Accurate anisotropy recovery from fluorophore mixtures using Multivariate Curve Resolution (MCR)

Casamayou-Boucau, Yannick; Ryder, Alan G.

doi:10.1016/j.aca.2017.11.031

Cited by 11 publications

(8 citation statements)

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“…EEM three-way arrays can be decomposed using bilinear methods such as MCR-ALS [39] or trilinear models like PARAFAC [33]. MCR-ALS offers the advantage to switch between bilinear, partial trilinear, and fully trilinear models [35], however, rotational ambiguities can be associated with the bilinear solutions [22,23,29,30,39,40]. Thus, different constraints and augmentation modes can yield different solutions from the correct solution [30].…”

Section: Data Analysis and Chemometric Methodsmentioning

confidence: 99%

“…Previously we showed that ARMES of Human Serum Albumin (HSA) using MCR modelling yielded multiple components [25], however these studies used thin film polarizers with no transmission below 300 nm which affected the TSFS data structure. Further developments of ARMES to enable short (<300 nm) wavelength excitation [26] and validation measurements have shown that one can accurately recover individual fluorophore emission in the absence of inner filter effects (IFE) and FRET [35]. These early studies also undertook MCR analysis on all four polarization measurements, mainly in an effort to confirm that the chemometric modelling was robust [26,36].…”

Section: Introductionmentioning

confidence: 99%

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Investigating native state fluorescence emission of Immunoglobulin G using polarized Excitation Emission Matrix (pEEM) spectroscopy and PARAFAC

Steiner-Browne

Elcoroaristizabal

Casamayou-Boucau

et al. 2019

Chemometrics and Intelligent Laboratory Systems

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Intrinsic fluorescence spectroscopy (IFS) measurements for protein structural analysis can be enhanced by the use of anisotropy resolved multidimensional emission spectroscopy (ARMES). ARMES attempts to overcome the tryptophan (Trp) and tyrosine (Tyr) spectral overlap problem and resolve emitting components by combining anisotropy measurements with chemometric analysis. Here we investigate for the first time the application of polarized excitation-emission matrix (pEEM) measurements and Parallel Factor (PARAFAC) analysis to study IFS from an Immunoglobulin G (IgG) type protein, rabbit IgG (rIgG), in its native state. Protein IFS is a non-trilinear system primarily because of Förster resonance energy transfer (FRET). Non-trilinearity is also caused by inner filter effects, and Rayleigh/Raman scattering, both of which can be corrected by data pre-processing. However, IFS FRET cannot be corrected for, and thus here we carefully evaluated the impact of various different data preprocessing methods on IFS data which used for PARAFAC. Care must be taken with data preprocessing and interpolation, as both had an impact on PARAFAC modelling and the recovered anisotropy values, with residual shot noise from the Rayleigh scatter which overlapped the emission blue edge being the root cause.pEEM spectra from thawed rIgG solutions (15-35 °C temperature range) were collected with an expectation being that this temperature range should cause sufficient emission variation to facilitate component resolution but without major structural changes. However, the only significant changes observed were of the overall intensity due to thermal motion induced quenching and this was confirmed by the PARAFAC scores. PARAFAC resolved one major component (>99%) for the emission data (polarized and unpolarized) which mostly represented the large Tyr-to-Trp hetero-FRET process, with a second, very weak component (<1%) apparently a contribution from directly excited Trp emission. PARAFAC scores recovered from normalized pEEM data showed minimal change which was further proof for negligible structural change. The results of this study serves as the starting point for the use of PARAFAC analysis of IFS from IgG type proteins and important processes such as denaturation and aggregation.

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Section: Data Analysis and Chemometric Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Investigating native state fluorescence emission of Immunoglobulin G using polarized Excitation Emission Matrix (pEEM) spectroscopy and PARAFAC

Steiner-Browne

Elcoroaristizabal

Casamayou-Boucau

et al. 2019

Chemometrics and Intelligent Laboratory Systems

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“…Still, MDF measurements generate large amount of spectral data that require careful interpretation and data reduction while retaining the essential information, which is generally accomplished using chemometric multivariate analysis tools. Curve resolution methods like PARAllel FACtor analysis (PARAFAC; Elcoroaristizabal et al, 2015; Murphy et al, 2013) or multivariate curve resolution‐alternating least squares (MCR‐ALS; Casamayou‐Boucau & Ryder, 2018; de Juan et al, 2014) can sometimes resolve the analyte of interest spectrum, from the background or interferents, but they are generally subjected to significant matrix effects in complex, biogenic solutions. Multivariate regression methods like unfolded (U‐PLS) or N‐way partial least squares (N‐PLS) usually provide better predictive performance, but they have problems where the relationship between signal and concentration is very nonlinear such as in bioreactors (Olivieri, 2018).…”

Section: Introductionmentioning

confidence: 99%

Development of a rapid polarized total synchronous fluorescence spectroscopy (pTSFS) method for protein quantification in a model bioreactor broth

Boateng

Elcoroaristizabal

Ryder

2021

Biotech & Bioengineering

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Protein quantification during bioprocess monitoring is essential for biopharmaceutical manufacturing and is complicated by the complex chemical composition of the bioreactor broth. Here we present the early‐stage development and optimization of a polarized total synchronous fluorescence spectroscopy (pTSFS) method for protein quantification in a hydrolysate‐protein model (mimics clarified bioreactor broth samples) using a standard benchtop laboratory fluorometer. We used UV transmitting polarizers to provide wider range pTSFS spectra for screening of the four different TSFS spectra generated by the measurement: parallel (||), perpendicular (⊥), unpolarized (T) intensity spectra and anisotropy maps. TSFS|| (parallel polarized) measurements were the best for protein quantification compared to standard unpolarized measurements and the Bradford assay. This was because TSFS|| spectra had a better analyte signal to noise ratio (SNR), due to the anisotropy of protein emission. This meant that protein signals were better resolved from the background emission of small molecule fluorophores in the cell culture media. SNR of >5000 was achieved for concentrations of bovine serum albumin/yeastolate 1.2/10 g L–1 with TSFS||. Optimization using genetic algorithm and interval partial least squares based variable selection enabled reduction of spectral resolution and number of excitation wavelengths required without degrading performance. This enables fast (<3.5 min) online/at‐line measurements, and the method had an LOD of 0.18 g L–1 and high accuracy with a predictive error of <9%.

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“…For proteins, where more information is needed one can use polarized EEM (pEEM) measurements and chemometric data analysis. When factor-based analysis can be implemented, this anisotropy resolved multidimensional emission spectroscopy [18,19] can be used to explore individual fluorophore emission contributions. .…”

Section: Introductionmentioning

confidence: 99%

Multi-attribute quality screening of immunoglobulin G using polarized Excitation Emission Matrix spectroscopy

Silva

Elcoroaristizabal

Ryder

2020

Analytica Chimica Acta

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Accurate anisotropy recovery from fluorophore mixtures using Multivariate Curve Resolution (MCR)

Cited by 11 publications

References 50 publications

Investigating native state fluorescence emission of Immunoglobulin G using polarized Excitation Emission Matrix (pEEM) spectroscopy and PARAFAC

Investigating native state fluorescence emission of Immunoglobulin G using polarized Excitation Emission Matrix (pEEM) spectroscopy and PARAFAC

Development of a rapid polarized total synchronous fluorescence spectroscopy (pTSFS) method for protein quantification in a model bioreactor broth

Multi-attribute quality screening of immunoglobulin G using polarized Excitation Emission Matrix spectroscopy

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