Intrinsic Fluorophores Research Articles

AN EX-VIVO SPECTRAL AND LIFETIME-DECAY ANALYSIS OF 5-ALA DERIVED PPIX flUORESCENCE: OBJECTIVE flUORESCENCE MAPPING IN GLIOMA SURGERY

Abstract AIMS 5ALA/Protoporphyrin IX (PpIX) fluorescence guided surgery (FGS) in high-grade glioma has been shown to increase resection and survival, despite this benefits are limited in low-grade glioma by low emission and subjective interpretation. Quantitative fluorescence could address these issues, however is challenging due to photobleaching and variations in tissue induced distortion and PpIX photochemical state that are poorly understood. Lifetime-decay measures intrinsic fluorophore properties and is not influenced by these factors, but is difficult to measure intra-operatively. We used paired optical and fluorescence spectroscopy with lifetime decay mapping to characterise PpIX emission within the glioma tissue microenvironment. METHOD Biopsies were collected from 20 patients undergoing glioma FGS, along with 4 controls from partial temporal lobectomies. Paired measurements of emission and lifetime were matched with optical property readings. Lifetime and emission ratios at 635 and 620nm were calculated (PpIX635/620), corresponding to the primary emission peak for the photochemical states of PpIX in tissue. Raw and optically corrected emission was correlated with lifetime decay. RESULTS 205 specimens were analysed by WHO grade (56 Grade4, 66 Grade3, 37 Grade2, 20 control). PpIX635/620 emission was 0.93 in control (CI 0.91-0.95), 1.03 in grade 2 tumours (CI 0.95-1.26), 1.56 in grade 3 (CI 1.22-1.91) and 6.99 (CI 5.78-8.20) in grade 4. PpIX635/620 lifetime correlated with emission (R2 =0.98, range 0.94-1.57). Correlation of emission with lifetime readings improved significantly following optical correction at 635nm (raw R2 =0.81, corrected R2 =0.90) and 620nm (raw R2 =0.44, corrected R2 =0.86). CONCLUSION PpIX620 and PpIX635 photochemical states are present in human glioma, with an increase in PpIX635/620 suggesting increased WHO grade. The ability to distinguish these forms holds the potential to provide real-time data for tumour differentiation and pathology. Accurate characterisation and correction of tissue induced distortion significantly improves the ability to detect, characterise and quantify PpIX intra-operatively.

Engineering the apparent quantum yield and emission rate of fluorophore molecules by coupling fluorophore dipoles with plasmon modes of gold using low frequency electric fields

Abstract Localized surface plasmons produced by gold and silver nanostructures have been utilized to enhance the intensity of fluorophore molecules. The issue with using nanostructure plasmons for fluorescence enhancement is their short-range nature (5–50 nm from the nanostructures), which limits accessibility to a few molecules. In addition, fluorophore dipoles needed to be aligned with the plasmon electric fields to maximize the fluorescence enhancement. To address these issues, we used low-frequency electric fields (<5 MHz) and commercially available nanorod and nanosphere samples and studied their effectiveness in enhancing the fluorescence of fluorophore-labeled short single-stranded DNA molecules (22 bases). We demonstrated that DNA molecules and nanorod particles can effectively be manipulated around the charging frequency of DNA molecules (∼3 MHz). Nanorod particles enhanced the fluorescence emission rate by ∼50-fold. When the 3 MHz electric field was introduced, the emission rate increased to over 700-fold. We also found that the introduction of a 3 MHz electric field aided the enhancement of the intrinsic quantum yield fluorophore molecules, which resulted in over a 1000-fold fluorescence enhancement. This enhancement was due to the very high electric produced by polarized DNA dipoles at 3 MHz, which resulted in a torque on fluorophore dipoles and subsequently aligning the fluorophore dipole axis with the plasmon electric field. At a fundamental level, our results demonstrate the role of the low-frequency electric field in the fluorophore–plasmon coupling. These findings can directly be applied to many fluorescence detection systems, including the development of biosensors.

Characterization and classification of pathogenic bacteria using native fluorescence and spectral deconvolution.

Identification and classification of pathogenic bacterial strains is of current interest for the early treatment of diseases. In this work, protein fluorescence from eight different pathogenic bacterial strains were characterized using steady state and time resolved fluorescence spectroscopy. The spectral deconvolution method was also employed to decompose the emission contribution from different intrinsic fluorophores and extracted various key parameters, such as intensity, emission maxima, emission line width of the fluorophores, and optical redox ratio. The change in average lifetime values across different bacterial strains exhibits good statistical significance (p ≤ 0.01). The variations in the photophysical characteristics of bacterial strains are due to the different conformational states of the proteins. The stepwise multiple linear discriminate analysis of fluorescence emission spectra at 280 nm excitation across eight different bacterial strains classifies the original groups and cross validated group with 100% and 99.5% accuracy, respectively.

