Vibrational Spectroscopy in Protein Research: From Purified Proteins to Aggregates and Assemblies

By Yukihiro Ozaki

Vibrational Spectroscopy in Protein Research offers a thorough discussion of vibrational spectroscopy in protein research, providing researchers with clear, practical guidance on methods employed, areas of application, and modes of analysis. With chapter contributions from international leaders in the field, the book addresses basic principles of vibrational spectroscopy in protein research, instrumentation and technologies available, sampling methods, quantitative analysis, origin of group frequencies, and qualitative interpretation. In addition to discussing vibrational spectroscopy for the analysis of purified proteins, chapter authors also examine its use in studying complex protein systems, including protein aggregates, fibrous proteins, membrane proteins and protein assemblies.

Emphasis throughout the book is placed on applications in human tissue, cell development, and disease analysis, with chapters dedicated to studies of molecular changes that occur during disease progression, as well as identifying changes in tissues and cells in disease studies.

• Provides thorough guidance in implementing cutting-edge vibrational spectroscopic methods from international leaders in the field
• Emphasizes in vivo, in situ and non-invasive analysis of proteins in biomedical and life science research more broadly
• Contains chapters that address vibrational spectroscopy for the study of simple purified proteins and protein aggregates, fibrous proteins, membrane proteins and protein assemblies

• Language: English
• Publisher: Academic Press
• Release date: May 19, 2020
• ISBN: 9780128186114

Book preview

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Preface

Yukihiro Ozaki, Malgorzata Baranska, Igor K. Lednev and Bayden R. Wood

Recently, a variety of state-of-the art vibrational spectroscopy techniques have been developed rapidly, for example, Raman imaging, Raman optical activity (ROA), surface-enhanced Raman scattering (SERS), nonlinear Raman spectroscopy, cascade laser IR spectroscopy, infrared (IR) imaging, near-infrared (NIR) imaging, and time-resolved vibrational spectroscopy. Moreover, several spectral analysis methods, such as quantum chemical calculations, chemometrics, and two-dimensional correlation spectroscopy, have also been advanced. These experimental and spectral analysis methods have all been extensively used for a variety of investigations of proteins.

The purpose of this book is to demonstrate the usefulness of these modern vibrational spectroscopic methods in protein research. This book places some emphasis on in vivo, in situ, and noninvasive studies of proteins. It also points to the imaging of biological systems including proteins; Raman, IR, and NIR imaging are introduced. Most of the chapters are concerned not only with simple purified proteins, but also with rather complicated protein systems, such as protein aggregates, fibrous proteins, membrane proteins, and protein assemblies. Moreover, the book can be connected with medical science to some extent.

The book outlines cutting-edge vibrational spectroscopy methods by the front-runners of this field. All the contributors are experts in both modern vibrational spectroscopy and protein research. A few new techniques, such as quantum cascade laser-based IR transmission spectroscopy and SERS with nanotag design, are introduced in this book.

This book aims at making a strong bridge between molecular spectroscopists and researchers in life sciences. Thus it is suitable for molecular spectroscopists who are interested in protein research and for protein scientists who are interested in molecular spectroscopy. This book is useful for scientists, engineers, and graduate students in various fields of protein research including chemistry, biological sciences, pharmaceutical science, agricultural science, and medicine. One can use this book as a text for a course, for example, at a graduate school.

We do hope this book can inspire readers to employ vibrational spectroscopy techniques for various investigations of proteins and/or to open new directions of protein research based on vibrational spectroscopy.

In closing, we would like to thank Dr. Aliaksandra Sikirzhytskaya for preparing the wonderful cover of this book and also to Mr. Peter Linsley and Ms. Sara Pianavilla of Elsevier for their continuous efforts in publishing this book.

November 2019

Chapter 1

ATR-FTIR spectroscopy and spectroscopic imaging of proteins

Bernadette Byrne, James W. Beattie, Cai Li Song and Sergei G. Kazarian,    Department of Chemical Engineering and Department of Life Sciences, Imperial College London, London, United Kingdom

Abstract

This chapter describes applications of attenuated total reflection (ATR)–Fourier transform infrared (FTIR) spectroscopy and ATR-FTIR spectroscopic imaging to studies of proteins. The latter method combines ATR-FTIR spectroscopy with an infrared array detector for obtaining both spatial and chemical information from protein samples. Two imaging modes, micro- and macro-ATR, provide a range of imaging fields of view and spatial resolutions. Micro-ATR-FTIR imaging has been successfully used to study hanging drop protein crystallization with high spatial resolution imaging, while macro-ATR-FTIR imaging provided new opportunities for in situ studies of protein crystallization and aggregation, as well as the effect of different wettability surface properties on protein adsorption and crystallization. Recent pioneering applications of ATR-FTIR spectroscopy involve analysis of protein ligand denaturation on chromatography columns, critical for improved industrial purification of biotherapeutics. These and other new high-throughput chemical imaging approaches are discussed in this chapter.

Keywords

Attenuated total reflection; ATR-FTIR imaging; FTIR spectroscopy; protein crystallization; protein aggregation kinetics

1.1 Introduction

1.1.1 Study of protein behavior—protein in solution, film, and tissue

Fourier transform infrared (FTIR) spectroscopy is an excellent tool to obtain structural and chemical information of proteins. There are multiple infrared spectroscopic methods that can be applied to gain key information on secondary structure and the behavior of proteins. One such method is the use of attenuated total reflection (ATR)–FTIR spectroscopy. ATR-FTIR spectroscopy has proven to be useful in the investigation of biomedical samples, protein crystallization, surface interaction with proteins, and behavior of proteins that undergo structural changes due to isolation methods, such as therapeutic antibodies [1–4].

