would like to thank Daiichi Sankyo Company Ltd. for funding this project through their TaNeDS program. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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