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Journal of Pharmaceutical and Biomedical Analysis 189 (2020) 113399

Contents lists available at ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

j o ur na l ho mepage: www.elsev ier .com/ locate / jpba

se of Raman spectroscopy and size-exclusion chromatographyoupled with HDX-MS spectroscopy for studying conformationalhanges of small proteins in solution

ui Wang a, Ian Mangion b, Alexey A. Makarov b,∗∗, Dmitry Kurouski a,c,∗

Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, USAMerck & Co., Inc., MRL, Analytical Research & Development, 33 Avenue Louis Pasteur, Boston, MA 02115, USAThe Institute for Quantum Science and Engineering, Texas A&M University, College Station, Texas, 77843, USA

r t i c l e i n f o

rticle history:eceived 21 March 2020eceived in revised form 19 May 2020ccepted 28 May 2020vailable online 10 June 2020

eywords:aman spectroscopyEC-HDX-MSbiquitin

a b s t r a c t

Protein-based drugs are a relatively new paradigm in modern therapeutics. These large molecule drugsoften have much higher specificity and selectivity compared to small molecules that have been usedin therapeutics for centuries. However, there are many analytical challenges associated with drug dis-covery and development of these new modalities. One of these analytical challenges concerns fast androbust assessment of peptides or small protein conformational structures in solution. In this study,we report a novel analytical approach that is based on Raman spectroscopy (RS) and size exclusionchromatography-hydrogen-deuterium exchange-mass spectrometry (SEC-HDX-MS) for probing confor-mational structures of proteins in solution. Specifically, we demonstrate that RS and SEC-HDX-MS can beused to probe temperature-induced changes in ubiquitin and insulin. We also show that a combination

nsulinrotein melting

of these techniques provides a more comprehensive analysis and comparison of peptide or small proteinconformational structures than by any one technique. Our results demonstrate that RS and SEC-HDX-MS allow for elucidation of sequential transformations in �-helix and �-sheet content of these proteins.These findings suggest that the proposed approach can be used for a fast investigation of changes inprotein or peptide secondary structures under different solution conditions.

© 2020 Elsevier B.V. All rights reserved.

. Introduction

In recent years, pharmaceutical industry efforts have includedeveloping peptide and protein-based therapeutics [1]. Peptidend protein-based therapeutics have many advantages in termsf specificity and selectivity; however, there are a lot of analyt-

cal challenges that come with drug discovery and developmentf these new modalities [2,3]. One of these analytical challengesoncerns fast and robust assessment of peptides or small proteinonformational structures in solution. Nowadays, the synthesis ofeptides for drug discovery efforts has evolved to include the simul-

aneous synthesis and purification of hundreds of compounds [4],hich makes speed of analysis paramount for guiding both lead

dentification and optimization processes.

∗ Corresponding author at: Department of Biochemistry and Biophysics, Texas&M University, College Station, TX 77843, USA.

∗∗ Corresponding author.E-mail addresses: alexey.makarov@merck.com (A.A. Makarov),

kurouski@tamu.edu (D. Kurouski).

ttps://doi.org/10.1016/j.jpba.2020.113399731-7085/© 2020 Elsevier B.V. All rights reserved.

Raman spectroscopy (RS) is a modern analytical techniquethat provides information about molecular vibrations and conse-quently the structure of the analyzed specimen. The Raman effectis based on inelastic light scattering of photons by molecules thatare being excited to higher vibrational or rotational states. RS hasbeen broadly used in various research fields ranging from forensicanalysis of bodily fluids and pesticides to food science and elec-trochemistry [5]. Our group recently demonstrated that Ramanspectroscopy can be used to detect and identify plant diseases[6–8]. Specifically, we were able to identify whether maize kernelswere healthy of infected by Aspergillus flavus, A. niger, Fusarium spp.,or Diplodia spp. with 100 % accuracy [6].

RS in general and deep UV resonance Raman (DUVRR) spec-troscopy in particular can be used to monitor conformationalchanges in proteins [9]. For instance, using RS, Kurouski et al.demonstrated that conformations of disulfide bonds of insulincould be determined upon protein aggregation in amyloid fib-

rils [9]. The researchers demonstrated that aggregation of insulinoccurred without scrambling of disulfides. The Lednev group pio-neered the use of DUVRR to investigate structural changes in insulin

https://doi.org/10.1016/j.jpba.2020.113399http://www.sciencedirect.com/science/journal/07317085http://www.elsevier.com/locate/jpbahttp://crossmark.crossref.org/dialog/?doi=10.1016/j.jpba.2020.113399&domain=pdfmailto:alexey.makarov@merck.commailto:dkurouski@tamu.eduhttps://doi.org/10.1016/j.jpba.2020.113399

2 harmaceutical and Biomedical Analysis 189 (2020) 113399

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Table 1Compounds used in the study.