ATR-FTIR spectroscopy is a powerful label-free technique that allows the generation of a spectrum containing absorption bands specific to the chemical species present in a measured sample. The ATR mode reflects IR radiation via an internal reflective element (IRE) which is in direct contact with the sample. This reflection of the IR radiation generates an evanescent wave at the interface between the sample and the IRE which probes the sample. The key parameter for evanescent wave generation is that the critical angle for internal reflection must be exceeded. This evanescent wave is reflected causing attenuation of the IR radiation exiting the IRE [5]. The IRE element consists of infrared transparent material that has a higher refractive index than the sample being analyzed. IREs such as diamond, Ge, Si, and ZnSe are used in the study of proteins as they have a higher refractive index than that of protein samples.

as shown by the Harrick equation (Eq. 1.1) below [6–8].

(1.1)

ATR-FTIR spectroscopy has a limited depth of penetration (c. 0.2–3 µm) due to the refractive index of the IRE crystal and the sample used. Therefore the spectrum generated is representative of the surface layer of the sample adjacent to the ATR crystal and not of the bulk phase [7,8]. It should be noted that the surface layer of the sample may not be representative of the bulk of a given sample [9].

1.1.2 Interaction of proteins with infrared—understanding amide bands

1.1.2.1 Interpreting secondary structures from amide bands

Proteins show clear interaction with infrared radiation through the generation of amide vibrational bands in measured spectra, the most prominent being the amide I, II, and III bands of protein spectra as illustrated by Fig. 1.1.

Figure 1.1 Example of a protein attenuated total reflection (ATR)–Fourier transform infrared (FTIR) spectrum highlighting the key amide I and II bands. The amide I band (1637 cm−1) is highlighted in green and the amide II band (1558 cm−1) is highlighted in yellow. The protein used to generate the spectrum is IgG4.

The amide I band is very useful for determining changes in the secondary structure of protein due to the fact that it corresponds to the vibrational modes of the chemical species that make up the peptide bonds of a protein. The amide I band typically absorbs at ≈1650 cm−1. In O stretch weakly coupled to C–N stretching as well as the N–H in-plane bending mode as delineated in green [1,10]. The exact position of the amide I band is determined by hydrogen bonding as well as the protein backbone conformation [11].

The amide II band is similarly very useful for determination of secondary structural changes in proteins, as differences in secondary structure components, such as disordered regions and aggregated β-sheets, are reflected in this band. The amide II band, made up of N–H in-plane bending and C–N stretching [1,10,11], typically absorbs at ≈1550 cm−1. In Fig. 1.1 this band is present at 1558 cm−1.

1.1.2.2 Qualitative and quantitative analysis

Qualitative and quantitative interpretations should both be used to interpret a measured infrared spectrum. Qualitative methods include assigning vibrational modes present in a spectrum using amide bands as previously shown in Fig. 1.1. This method allows for changes in protein structure, such as changes that occur when a protein aggregates, to be described. By comparing peak height of the spectral band and its position to a difference spectrum at a given wave number (i.e., amide I) certain structural features including formation of intermolecular β-sheet aggregates can be detected [1].

ATR-FTIR spectroscopic measurements can be interpreted quantitatively to determine the concentration of analytes. Protein concentrations can be determined by integration of the amide II band and through generation of a calibration curve of the integrated absorbance. For example, the protein A ligand concentration in immunoaffinity resin adjacent to an IRE was determined in this manner, as shown in Fig. 1.2A. This quantification method provides a viable alternative to using UV spectroscopy to quantify protein [12]. This method of quantification also allows for application of ATR-FTIR spectroscopy as a means of determining binding capacity of the stationary phase (of protein A resin) as denoted in Fig. 1.2B [12].

Figure 1.2 (A) Standard addition curve used for protein A local concentration quantification. (B) Difference in local protein concentration measured by attenuated total reflection (ATR)–Fourier transform infrared (FTIR) spectroscopy as a function of the static binding capacity (q). (C) In-column protein concentration difference calculated from ATR-FTIR-based partial least squared (PLS) regression (red) after elution and cleaning in place (CIP) for different conditions. Source: Reproduced with permission from Springer Nature (M. Boulet-Audet, B. Byrne, S.G. Kazarian, Cleaning-in-place of immunoaffinity resins monitored by in situ ATR-FTIR spectroscopy, Anal. Bioanal. Chem. 407 (2015) 7111–7122; M. Boulet-Audet, S.G. Kazarian, B. Byrne, In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies, Sci. Rep. 6 (2016) 30526).

An additional method of quantifying ATR-FTIR spectra is to use a calibration curve as the basis of a partial least squared (PLS) regression method. The PLS method used in Fig. 1.2B utilizes the range of 1400–1800 cm−1 to quantify protein concentration of an immunoaffinity resin accurately with a confidence interval of 95% [13].

1.1.2.3 Challenges—interference of water spectral bands

It is well documented that protein analysis by FTIR spectroscopy is limited by the strong absorption of IR radiation by water, particularly for measurements in transmission. ATR-FTIR spectroscopy probes only a few micrometers of a sample that is directly in contact with the ATR crystal, removing the issue of complete absorption of the IR radiation by the aqueous phase of a sample. This also provides an additional advantage as usually all spectral bands in ATR-FTIR spectroscopy are on-scale, the exception to this is when accessories with many internal reflections are used.

