Microfluidic Modulation Spectroscopy (MMS) is a novel spectroscopic tool that directly addresses the limitations of current technologies by facilitating direct, label-free analysis of secondary structure over a wide concentration range (0.1 to 200 mg/mL) in complex formulations without the need for buffer exchange or dilution. MMS utilizes a tunable Quantum Cascade Laser that is at least 1000 times more intense than conventional light sources, allowing for optimal signal-to-noise ratio to achieve ultra-high sensitivity. This sensitivity allows MMS to see changes missed by other technologies and gives you greater confidence in your biophysical characterization. Now with the power of the Aurora and Apollo MMS systems, the sample (biomolecule-in-buffer) solution and a matching buffer reference stream are introduced into the transmission cell under continuous flow and then rapidly modulated (1–5 Hz) across the laser beam path. This produces nearly drift-free, background-compensated, differential absorbance scans of the Amide I band (1700-1600 cm-1) and allows changes in HOS to be easily measured and compared.
Microfluidic Modulation Spectroscopy is a novel tool that can strengthen a bioanalytical tool kit by providing critical insight through all phases of drug development. This technique enhances current toolkits with the ability to monitor 5 key biomolecule characteristics including aggregation, quantitation, structure, stability, and similarity. MMS provides the following benefits:
- A reduction in time and cost through automation (24/96 well formats).
- Improvement in product quality with measurements of previously undetectable changes in biomolecule structure.
- De-risking of downstream product failures through earlier up-stream monitoring.
- Generation of quality data for confident decision-making in order to accelerate development timelines.
Are there benefits to using absorption methods such as MMS, FTIR or CD complementary to scattering methods like DLS?
Yes! In addition to particle sizes interrogated with DLS, absorption methods provide fundamental structural insights. In the case of proteins, this helps to understand underlying mechanisms that may stabilize or destabilize protein formulations, for example as a response to external stresses or in course of binding events.
What is the difference between absorption spectroscopy and scattering techniques?
In absorption spectroscopy, the interaction of matter with EMR is interrogated by sending radiation through a sample and recording the portion that is transmitted. The difference between incoming and transmitted light, i.e. absorption, provides fundamental information about the nature of that sample. As detailed above, the information obtained from this kind of experiment depends on the frequency range of the EMR used. As an example, IR absorption spectroscopy probes molecular vibrations which may provide structural and dynamic information about the studied sample. In MMS, the frequency window used is the so-called Amide I band which allows for secondary structural decomposition of protein samples.
In contrast to transmission absorption methods, scattering techniques record the light that is scattered from samples constituent. There are mainly two different scattering mechanisms, namely elastic (Rayleigh) and inelastic scattering. Inelastic can also be used to get spectral information about a sample due to absorption or emission. An example of that is Stokes/Anti-Stokes Raman spectroscopy. Rayleigh scattering can be used to study the size of small particles by recording the fluctuation of scattered light resulting from Brownian motion of particles in solution. This principle is used in Dynamic Light Scattering (DLS) to study the formation of protein aggregates by identifying particle size distributions. A detailed introduction to DLS is given in this video: Dynamic Light Scattering – Structure Matters (structure-matters.com)
How does circular dichroism compare to MMS as a protein spectroscopy technique?
Both circular dichroism (CD) and MMS are spectroscopic techniques that measure protein secondary structures. Both these techniques are known for their high sensitivities and the ability to provide accurate assessment of the secondary structures. These techniques differ by their principles. Because proteins are chiral molecules, they interact with circularly polarized light, and the different secondary structures in protein give rise to differential absorbance upon interactions with left-handed and right-handed polarized light. CD uses this principle to detect the different secondary structures in proteins. MMS on the other hand, probes the vibrationa lresponses of the backbone carbonyl groups of the protein. For CD, the signal quality depends on the total absorbance of a sample. Because of this limitation, the concentration range for CD is narrow (0.01-1 mg/mL) and the buffer compatibility is poor. In contrast, because MMS uses IR spectroscopy andallows for real-time buffer subtraction, it allows a wide range of absorbance and is not limited by buffer signals. MMS can measure samples at a wide concentration range (0.1-200 mg/mL) and is compatible with most buffers and excipients. A direct comparison between CD, FTIR and MMS is given in this application note:
How does Fourier Transform Infrared compare to MMS as a protein spectroscopy technique?