Two-photon autofluorescence lifetime assay of rabbit photoreceptors and retinal pigment epithelium during light-dark visual cycles in rabbit retina

Two-photon excited fluorescence (TPEF) is a powerful technique that enables the examination of intrinsic retinal fluorophores involved in cellular metabolism and the visual cycle. Although previous intensity-based TPEF studies in non-human primates have successfully imaged several classes of retinal cells and elucidated aspects of both rod and cone photoreceptor function, fluorescence lifetime imaging (FLIM) of the retinal cells under light-dark visual cycle has yet to be fully exploited. Here we demonstrate a FLIM assay of photoreceptors and retinal pigment epithelium (RPE) that reveals key insights into retinal physiology and adaptation. We found that photoreceptor fluorescence lifetimes increase and decrease in sync with light and dark exposure, respectively. This is likely due to changes in all-trans-retinol and all-trans-retinal levels in the outer segments, mediated by phototransduction and visual cycle activity. During light exposure, RPE fluorescence lifetime was observed to increase steadily over time, as a result of all-trans-retinol accumulation during the visual cycle and decreasing metabolism caused by the lack of normal perfusion of the sample. Our system can measure the fluorescence lifetime of intrinsic retinal fluorophores on a cellular scale, revealing differences in lifetime between retinal cell classes under different conditions of light and dark exposure.

Inhibition and Mechanism of Protein Nonenzymatic Glycation by Lactobacillus fermentum.

Lactobacillus fermentum (L. fermentum) was first evaluated as a potential advanced glycation end-product (AGE) formation inhibitor by establishing a bovine serum albumin (BSA) + glucose (glu) glycation model in the present study. The results showed that the highest inhibition rates of pentosidine and total fluorescent AGEs by L. fermentum were approximately 51.67% and 77.22%, respectively, which were higher than that of aminoguanidine (AG). Mechanistic analysis showed that L. fermentum could capture methylglyoxal and glyoxal, inhibit carbonyl and sulfhydryl oxidation, reduce the binding of glucose and amino groups, increase total phenolic content and antioxidant activity, and release intracellular substances to scavenge free radicals; these abilities were the basis of the antiglycation mechanism of L. fermentum. In addition, L. fermentum significantly prevented conformational changes in proteins during glycation, reduced protein cross-linking by 35.67%, and protected the intrinsic fluorophore. Therefore, the inhibition of L. fermentum on glycation mainly occurs through antioxidation, the capture of dicarbonyl compounds, and the protection of the BSA structure. These findings collectively suggest that Lactobacillus is an inhibitor of protein glycation and AGE formation and has the potential for nutraceutical applications.

Differential scanning fluorimetry to assess PFAS binding to bovine serum albumin protein.

The rapid screening of protein binding affinity for poly- and perfluoroalkyl substances (PFAS) benefits risk assessment and fate and transport modelling. PFAS are known to bioaccumulate in livestock through contaminated food and water. One excretion pathway is through milk, which may be facilitated by binding to milk proteins such as bovine serum albumin (BSA). We report a label-free differential scanning fluorimetry approach to determine PFAS-BSA binding over a broad temperature range. This method utilizes the tryptophan residue within the protein binding pocket as an intrinsic fluorophore, eliminating the need for fluorophore labels that may influence binding. BSA association constants were determined by (a) an equilibrium-based model at the melting temperature of BSA and (b) the Hill adsorption model to account for temperature dependent binding and binding cooperativity. Differences in binding between PFAS and fatty acid analogs revealed that a combination of size and hydrophobicity drives PFAS binding.

Fluorescent Machine Learning Aided Classification of Pathogenic Bacteria Using the Excitation Emission Matrix

The rapid identification of pathogenic bacterial strains is becoming a challenging task as it causes many hospital associated infections. Many have reported on the use of fluorescence spectroscopy as an alternative to characterize bacteria. As bacteria possess intrinsic fluorophores, attempts were made to characterize eight strains using excitation emission matrix (EEM) measurements. From the results of parallel factor analysis (PARAFAC), four fluorophores, tryptophan, anthranilic acid, nicotinamide adenine dinucleotide, and flavin adenine dinucleotide, were identified with varying distributions. The data obtained from PARAFAC analysis were subjected to hierarchical cluster analysis (HCA) and linear discriminant analysis (LDA). This study demonstrates the potential of EEM technique to classify bacteria with 100% accuracy.