However, water remains a challenge in spectral analysis even after removal of water saturation, since the amide I band often overlaps with the water bending mode (≈1650 cm−1). This can cause issues such as oversubtraction and thus the loss of key structural features of a desired protein analyte. A solution to this issue is to use deuterated water to shift the bending band away from the amide I band. However this causes the generated spectrum to be unrepresentative of the native protein structure because of isotope exchange; structural changes under these conditions are indicated by a shift in the amide II band from ≈1550 cm−1 to ≈1450 cm−1 [10]. To ensure correct subtraction of the background buffer spectrum from the sample spectrum specific criteria must be met. The subtraction coefficient should be determined by using a least squares method on the combination band of water (2150 cm−1) in protein samples to remove variations of human bias in the subtraction of water over multiple samples. In addition, the ratio of amide I to amide II absorbance should not be higher than 1.5 in a measured spectrum of a protein sample [14].

1.1.2.4 Comparison between transmission and ATR spectroscopic analysis of proteins

Transmission spectra are generated by passing infrared light through a sample typically held between an IR transparent window such as CaF2. A significant disadvantage of the transmission method when measuring solutions of proteins is that proteins are in an aqueous phase. As previously mentioned water strongly absorbs IR radiation, thus only a very small path length (≈8 µm) can be used to minimize the risk of completely absorbing all the IR radiation being passed through the sample before it reaches the detector [15]. Similarly, longer path lengths through the solution of protein result in spectral bands being off-scale, making the spectroscopic analysis very challenging or even impossible.

Employing ATR rather than transmission methods is preferable when measuring aqueous samples due to the interaction of the reflected IR radiation with only the surface layer of a sample (0.2–3 µm) which is in direct contact with the ATR element, ensuring that water does not saturate the spectrum. ATR methodologies require minimal sample preparation when compared to transmission mode as samples can simply be placed on the ATR element for measurement. The evanescent wave probing of the surface layer of the sample and the technique’s ability to be combined with other methods to alter protein structure such as changing pH, temperature, and surface properties make this technique ideal for characterization of proteins [1,4].

However, ATR methodologies are less sensitive than transmission methodologies because of the smaller path length used, resulting in a lower signal to noise ratio, making it harder to discern spectral noise from actual vibrational modes present. As a minimum, spectral bands should be three times stronger in absorbance than that of spectrum noise [16]; this can limit the data that is acceptable to be interpreted for a given spectrum [15].

1.1.3 The significance of study of protein crystallization and aggregation with new vibrational spectroscopic methods

A particularly useful advance to the analysis of proteins using IR spectroscopy has been the integration of a focal plane array (FPA) to FTIR spectroscopic measurements. The use of a FPA allows thousands of spectra to be simultaneously measured in a timescale of seconds without sample preparation methods such as the addition of protein stains [1,4]. This spectroscopic imaging method, an inherently high-throughput approach, allows for the drawbacks of low-throughput methods such as traditional point to point mapping of samples to be overcome and for real-time monitoring of structural changes in proteins to be carried out [2,3,12].

The FPA is a multichannel detector that creates images detailing high absorbance and low absorbance of corresponding vibrational modes, for example, those that are used to determine protein structure such as amide I and II bands [16]. This generation of a chemically specific Heat map allows multiple components in a sample to be analyzed simultaneously [1,12,17].

The ability of the FPA to differentiate substances within a multicomponent system is shown in Fig. 1.3. The plotting distribution of integrated absorbance of chemically specific bands (i.e., amide II) allows chemical images to be generated as a nondestructive method of assessing a particular protein crystallization condition and to help identify whether crystals formed for X-ray crystallography are protein or salt [2,4]

Figure 1.3 Attenuated total reflection (ATR)–Fourier transform infrared (FTIR) image (the imaged area is approximately 2.5×3.6 mm²) (A) of the protein crystal formed on the measuring surface 20 h after introduction of the crystallizing agent. Red represents high protein concentration. The optical microscopy image (B) shows the protein crystals formed coincide well with the location of the red spots on the ATR-FTIR image. (C) Result of overlaying images (A) and (B). Five out of 50 crystals (highlighted with arrows) were not captured in the ATR-FTIR image, and three crystals (circled) were misallocated. Source: Reproduced with permission from K.L.A. Chan, et al., Attenuated total reflection-FT-IR spectroscopic imaging of protein crystallization, Anal. Chem. 81 (10) (2009) 3769–3775. Copyright 2009 American Chemical Society.

1.2 ATR-FTIR spectroscopic imaging of proteins

1.2.1 Macro-ATR spectroscopic imaging

1.2.1.1 High-throughput measurements: protein crystallization growth, aggregation, study of protein adsorption by functionalizing ATR crystal surfaces

X-ray crystallography remains the most common method of protein structure determination. At the time of writing this technique is responsible for ~90% of all the structures deposited in the protein databank (www.rcsb.pdb.org). However, determination of structures using X-ray crystallography requires the formation of protein crystals, a process dependent on extensive and time-consuming screening of crystallization conditions requiring large amounts of protein [18]. Any crystals obtained have to be confirmed as protein and tested for ability to diffract X-rays.

The most widely used crystallization technique is vapor diffusion using either a hanging drop or sitting drop setup involving equilibration of a droplet of protein and precipitant solution with a reservoir containing a higher concentration of precipitant [19]. During equilibration the protein concentration in the droplet increases and, if conditions are correct, nucleation and ultimately crystallization occur. Other techniques, including microbatch under oil [20] and lipidic cubic phase [21], have been used successfully. Given the comparatively stochastic nature of the crystallization process, structure determination of proteins using this approach remains a very inefficient process. Methods to improve control of the crystallization process would be of great benefit to the community.