Compoundname

Number of solventinaccessible labile protons**

Calculated total number oflabile protons*

Insulin 15 91Ubiquitin 33 154

* Calculated based on pdb files 4I5Z and 2MJB respectively for insulin and ubiq-uitin.

** Solvent accessibility was calculated in Biovia Discovery Studio v.17.1.0.16143:

R. Wang, I. Mangion, A.A. Makarov et al. / Journal of P

nd other amyloid associated proteins that were taking place uponmyloid fibril formation [10–12]. Also, the Lednev group demon-trated that DUVRR could be used to probe structures of amyloidbril polymorphs, aggregates that are formed from the same pro-

ein under the same or slightly different experimental conditions13].

A typical Raman spectrum of a protein is composed of contri-utions from two major types of modes, vibrations of polypeptideackbone (amide bands) and amino acid residue side chains [14].mide modes include: the amide I vibration (1640−1680 cm−1),hich primarily represents C O stretching and a small amount of

ut-of-phase C N stretching; the amide II vibration (∼1550 cm−1),hich consists of an out-of-phase combination of C N stretching

nd NH bending motions; and the amide III vibration (1200−1340m−1), a complex vibration mode which involves C N stretchingnd NH bending [15]. Analysis of amide I band is the most com-only used approach for elucidation of structural changes that

s taking place in proteins. This is in part due to the overlay ofhe amide II and amide III bands with the vibrational frequen-ies of certain stretching modes, such as C C, CN and CH2, thatubstantially complicates their assignment and interpretation. Theosition of the amide I band depends on the conformation ofhe polypeptide backbone and intra- and intermolecular hydro-en bonds of the protein specimen. The amide I band (locatedn the 1665−1680 cm−1 range) corresponds to a �-sheet struc-ure, while �-helical protein secondary structure corresponds tohe amide I band (located in the 1640−1654 cm−1 range). Themide I vibration located in the 1654−1665 cm−1 range is typicallyssigned to unordered or disordered protein secondary structures15].

Hydrogen deuterium exchange (HDX) is an established method-logy for studying protein higher-order structure [16–18]. Labilerotons can be exchanged to deuterons in amides, alcohols, car-oxylic acids, or amines and the exchange is observable by MS andMR [19]. Intrinsic H/D exchange rate depends on pH and temper-ture, for example in aqueous solution (at 25 ◦C and neutral pH)xchange occurs in a second-time scale [20]. There is an exchange-ble NH proton in every peptide bond of a protein (except proline),hich makes HDX is a very useful technique for studying protein

igher-order structure, dynamics and folding [21]. The utility ofhis technique has been recognized and workflows based on HDX

ethodology have been robustly implemented in the pharmaceuti-al and biopharmaceutical industries [22–24]. It was demonstratedhat size-exclusion chromatography (SEC) coupled with differentialDX can be successfully used to compare global conformational

hanges of proteins under different solution conditions in manypplications [24,25].

In this study we report an evaluation of the approach thatombines two orthogonal techniques, Raman spectroscopy andEC-HDX-MS, to investigate protein conformational structures inolution. Combination of these techniques aims to provide a moreomprehensive analysis and comparison of peptide and proteinonformational structures than by any one technique. The pro-osed approach can be applied for a fast investigation of proteinigher-order structure under different solution conditions, as wells for the variant selection of peptide and small protein drug can-idates based on conformational structure comparison during lead

dentification and optimization processes.

. Materials and methods

.1. Materials

Bovine recombinant insulin (bovine) and ubiquitin were pur-hased from Sigma-Aldrich Inc. (St. Louis, MO). Ultrapure water

solvent inaccessible was defined as less than 10 % of solvent accessible surface (SAS),based on approach by the DCLM method.

was obtained from a Milli-Q Gradient A10 from Millipore (Bed-ford, MA, USA). Trifluoroacetic acid (TFA), ammonium hydroxideand acetonitrile (ACN) HPLC grade were purchased from FisherScientific (Fair Lawn, NJ). The insulin and ubiquitin stock solu-tion was prepared by dissolving the protein powder into theDI water with pH adjusted to pH 1.0 and 7.0 respectively. Thefinal concentration of the insulin and ubiquitin stock solutionwas 140 and 100 mg/mL respectively. To perform the spectro-scopic study, 1 mL of the insulin/ubiquitin stock solution wasadded to a capped glass vial, the vial was then placed in a self-made heater, whose temperature was controlled by circulatingwater from a thermostat. Besides, the temperature in the heaterwas also monitored by thermometer. The protein sample withthe heater was placed on a piece of coverslip for Raman measur-ing.