Fourier Transform Infrared (FTIR) and MMS are both IR-based spectroscopic techniques, meaning that the way they probe the proteins (backbone carbonyl groups) is the same. Therefore, both techniques measure the secondary structure of proteins. The main differences between FTIR and MMS are the sensitivity and concentration range. With the high-power quantum cascade and the microfluidic flow cell that allows for real-time buffer subtraction, MMS largely improves the signal-to-noise ratio and provides 30x sensitivity compared to FTIR (Kendrick, B. S.; Gabrielson, J. P.; Solsberg, C. W.; Ma, E.; Wang, L. Determining Spectroscopic Quantitation Limits for Misfolded Structures. Journal of Pharmaceutical Sciences 2020, 109 (1), 933–936. https://doi.org/10.1016/j.xphs.2019.09.004).
Typically, FTIR requires high concentration of proteins (10 mg/mL) for measurements, but MMS can measure proteins at a range from 0.1 to >200 mg/mL. In addition, compared to FTIR’s manual workflows, MMS is a fully automated technique. An entire workflow from high throughput (up to 96-well plate) sample measurement to cleaning, baselining, and data analysis can be done with a single mouse click using MMS.
What are some key factors to consider when quantifying protein concentration using IR spectroscopy vs other spectroscopic methods?
IR spectroscopy probes the amide carbonyl group in the protein backbone, and as a result the IR absorbance is directly proportional to the number of amide groups in a protein. IR absorbance is therefore a direct measurement of the overall mass of the protein. One important factor to consider is that because the backbone carbonyl groups can form different hydrogen bonds depending on the type of secondary structures they are in, the IR absorption peak can be in a range, between 1600 and 1700 cm-1,or 5882 and 6250 nm. UV-vis spectroscopy on the other hand, probes the aromatic groups in the protein which mainly come from the side chains of specific amino acids such as tryptophan, tyrosine, and phenylalanine. These groups have a peak absorption at 280 nm, commonly known as A280. The major limitation using A280 to quantify protein concentration is that as the size of the protein gets smaller, the portion of these aromatic amino acids in a protein become more inconsistent. For small peptides that lack certain aromatic amino acids, UV-vis becomes inaccurate or even impossible to quantify concentrations.
Why is it important to use spectroscopy to measure protein concentration alongside protein structure?
Other than providing structural information about the proteins, spectroscopy allows a quick and easy way to quantify protein concentration using the Beer-Lambert Law, or Beer’s Law. For proteins in solution, Beer’s Law states that when light passes through the solution, the absorbance is proportional to the concentration of the protein, given that the optical path length and the absorptivity of the protein stay constant. This gives spectroscopic techniques advantages to not only analyze protein structures but also provide accurate quantification of protein concentrations.
Is Microfluidic Modulation Spectroscopy used only to characterize protein?
MMS can be used to characterize any biomolecules that have distinct absorbance in the amide I region of the IR spectrum, for example: nucleic acids and AAVs are both emerging applications areas for MMS.
Stay tuned for upcoming app notes in these areas!
Is MMS considered a protein spectroscopy technique?
Yes! MMS uses mid-Infrared light to detect protein structure. Specifically, we use the amide I band in the IR spectrum to detect the carbonyl stretching vibration in the protein backbone. This vibration will be very different if the carbonyl bond is making hydrogen bonds as part of an alpha-helix or beta-sheet secondary structure and we can quantitate the percentages of different secondary structural motifs based on this principle.
What are some of the primary applications of protein spectroscopy?
There are many different applications depending on the technique being used. Here are some examples:
UV-vis spectroscopy is used to quantitate protein concentration by using visible and ultraviolet light.
Microfluidic modulation spectroscopy (MMS) and Fourier transform infrared spectroscopy (FTIR) both use infrared light and determine protein secondary structure in the amide I band, a spectral window within the MIR region. This region is sensitive to the hydrogen bonds in the protein backbone.
Circular dichroism (CD) spectroscopy uses circularly polarized light to determine protein secondary structure using chirality.
Fluorescence spectroscopy uses intrinsic fluorescence of proteins caused by the aromatic amino acids tryptophan, tyrosine, and phenylalanine. This technique probes tertiary structure as these amino acids are hydrophobic and often hidden in the core of the protein. As a protein is unfolded, these amino acids will become solvent exposed and excitable using specific wavelengths of light.
Nuclear magnetic resonance (NMR) spectroscopy uses radio frequency pulses to excite the nuclei within samples. This is most commonly used in small molecule work, but 2-dimensional NMR can be used to study protein structure to get high resolution information about the 3-dimensional tertiary and quaternary structure.
What is meant by the term "protein spectroscopy"?
Protein Spectroscopy refers to the study of proteins by using their interaction or emission of electromagnetic radiation (EMR).
This includes the use of x-rays, ultraviolet and visible light, infrared light, microwaves, and radio waves. Some examples of protein spectroscopy techniques are: UV-vis, CD, MMS, FTIR, Fluorescence, and NMR.