A macro-ATR-FTIR spectroscopic imaging approach [2] has been used to study protein crystallization under oil with either single or multiple crystallization droplets applied directly to a ZnSe ATR accessory. Using this setup crystals (>40 µm in size) of lysozyme and thaumatin formed within 1 day and were clearly detectable as ATR-FTIR chemical images which corresponded to crystals visible under a light microscope (Fig. 1.3B). Higher spatial resolution was possible when using a diamond ATR accessory [22] allowing detection of very small crystals (12 µm). Using this approach an integral membrane protein was submitted to crystallization trials and although crystals visible under a light microscope were obtained, ATR-FTIR imaging revealed that these were nonproteinaceous.

Currently there are a number of methods to determine if a crystal is protein. Testing the crystals in an X-ray beam can be performed, however, this requires crystal manipulation and is wasteful of beam time. Protein crystals typically fluoresce strongly under a UV microscope and such an approach is widely used, however, it is not completely reliable in terms of differentiation of protein and nonprotein crystals [23]. Dye-based fluorescence methods have also been reported [24]. Thus ATR-FTIR chemical imaging, which is label-free, nondestructive, and able to very reliably differentiate between protein and nonprotein crystals, represents a viable alternative to these approaches. The Chan et al. study [2] described above focused on crystallization under oil which, while used by researchers in the field, is not one of the most common crystallization techniques.

Glassford and colleagues [3] further applied micro-ATR-FTIR chemical imaging, a technique with higher spatial resolution than macro-ATR-FTIR, to study the crystallization of a number of proteins—in this case, the much more widely used hanging drop approach with the setup shown in Fig. 1.4A with drops of protein solution placed directly on the removable germanium crystal and the reservoir placed below. Using this setup it was possible to both image lysozyme crystals and observe them as they grew (Fig. 1.4B).

Figure 1.4 (A) Micro-attenuated total reflection (ATR)–Fourier transform infrared (FTIR) imaging setup for detection of protein crystals in a hanging drop. (B) Micro-ATR images of a lysozyme crystal obtained after 80, 110, and 140 min, and 20 h [3] indicated by letters C, D, E and F respectively. (C) Spectra for these times as well as 0 and 40 min indicated by the letters A and B respectively. An additional light microscopic image shows typical lysozyme crystals with the approximate imaging area indicated by the dashed box. Source: Reproduced from S.E. Glassford, et al., Micro ATR FTIR imaging of hanging drop protein crystallisation, Vibrat. Spectrosc. 63 (2012) 492–498 with permission from Elsevier.

The small area measured in micro-ATR-FTIR imaging means that it might not be possible to always capture individual nucleations and follow crystal growth. However, because of the high spatial resolution of micro-ATR-FTIR imaging, it is possible to detect crystals of thaumatin and lobster α-crustacyanin as small as 6 µm, much smaller than those detectable using macro-ATR-FTIR spectroscopic imaging [2].

Clearly many factors can affect protein crystallization including the physical properties of the vessel that is being used. Another study utilized ATR-FTIR imaging with a Si ATR element to explore the effect that surface gradient property has on protein crystallization [4]. A gradient of octyltrichlorosilane (OTS), a hydrophobic molecule, was generated on the surface of the ATR element which had been coated with a thin layer of gold (Fig. 1.5A). Gradient formation was monitored by ATR-FTIR spectroscopic imaging (Fig. 1.5B).

Figure 1.5 (A) Overall experimental setup used for the study of gradient surface property on protein crystallization. FPA=focal plane array detector used to measure thousands of spectra in a short period of time. (B) Generation of the OTS gradient on the surface of the attenuated total reflection (ATR) element as assessed by ATR–Fourier transform infrared (FTIR) imaging with images taken at different time points as indicated. The OTS reservoir is at the top of the images. (C) ATR-FTIR imaging of the lysozyme protein crystals which grew on the ATR element with the corresponding light microscopic image. Source: Reproduced with permission from S. Glassford, et al., Chemical imaging of protein adsorption and crystallization on a wettability gradient surface, Langmuir, 28 (6) (2012) 3174–3179. Copyright 2012 American Chemical Society.

Using this setup it was possible to grow lysozyme crystals on the Si ATR element and ATR-FTIR imaging revealed that there was a clear preference for crystal growth on the region of the ATR element with higher concentrations of octyltrichlorosilane and thus on the more hydrophobic regions of the surface (Fig. 1.5C). Although it should be noted that fewer crystals grew on the least hydrophobic areas of the ATR element, those crystals that did grow were of similar dimensions to those that grew at higher density on the more hydrophobic regions of the ATR element. This study only explored crystallization of one protein on this hydrophobic gradient. Clearly it will be interesting to also assess the effects of this surface gradient on crystallization of other proteins including, for example, integral membrane proteins. Such gradient surface properties also have the potential for facilitating adhesion and study of live cells.

1.2.1.2 Eliminating anomalous dispersion with varying angle-macro-ATR

Anomalous dispersion, which poses a challenge for quantitative and qualitative analysis when present in a spectrum, is often observed in the ATR-FTIR measurement when the refractive index of the sample is close to the wavelength of an absorbance band. This dispersion artifact manifests itself as a shift in the strong absorbing spectral bands and could exhibit a derivative-like baseline shape near the bands [25].