2.2. Size exclusion chromatography-hydrogen-deuteriumexchange-mass spectrometry (SEC-HDX-MS)

SEC-HDX-MS was performed using a Synapt G1 HDMS massspectrometer with an Acquity UPLCTM system (Waters Corp., Mil-ford, MA, USA), with Waters MassLynx V4.1 software for instrumentcontrol and data processing. An Acquity UPLC® Protein BEH SEC-125 4.6 × 150 mm, 1.7 �m column with 125 Å pore size from Waterswas used in all experiments. The aqueous mobile phase (A) was 50mM ammonium formate adjusted to pH 2.0 with TFA or to pH 5.5with ammonium hydroxide and the organic mobile phase (B) wasacetonitrile. Sample solutions of the proteins used for experimentswere prepared at about 0.2 mg/mL in 0.1 v/v % TFA (insulin) or 50mM ammonium formate adjusted to pH 5.5 (ubiquitin) in water orin deuterium oxide. All analytes were completely dissolved beforeinjections. To label insulin and ubiquitin for SEC-HDX experiments,protein samples in deuterium oxide were incubated for 24 h at 40◦C. The SEC-HDX experiments were conducted in the similar man-ner as previously reported. [24,26] The SEC conditions consisted ofa 5 % B isocratic run at 0.250 mL/min with total run time 10 min.The column conditions mimicked the same temperature inducedunfolding as in Raman Spectroscopy experiments. The MaxEnt1module was used for all mass spectra deconvolution. Note that inthis study, we used an all-or-nothing approach and our SEC methodwas developed to eliminate exchange restrictions based on intrin-sic rate of hydrogen-deuterium exchange for insulin and ubiquitin[24].

Protein solvent accessibility was estimated for the proteins usedin this study with the computational chemistry software Biovia Dis-covery Studio v.17.1.0.16143 (Dassault Systemes Biovia) with inputpdb (Protein Data Bank) files 2MJB for ubiquitin and 4I5Z for insulin(Table 1). The solvent inaccessible was defined as less than 10 %of solvent accessible surface (SAS) modeled by the double cubiclattice method (DCLM) [27]. The number of labile protons was cal-

culated for the solvent-inaccessible part of the protein sequence(as determined by the software), based on the respective pdbfiles.

R. Wang, I. Mangion, A.A. Makarov et al. / Journal of Pharmaceutical and Biomedical Analysis 189 (2020) 113399 3

Table 2Vibrational bands in the spectra of ubiquitin and insulin and their assignments.

Frequency (cm−1) Assignment [9,14]

646 Tyrosine832 Tyrosine859 Tyrosine1007−1011 Phenylalanine1355 CH deformation1453 CH2 deformation

2

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Fig. 1. A. Raman spectra (� =785 nm; P =15 mW; T = 60 s) collected from bovine

1620 Tyrosine, phenylalanine1667−1669 Amide I

.3. Spectroscopy

Raman spectra were collected on an inverted microscope (NikonE-2000U) with 20X dry Nikon objective (NA = 0.45). A single lon-itudinal diode mode laser (Necsel, CA, USA) was used to generate

785 nm excitation. Spectral acquisition time was 60 s with laserower at the sample of 15 mW. Three samples were tested in par-llel for both insulin and ubiquitin. The signal was collected in aackscattering configuration and directed to a confocal IsoPlaneCT 320 Raman spectrometer (Princeton Instruments, NJ, USA)quipped with a 600 groove/mm grating blazed at 750 nm. Prioro entering the spectrograph Rayleigh scattering was filtered withP02−785RE-25 long-pass filter (Semrock, NY, USA). The dispersedight was then sent to PIXIS:400BR CCD (Princeton Instruments, NJ,SA). A motorized stage H117P2TE (Prior, MA, USA) controlled byrior Proscan II was used to move the sample relative to the incident

aser beam. All data was processed using GRAMS/AI 7.0 (Thermoalactic, NH, USA). The reported spectra are baseline corrected.

. Results and discussion

.1. Temperature dependent Raman spectra of ubiquitin inolution

Solution of bovine ubiquitin (100 mg/mL, pH 7.0) was heatedrom 30 to 60 ◦C increment of 10 ◦C for each data point. Ramanpectra collected from these solutions exhibited three clearly dis-inct vibrational bands at 1007, 1453 and 1667 cm−1, which can bessigned to phenylalanine (Phe), CH2 and amide I vibration respec-ively, Table 2, Fig. 1, A. For comparison of changes in intensitiesf vibrational bands, we normalized all collected spectra on 1453m−1 peak (CH2 vibration). This band has been chosen for normal-zation of Raman spectra because CH2 groups are present in manymino acids located across the sequence of ubiquitin. Thus, suchormalization is unbiased to the specific amino acid.

We have found that intensity of the amide I band graduallyecreases with an increase in the solution temperature indicat-

ng deformation or melting of the peptide secondary structure,ig. 1, B. We have also found that intensity of Phe remained nearlynchanged at low (30 ◦C) and high (60 ◦C) temperatures, Fig. 1, A,ig. S1. This indicates that local environment of this amino acid didot change upon peptide melting [9].

Next, ubiquitin solution was gradually cooled down with anncrement of 10 ◦C for each data point, Fig. 2.

We have found that intensity of amide I band gradually increaseith an increase in the solution temperature reversible conforma-

ional changes in the peptide secondary structure, Fig. 2, B. Thesepectral changes suggest about re-folding of ubiquitin that is tak-ng place upon depression of the peptide temperature. We havelso found that intensity of Phe remained nearly unchanged at low

30 ◦C) and high (60 ◦C) temperatures, Fig. 2, A, Fig. S2.

We have plotted a temperature dependent change in the inten-ity of amide I band (1590−1730 cm−1) to demonstrate that RSan be used for quantitative prediction of conformational changes

ubiquitin (100 mg/mL; pH 7.0) at different temperatures (30-60 ◦C). Normalizationwas done based on 1453 cm−1 peak (CH2 vibration). B. Amide I region of thesespectra.

in the protein secondary structure, Fig. 3. Our results demonstratethat RS can be used to predict a degree of temperature-inducedmelting of ubiquitin secondary structure. These data also showthat RS can be used to probe the degree of ubiquitin re-foldingthat is taking place at a low temperature (50−30 ◦C) of the peptidesolution.

RS offers a precision analysis of conformational changes that aretaking place in different secondary structure elements of proteins[9]. As was discussed above, amide I band consists of individualcontributions of �-sheet structure (1665−1680 cm−1) and �-helix(1640−1654 cm−1) [9,28]. Thus, precise analysis of amide I bandcan be used to investigate changes in the content of these secondarystructures that are taking place upon temperature-induced meltingand re-folding of proteins.

We fitted amide I in the spectra collected from ubiquitin solutionat different temperatures to investigate conformational changesthat are taking place in the �-helix (1643 cm−1) and �-sheet (1670cm−1) of ubiquitin upon heating from 30 to 60 ◦C, Fig. 4

We have found that a ratio of �-helix to �-sheet increased from1.46 to 3.26 as the peptide solution was heated from 30 to 40 ◦C.These results suggest that �-helix melted faster than �-sheet in thistemperature range. Deconvolution of Raman spectra of ubiquitin at50 ◦C suggest even further degradation of �-helical component ofthis peptide, whereas the content of �-sheet does not experiencesimilar changes. This quantitative analysis was limited to the spec-trum of ubiquitin at 60 ◦C due to low signal-to-noise of the amide Iregion. This suggests significant loss of the peptide secondary struc-

tures at this temperature. Nevertheless, one can notice that the�-sheet component nearly disappeared in the Raman spectrum ofubiquitin collected at 60 ◦C suggesting that melting of �-sheet inubiquitin is taking place between 50 and 60 ◦C.

4 R. Wang, I. Mangion, A.A. Makarov et al. / Journal of Pharmaceutical and Biomedical Analysis 189 (2020) 113399

Fig. 2. A. Raman spectra (� =785 nm; P =15 mW; T = 60 s) collected from bovineubiquitin (100 mg/mL; pH 7.0) at different temperatures (60-30 ◦C). Normalizationwas done based on 1453 cm−1 peak (CH2 vibration). B. Amide I region of thesespectra.