The influence of anomalous dispersion on the shifting of the amide bands was reported by Boulet-Audet et al. in a study on protein film conducted on an ATR accessory. It should be noted that the band shift was significantly reduced when a germanium ATR element was used instead of a diamond because of its higher refractive index [26]. Since a shift in the amide I band has serious implications for interpretation of the secondary structure of the protein it is necessary to correct for this. The researchers accounted for the band distortion resulting from dispersion by quantification through determination of the optical constant using ATR [26]. However, Kazarian and coworkers also found that the spectral distortion could be minimized simply by increasing the angle of incidence of the IR beam using specially made apertures [27,28]. This is particularly useful in cases where a diamond ATR element is preferable, for example, where chemical inertness and high resilience to pressure of the ATR element are required.

The use of apertures to physically alter the angle of incidence of the IR beam for ATR-FTIR imaging with a diamond Golden Gate accessory has successfully shown that distortion of spectral bands can be effectively minimized by increasing the angle of incidence [27,28]. The apertures are designed to have various positions that can be fitted onto the condenser lens of the diamond imaging optics, allowing light to pass through at a specific angle of incidence [28].

Comparison of the lysozyme spectrum obtained using a diamond ATR at different angles of incidence reveals that at 37 degrees, the smallest angle of incidence, the amide I peak is at 1636 cm−1 compared to 1645 cm−1 at 45 degrees (Fig. 1.6) [15]. Similarly, the amide II peak also shifted from 1515 to 1535 cm−1 with increasing angle of incidence. Both amide bands shifted toward higher wave number as the angle of incidence increased (Fig. 1.6).

Figure 1.6 Attenuated total reflection (ATR)–Fourier transform infrared (FTIR) spectra of lysozyme film at various angles of incidence. Source: Reproduced from S.E. Glassford, Applications of ATR-FTIR spectroscopic imaging to proteins, in: Chemical Engineering, Imperial College London, London, 2013, p. 158.

It is not possible to completely remove all the spectral distortion due to the limitation in the range of allowed angles of incidence possible with the arrangement and alignment of the Golden Gate IRE. However, as the angle of incidence increases, absorbance of the amide bands is observed. This is because of the shallower depth of penetration of the evanescent wave at a greater angle of incidence of the IR beam. Due to the improved performance offered by variable angle ATR-FTIR measurement, it has been employed extensively for investigation of absorbance change in the z-direction, also known as depth-profiling [29–31], which has the potential to allow study of aspects of protein behavior including protein aggregation.

1.2.1.3 High-throughput analysis of aggregation of a monoclonal antibody by macro-ATR-FTIR spectroscopic imaging

Protein aggregation can have major impact on the use of biotherapeutics including monoclonal antibodies (mAbs). Aggregation of a therapeutic mAb is associated with reduced efficacy and can lead to highly undesirable immunological responses when administered [32,33]. It is thus important to have an effective means of assessing the propensity of a protein sample to aggregate. There are a number of methods for studying protein aggregation including size-exclusion chromatography (SEC), dynamic light scattering, and circular dichroism [34], however, many of the available methods are unable to observe changes in aggregation in protein samples in situ, or are limited in their ability to quantify aggregation or limited in the range of conditions which can be tested. Protein aggregation of purified protein in vitro can be effectively analyzed using macro-ATR-FTIR spectroscopic imaging. Boulet-Audet et al. utilized a high-throughput approach to study the aggregation of mAbs in a range of different pH and ionic strength conditions, including the conditions experienced by mAbs during the isolation process [1]. In this case PDMS wells were cast allowing 12 samples to be analyzed simultaneously. The wells were mounted on to a ZnSe ATR crystal, which was used in preference to a germanium crystal due to the greater absorbance of the bands produced. Data analysis was performed in protein samples in a range of buffer conditions while the protein was being heated. Heating is commonly used as a stress to induce aggregation in a timescale more suited to laboratory experiments. The formation, upon heating, of aggregates and/or precipitates which are heavier than the monodispersed protein in the bulk solution results in a larger amount of protein being in contact with the ATR crystal (Fig. 1.7). Thus the relative stabilities of the protein in the different conditions can be readily compared. However, the real power in ATR-FTIR spectroscopy is that in addition to protein stability analysis, the technique also reveals key changes in the secondary structure of the mAb that lead to aggregation in a high-throughput manner over a period of about 30 minutes [1] (Fig. 1.8).

Figure 1.7 ATR-FTIR spectroscopic images generated using the amide II band area after 2000 s. The unit of the scale bar is integrated absorbance in cm−1. The polygons represent the integrated region of interest of each well. The field of view covered approximately 9.8 mm along the Y axis and 7.0 mm along the X axis. Source: Reproduced with permission from M. Boulet-Audet, et al. High-throughput thermal stability analysis of a monoclonal antibody by attenuated total reflection FT-IR spectroscopic imaging. Anal. Chem. 86 (19) (2014) 9786-9793. Copyright 2014 American Chemical Society.

Figure 1.8 (A) Attenuated total reflection (ATR)–Fourier transform infrared (FTIR) spectra of a dried film of native IgG from a demineralized solution (blue), a dried film from thermally induced aggregated IgG solution (red), and the difference between them (green). The dried native film was normalized for comparison using the average absorbance between 1300 and 1350 cm−1. (B) ATR-FTIR spectra of buffer only (blue), 1 mg/mL native IgG solution (light green), precipitated aggregates induced by heating of a 1 mg/mL IgG solution (red), difference between the native IgG solution and the buffer (dark green), its second derivative (teal), as well as the difference between the aggregated IgG and the buffer (fuchsia) and its second derivative (pink). (C) Schematics illustrating the native IgG (green box) and the increased local concentration in the volume probed by ATR-FTIR spectroscopy caused by heat-induced protein aggregates precipitation (red box). Source: Reproduced with permission from M. Boulet-Audet, et al. High-throughput thermal stability analysis of a monoclonal antibody by attenuated total reflection FT-IR spectroscopic imaging. Anal. Chem. 86 (19) (2014) 9786–9793. Copyright 2014 American Chemical Society.