Fig. 3. Temperature dependent change in the intensity of amide I band (1590-1730

Fig. 4. Deconvolution of amide I band of ubiquitin in Raman spectra colle

cm−1) of ubiquitin upon heating (black) and cooling (red). Corresponding changesin HDX profile of ubiquitin upon heating (blue). SEC-HDX-MS solvent: 95 % aqueous50 mM ammonium formate adjusted to pH 5.5/ 5% acetonitrile.

SEC-HDX-MS was used as an orthogonal technique to screenfor global conformational changes in protein (ubiquitin or insulin)structure upon temperature increase. In this study, deuterium-to-hydrogen exchange was performed on SEC column in thesolution environments matching conditions in experiments usingRS, and the overall degree of exchange was measured by MSdetection on-line. We have previously reported on-line screen-ing for global protein conformational changes in solution usingcombined SEC-HDX methodology [24–2629,30]. The conforma-tional structure changes were compared based on the number oflabile protons which remained un-exchanged (solvent inaccessi-ble) in the protein structure. The number of labile protons which

remained un-exchanged (�HDX) was measured based on themolecular mass difference between the deuterium-oxide-labeledprotein after deuterium-to-hydrogen exchange (at specific condi-tions) and the molecular mass of the same protein not exposed

cted at four different heating temperatures (30, 40, 50 and 60 ◦C).

R. Wang, I. Mangion, A.A. Makarov et al. / Journal of Pharmaceutical and Biomedical Analysis 189 (2020) 113399 5

Fid

tctamdasoribun

3

3c8v8ctcbvls

di

ig. 5. A. Raman spectra (� =785 nm; P =15 mW; T = 60 s) collected from bovinensulin (140 mg/mL; pH 1.0) at different temperatures (30-60 ◦C). Normalization wasone based on 1454 cm−1 peak (CH2 vibration). B. Amide I region of these spectra.

o deuterium oxide (under the same conditions). The %�HDX wasalculated based on the total number of labile protons in the pro-ein structure (Table 1). Fig. 3 demonstrates %�HDX for ubiquitint different temperatures, which indicated the ubiquitin confor-ational structure unfolding upon temperature increase (%�HDX

ecreased upon temperature increase). Interestingly the slope ofmide I band intensity upon heating was different than %�HDXlope upon heating (Fig. 3). This %�HDX slope indicates the speedf overall conformational changes in solution. This may corrobo-ate observations based on RS data that a ratio of �-helix to �-sheetncreased upon ubiquitin heating. Since %�HDX reflected the com-ined result of global conformational structure stability, the slowernfolding of �-sheet (demonstrated by RS) resulted in less steep-ess of the %�HDX slope for ubiquitin.

.2. Temperature dependent Raman spectra of insulin in solution

Solution of bovine insulin (140 mg/mL, pH 1.0) was heated from0 to 60 ◦C increment of 10 ◦C for each data point. Raman spectraollected from these solutions exhibited vibrational bands at 646,32, 859, 1011, 1355, 1453, 1620 and 1669 cm−1, Table 2. Theseibrational bands can be assigned to tyrosine (Tyr) (646, 832 and59 cm−1), Phe (1011 cm−1) and both of these amino acids (1620m−1), as well as CH (1355 cm−1) and CH2 (1453 cm−1) vibra-ions. We have also observed an amide I band centered at 1669m−1. For comparison of changes in intensities of these vibrationalands, we normalized all collected spectra on 1453 cm−1 peak (CH2ibration). This chemical moiety presents in nearly all classes of bio-ogical molecules. Therefore, normalization on CH2 vibration makes

pectral interpretation the least biased, Fig. 5.

We have found that the intensity of amide I band graduallyecreased with an increase in the solution temperature indicat-

ng deformation or melting of insulin secondary structure, Fig. 5,

Fig. 6. Temperature dependent change in the intensity of amide I band (1590-1730cm−1) of insulin upon heating (black) and cooling (red). Corresponding changes inHDX profile of ubiquitin upon heating (blue). SEC-HDX-MS solvent: 95 % aqueous50 mM ammonium formate adjusted to pH 2.0/ 5 % acetonitrile.

B. We have also found that the intensity of Tyr and Phe remainednearly unchanged at low (30 ◦C) and high (60 ◦C) temperatures,Fig. 1, A, Fig. S3 and S4. This indicates that the local environment ofthis amino acid did not change upon peptide melting.