1.2.1.4 Protein purification: cleaning-in-place for immunoaffinity resin and in-column ATR-FTIR spectroscopy

Another field in which ATR-FTIR spectroscopy has obtained novel insights is protein purification. mAbs, proteins widely employed for treatment of a range of cancers and chronic diseases such as rheumatoid arthritis, constitute a fast-growing area of biopharmaceuticals [35]. However, the high cost associated with their production has a major impact on the production scale, profitability, and, ultimately, patient availability of mAbs [36]. Antibody purification is a key part in the mAb production pipeline and involves an ultrafiltration step to concentrate the tissue culture media containing the mAbs and a number of chromatography steps. The most effective purification step employs affinity chromatography, utilizing the very specific interaction between mAbs and protein A [37] at pH 7, with the mAbs eluting in buffer at pH 3. This protein A affinity chromatography step is a major contributor to the cost of mAb production, since the resin used accounts for more than 50% of the total production expense [36,38]. A particular problem is the relatively low life span of the resin, thought to be due to nonspecific binding of contaminants and loss of the protein A ligand. Such contaminants can block pores or hinder the access to surface ligands. To prevent contaminant buildup, washing the resin with alkaline solutions is often adopted as part of the resin regeneration protocol [cleaning in place (CIP)]; these protocols increase resin life span, however, harsh regeneration conditions also contribute to degradation or leaching of the ligands over time. Changes in the binding capacity of the resin can be assessed in a number of ways but most of these are indirect. A mechanistic approach consists of measurement of Langmuir adsorption isotherms [38], with other methods relying on sodium dodecyl sulfate polyacrylamide gel electrophoresis and analytical SEC in an attempt to analyze eluate samples for the presence of leaked protein A and host cell protein content [39].

A better understanding of the issues associated with reduction in binding capacity through more direct ATR-FTIR spectroscopic assessment of on-column changes during both protein isolation steps and the CIP procedures used to remove bound contaminants is still needed.

Since ATR is a surface-sensitive technique that relies on close contact between sample (e.g., resin agarose beads) and internal reflection element (IRE, e.g., diamond), the beads were pressed onto the IRE under a controlled load. A plunger was used to exert pressure and a load cell was employed to precisely control the load applied. An optimal load maintains effective contact between the beads and the IRE without damaging the beads or affecting flow across the IRE [12]. Fig. 1.9A and B show schematics of the experimental setup. Using this setup it was possible to detect the protein A ligand on the resin beads and mAb binding, as well as changes in protein conformation and proteolysis caused by addition of a high concentration of NaOH (Fig. 1.9C) [12].

Figure 1.9 Experimental setup utilized for the analysis of protein A resin with ATR-FTIR spectroscopy and imaging (A). Zoomed in view of the setup (B); a PDMS cell is clamped to the ATR accessory using an acrylic top plate and to exert pressure on the resin beads a plunger is utilized. To measure the force applied, a load cell is placed on top of the plunger. (C) Resin in equilibrium is injected on the IRE surface and pressed under controlled load; ATR-FTIR spectra of resin beads with/without mAb (IgG) are measured as a function of denaturant concentration (NaOH). Source: Reproduced with permission from Springer Nature (M. Boulet-Audet, B. Byrne, S.G. Kazarian, Cleaning-in-place of immunoaffinity resins monitored by in situ ATR-FTIR spectroscopy, Anal. Bioanal. Chem. 407 (2015) 7111–7122. M. Boulet-Audet, S.G. Kazarian, B. Byrne, In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies, Sci. Rep. 6 (2016) 30526).

The resin is usually washed with alkaline solutions according to one of a number of established CIP protocols to prevent buildup of contaminants and thus a decrease in binding capacity. The ATR-FTIR spectroscopic approach described above allowed comparative analysis of a range of different CIP protocols to assess both their relative effectiveness at removing contaminants and their effects on the protein A ligand [12].

More recent studies explored mAb purification under dynamic flow during protein A affinity chromatography by direct monitoring using in-column ATR-FTIR spectroscopy whilst also monitoring eluates by standard UV spectroscopy [13]. This utilized a modified version of the setup shown in Fig. 1.9A and B, allowing controlled and dynamic alteration of the buffer surrounding the beads on the IRE, allowing mAb binding, elution, and CIP cycles to be continuously monitored [13]. ATR-FTIR spectra revealed that protein A leaches from the column during CIP and that even after CIP, contaminants remain bound to the column reducing the binding capacity of the resin [13]. The types of contaminants bound to protein A columns have been a key unknown for a very long time and this has limited the development of novel protocols and approaches to reduce contaminant binding in industrial settings. One major advantage to ATR-FTIR spectroscopy is that since each species in a sample has a distinct chemical fingerprint it is possible to discern what types of molecules are in a particular sample. The analysis of the resin beads indicated that no lipid or DNA was detectable, thus it is likely that most if not all the contaminant material is proteinaceous in nature [13].