A decrease in temperature of insulin solution reflected in theincrease in the intensity of amide I band. This suggests aboutreversible conformational changes (re-folding) in the insulin sec-ondary structure. We have also found that intensity of Tyr and Pheremained nearly unchanged at low (30 ◦C) and high (60 ◦C) tem-peratures. For the clear visualization of these spectral changes, wehave plotted a temperature dependent change in the intensity ofamide I band (1590−1730 cm−1), Fig. 6. These results demonstratethat RS can be used to predict a degree of temperature-inducedmelting of insulin secondary structure of insulin. These data alsoshow that RS can be used to probe the degree of insulin re-foldingthat is taking place at low temperature of the peptide solution.

We performed detailed analysis of changes in amide I band ofRaman spectra collected from insulin heated at 30, 40, 50 and 60◦C, Fig. 7. We have found that at 30 ◦C, presence of both �-helix(1631 cm−1) and �-sheet (1668 cm−1) with the ratio of 0.2 hasbeen observed. At 40 ◦C, the ratio of �-helix to �-sheet became 0.85suggesting that substantial amount of peptide �-sheet denatured atthis temperature. We have found that at 50 ◦C, �-helical componentof insulin nearly completely disappeared, whereas remaining �-sheet was preserved. We were not able to perform this quantitativeanalysis of changes in secondary structures of insulin that weretaken place at 60 ◦C due to low signal-to-noise of the amide I region.

These results demonstrate that RS can be used to probe sequen-tial changes in both �-helix and �-sheet content of insulin that aretaking place upon temperature induced peptide melting.

The SEC-HDX-MS experiment for insulin was performed on col-umn in solution environments matching those conditions in theexperiment using Raman Spectroscopy (Fig. 6). Upon heating, theSEC-HDX conformational screening for insulin corroborated theobservations by RS. Indeed, the slope of insulin’s %�HDX uponheating was almost identical to amide I intensity slope by RS upontemperature increase. This is an indication that overall insulinstructure unfolding upon heating was mostly based on �-helixunfolding, which resulted in a direct correlation between globalunfolding (by SEC-HDX) and specific �-helix unfolding (by RS).

4. Conclusions

The manuscript reports the experimental demonstration ofcombination of RS with SEC-HDX-MS for a fast elucidation oftemperature-induced changes in two model proteins namelyinsulin and ubiquitin. Our results demonstrated complementar-

6 R. Wang, I. Mangion, A.A. Makarov et al. / Journal of Pharmaceutical and Biomedical Analysis 189 (2020) 113399

collec

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Fig. 7. Deconvolution of amide I band of insulin in Raman spectra

ty of RS and SEC-HDX-MS in terms of sensing of changes inrotein secondary structure. We also show that in addition tohis temperature-induced changes, RS allowed for elucidation ofequential transformations in �-helix and �-sheet content of theseroteins. Combination of these techniques was able to provide aore comprehensive analysis and comparison of protein confor-ational structures in solution than by any single technique. The

roposed approach can be applied for a fast investigation of pro-ein higher-order structure in solution, as well as for the variantelection of peptide or small protein drug candidates based ononformational comparison during lead identification and opti-ization processes.

ompliance with ethical standards

None.

RediT authorship contribution statement

Rui Wang: Conceptualization, Methodology, Investigation,riting - original draft, Visualization, Writing - review & edit-

ng. Ian Mangion: Supervision, Project administration, Writing -eview & editing. Alexey A. Makarov: Conceptualization, Method-logy, Investigation, Writing - original draft, Visualization, Writing

review & editing, Supervision. Dmitry Kurouski: Conceptu-lization, Methodology, Investigation, Writing - original draft,isualization, Writing - review & editing, Supervision.

eclaration of Competing Interests

The authors declare that they have no known competing finan-ial interests or personal relationships that could have appeared tonfluence the work reported in this paper.

ted at four different heating temperatures (30, 40, 50 and 60 ◦C).

Acknowledgements

We are grateful to Merck for povided financial support.

Appendix A. Supplementary data

Supplementary material related to this article can be found,in the online version, at doi:https://doi.org/10.1016/j.jpba.2020.113399.

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Use of Raman spectroscopy and size-exclusion chromatography coupled with HDX-MS spectroscopy for studying conformational c...1 Introduction2 Materials and methods2.1 Materials2.2 Size exclusion chromatography-hydrogen-deuterium exchange-mass spectrometry (SEC-HDX-MS)2.3 Spectroscopy

3 Results and discussion3.1 Temperature dependent Raman spectra of ubiquitin in solution3.2 Temperature dependent Raman spectra of insulin in solution

4 ConclusionsCompliance with ethical standardsCRediT authorship contribution statementDeclaration of Competing InterestsAcknowledgementsAppendix A Supplementary dataReferences