Further optimization of the purification and CIP protocols are possible using this type of setup, since even small improvements in the life span of the protein A columns has potential to significantly reduce the cost of industrial scale production of therapeutic mAbs.

1.2.2 Micro-FTIR spectroscopic imaging

1.2.2.1 Association with disease: time-resolved imaging of protein aggregation in living cells

FTIR microspectroscopy has been used to study protein aggregates in tissues and cells in vivo, in vitro, and in situ to better understand a wide range of protein-folding diseases. Protein aggregates are usually detected based on the presence of a specific marker band, such as the amide I band or amide II band, as a result of the formation of intermolecular β-sheet structures visible in the FTIR spectrum [40].

A study was conducted by Mitri et al., whereby the response of live mammary breast adenocarcinoma cell lines to severe heat-shock was monitored and recorded using FTIR microspectroscopy. As a result of the stress induced on the live cells, persistent intracellular accumulation of extended β-sheet protein aggregates was detected [41]. Thus the capability of the IR microscopy for real-time measurement of in-depth variation of the cellular biochemical profile was confirmed. This IR microspectroscopic approach has also been applied to explore the kinetics of the formation of inclusion bodies within growing Escherichia coli cells under various expression conditions [42]. In this case, the spectroscopic analysis suggested that the recombinant protein is more prone to aggregate at 37°C. Interestingly, it is also possible to obtain complementary information on cell processes that accompany protein aggregation, including, for example, the effects on cell membranes from further analysis of the IR spectra obtained, such as the effects of recombinant protein misfolding and aggregation on bacterial membranes [43].

A further study by Ami et al. used an FTIR microscope to screen cardiac tissues for the presence of characteristic amyloid deposits [44]. These results demonstrated that tissue analysis by FTIR spectroscopy not only aids in diagnosis, but could also detect the presence of fibrillar aggregates in clinical specimens. Futhermore, this approach can also provide meaningful spectra allowing analysis of aggregate core properties, including the strength of the hydrogen bonds, which affect the compactness of aggregate structure and thus may affect their functions.

The challenge of examining small aggregates can be overcome by the use of a synchrotron-based IR microscope. One of the earliest uses of this approach investigated the secondary structure of Aβ protein directly within the brain tissue samples of a deceased subject suffering from Alzheimer’s [45]. They found that the spectra obtained in vivo and in situ are able to associate protein behavior with the surrounding environment. Since then, synchrotron-based FTIR study in Alzheimer’s disease has also revealed that the density of dense-core amyloid plaques is approximately 1.5 times higher than the surrounding brain tissue (Fig. 1.10) [46].

Figure 1.10 (A) Thioflavin S-stained PSAPP mouse brain tissue showing three plaques. (B) Infrared image of the same tissue showing the distribution of protein measured by the Amide II band. (C) Infrared spectra collected from the areas marked with asterisks in (A) and (B), showing the relative amount of protein in the center of a plaque (black) and the surrounding tissue (red). All scale bars are 5 μm [46]. Source: Reproduced with permission from Elsevier.

More recently, time-lapse infrared imaging has been applied to aggregate formation in a cell culture model of amyotrophic lateral sclerosis (ALS). ALS is a neurodegenerative disease that causes death of neurons controlling voluntary actions and is associated with mutations in the antioxidant protein copper–zinc superoxide dismutase (SOD1) [47]. With the ability of FTIR spectroscopy to distinguish between parallel and antiparallel β-sheet structure, real-time imaging of SOD1 aggregation in the cell culture model provided insight into structural intermediates, timescale, and mechanisms of cell toxicity triggered by aggregation.

1.3 Further applications

1.3.1 Monitoring low-concentration protein conformational change with QCL spectroscopy; potential of micro-ATR-FTIR imaging for analysis of tissues

This chapter has highlighted ATR-FTIR spectroscopic imaging as a powerful and versatile technique for the study of protein crystallization and aggregation using isolated protein samples, as well as protein aggregation in in vivo tissues and cell samples and protein behavior on a chromatographic column. Future developments in this field will only enhance the potential of infrared imaging in enabling the further understanding of protein behavior. One of the limitations of ATR-FTIR spectroscopy and spectroscopic imaging is relatively low sensitivity of detection because of the relatively short path lengths used, particularly for imaging where a single reflection IRE is employed. This limitation can be overcome through the use of much more powerful infrared sources, such as a quantum cascade laser (QCL), rather than the thermal sources used in conventional spectrometers. QCLs, which have been applied to study tissues, are tunable laser sources which output discrete frequencies of infrared light brighter than a synchrotron but small enough for benchtop instruments [48].

With conventional ATR-FTIR spectroscopy protein of low concentration may be undetectable; however, QCL-based infrared spectroscopy has been demonstrated to measure protein at a concentration as low as 0.25 mg/mL in transmission mode [49]. The observed results of the novel setup agree with those obtained from conventional infrared spectroscopic measurement. The robust measurement of protein at low concentrations is made possible with QCL because such sources produce mid-infrared light several orders of magnitude brighter than that from a silicon carbide heating element or synchrotron. The most notable recent studies on protein samples have utilized QCL infrared spectroscopy to capture dynamic changes in the formation of β-sheet aggregates at varying pH values and protein concentrations [50], and the screening of bovine milk [51]. In addition, a time-resolved study of enzymatic reactions was accomplished with a novel dual-comb QCL system [52]. In the future we expect the adoption of QCLs will allow the application of ATR-FTIR spectroscopy and spectroscopic imaging to an increasingly wide range of protein studies.

As more work progresses in this field, improved in-depth understanding of protein structure is inevitable. Not only the secondary structure of proteins can be explored, but it is envisioned that nano-FTIR spectroscopy will provide insights into the quaternary structure of proteins. This type of spectroscopy circumvents the diffraction-limited resolution of FTIR microscopy by focusing infrared light onto a metalized AFM tip, similar to tip-enhanced Raman scattering spectroscopy. Infrared images of nanoscale spatial resolution as low as 20 nm can be recorded. Amenabar et al. [53] applied nano-FTIR to study 3-nm-thin individual insulin fibrils, a widely used model system for neurodegenerative disorders, including Alzheimer’s and Parkinson’s disease. Their results not only demonstrated that nano-FTIR has a high sensitivity to differentiate individual protein complexes, but also revealed that the presence of α-helices in the shell of insulin fibrils is likely to contribute to aggregation of the fibrils [53]. Protein microarrays are pushing the boundaries of high-throughput analysis of proteins. Coupled with IR microscopic imaging, the sensitivity of the microarrays was tested with lysozyme, albumin, and hemoglobin solutions in a total of 45 spots, each with an approximate diameter of 100 µm, recorded simultaneously over a few minutes. Despite the minute amount of protein in each spot, high-quality spectra were obtained from single monolayers of the different protein with FTIR microspectroscopy. These findings demonstrate that it is possible for IR imaging to be employed for the detection of protein binding to other proteins, substrates, etc. [54]. In future, there are opportunities for still further development of the technology in terms of the methodology used for capture and analysis of spectra, as well as improvements in instrumentation which will increase both sensitivity and the breadth of investigations that can be carried out on proteins. Additionally, future developments in standard protocol and chemometric methods may render spectral analysis of proteins quicker and easier.

Finally, a capability, developed in the authors’ laboratory [30,31,55], of ATR-FTIR spectroscopic imaging with variable angle of incidence to obtain a depth profile of samples may prove to be useful for assessment of processes at the surface layers of the resins in stationary chromatographic columns. This depth profiling capability with the sub-micrometer spatial resolution (the spatial resolution in z-direction when depth profiling is not limited by diffraction of light, unlike the resolution in the x and y directions) reveals chemical information by studying the variation in a sample's composition as a function of depth in a nondestructive way.

1.4 Conclusions

The research presented in this chapter summarizes the applicability of ATR-FTIR spectroscopic imaging and ATR-FTIR spectroscopy to a range of different protein systems and presents new opportunities for applications of ATR-FTIR imaging to challenges in this field. Overall, spectroscopic imaging capability could have benefits for research in structural proteomics and contribute to enhancement of crystallization conditions and optimization of protein isolation protocols. In particular, this powerful analytical and research tool has significant potential for the study of a range of different proteins and protein complexes in addition to important biopharmaceutical and bioprocessing targets. This chapter presented novel approaches, such as macro-ATR-FTIR imaging, for high-throughput protein analysis of both protein crystallization and aggregation; this approach is also well-suited to study the effects of wettability gradient surfaces on protein adsorption and crystallization. Micro-ATR-FTIR imaging can be used to study hanging drop protein crystallization with high spatial resolution.

A significant breakthrough has been the application of ATR-FTIR spectroscopy to the study of loss in protein A resin binding capacity. This approach allowed monitoring of both ligand conformation and the amount of adsorbed protein on the beads and as a result demonstrated ligand denaturation and proteolysis caused by CIP protocols and irreversible protein contaminant binding, as described in this chapter.

The importance of the understanding of resin aging in stationary chromatography columns was recently highlighted in a discussion in Trends in Biotechnology [56]. It has been suggested that it is necessary to monitor the events that result in efficiency loss in chromatography columns. At present there is no established procedure for this as an industry standard [56]. Therefore the advanced FTIR spectroscopic approaches [57], discussed in this chapter, to monitor column aging during operation may contribute to the understanding of column aging and help to establish procedures in industry pipelines.

Acknowledgments

The authors wish to thank the BBRSC and the Bioprocessing Research Industry Club (BRIC) for funding this research (BB/K0111030/1 and BB/R019533/1). We also thank BBSRC for BBSRC Targeted Priority Studentships in the area of Bioprocessing and for iCASE Studentships.

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Chapter 2

Light-induced difference Fourier-transform infrared spectroscopy of photoreceptive proteins

Hideki Kandori and Kota Katayama,    Department of Life Science and Applied Chemistry and OptoBioTechnology Research Center, Nagoya Institute of Technology, Nagoya, Japan

Abstract

Light-induced difference Fourier-transform infrared (FTIR) spectroscopy is a powerful, sensitive, and informative method for studying protein structural changes in photoreceptive proteins. Strong absorption of water in the IR region is always an issue in this method. However, if water content in the sample is controlled during measurements, this method can provide detailed structural information on a single protein-bound water molecule. We have established three sample preparation methods, including hydrated film, redissolved sample, and concentrated solution. The hydrated film-based method is preferably used for the FTIR measurements of many photoreceptive proteins such as microbial and animal rhodopsins, LOV and BLUF domains. In this method, accurate difference FTIR spectra are obtained in the whole mid-IR region (4000–800 cm−1). On the other hand, redissolved sample- and concentrated solution-based methods are used for the measurements with enzymatic turnover in photolyase, and for samples less tolerant to drying. In this chapter, we describe how light-induced difference FTIR spectroscopy was applied to study photoreceptive proteins, along with a summary of the outcome on our understanding of microbial rhodopsins, animal rhodopsins, and