Intrinsic Burst-Blinking Nanographenes for Super-Resolution Bioimaging.

Single-molecule localization microscopy (SMLM) is a powerful technique to achieve super-resolution imaging beyond the diffraction limit. Although various types of blinking fluorophores are currently considered for SMLM, intrinsic blinking fluorophores remain rare at the single-molecule level. Here, we report the synthesis of nanographene-based intrinsic burst-blinking fluorophores for highly versatile SMLM. We image amyloid fibrils in air and in various pH solutions without any additive and lysosome dynamics in live mammalian cells under physiological conditions. In addition, the single-molecule labeling of nascent proteins in primary sensory neurons was achieved with azide-functionalized nanographenes via click chemistry. SMLM imaging reveals higher local translation at axonal branching with unprecedented detail, while the size of translation foci remained similar throughout the entire network. These various results demonstrate the potential of nanographene-based fluorophores to drastically expand the applicability of super-resolution imaging.

Emulsifying properties of O/W emulsion stabilized by soy protein isolate and γ-polyglutamic acid electrostatic complex.

In order to improve the emulsifying properties of soy protein around isoelectric point, soy protein isolate (SPI) and γ-polyglutamic acid (γ-PGA) complexes were prepared by electrostatic interaction. The formation of SPI-γ-PGA electrostatic complex and emulsifying properties were investigated by monitoring turbidity, zeta potential, intrinsic fluorophores, emulsion characterization, and microstructure observation. The results showed that the formation of SPI-γ-PGA electrostatic complex was identified through turbidimetric analysis and zeta-potential measurement. Intrinsic fluorescence spectrum indicated internal structure changes of electrostatic complexes. Furthermore, SPI-γ-PGA complex-stabilized emulsions showed better stability with small droplet sizes and slow growth as well as the uniform microstructure around the isoelectric point (pH 4.0-5.0) than SPI-formed emulsions. Under the different thermal treatments and ionic strengths, emulsions stabilized by SPI-γ-PGA-soluble complex resulted in improved emulsion stability to environmental stresses. This may be attributed to the increased steric repulsion and electrostatic repulsion by SPI-γ-PGA complexes at oil-water interfaces. The findings derived from this research would provide theoretical reference about SPI-γ-PGA electrostatic complex that can be applied in acid beverages and developed a novel plant-based sustainable stabilizer for emulsions. PRACTICAL APPLICATION: The electrostatic interaction between SPI and γ-PGA improved the emulsifying characteristics of soy protein around isoelectric point. The results derived from this research would expand applications of SPI-γ-PGA-soluble electrostatic complex that can be applied in acid beverages, as well as a novel plant-based sustainable stabilizer for emulsions.

Study of relations between chemical, colour and fluorescence properties of raw and dried meat powders of cow and yak (Bos grunniens)

Gluteus medius, Longissimus thoracis, and Semitendinosus muscles from the cow and yak (Bos grunniens) reared in the Kyrgyz Republic were dried by convective (hot air) drying and freeze drying. The dried muscles were grinded into fine powders and along with raw muscles were evaluated for water activity (aW), chemical composition (moisture, fat, protein), colour characteristics (L, a, and b values), fluorescence of intrinsic fluorophores (tryptophan, vitamin A, and riboflavin). Processing of measured data tables using common components and specific weights analysis (CCSWA) showed close relations among the chemical, colour, and fluorescence data. CCSWA discriminated muscles depending on chemical composition, animal type, and drying technique applied based on the chemical properties, colour characteristics, and fluorescence spectra. The freeze drying was approved as a preferable dehydration technique comparing with convective drying as the one causing less discoloration to meat. Partial Least Squares Regression (PLSR) models developed using fluorescence spectra allowed accurate quantitative predicting of water activity (aW), moisture, fat, and protein contents, and colour characteristics (L, a, and b values).

Anatomy-Specific Classification Model Using Label-Free FLIm to Aid Intraoperative Surgical Guidance of Head and Neck Cancer.

Intraoperative identification of head and neck cancer tissue is essential to achieve complete tumor resection and mitigate tumor recurrence. Mesoscopic fluorescence lifetime imaging (FLIm) of intrinsic tissue fluorophores emission has demonstrated the potential to demarcate the extent of the tumor in patients undergoing surgical procedures of the oral cavity and the oropharynx. Here, we report FLIm-based classification methods using standard machine learning models that account for the diverse anatomical and biochemical composition across the head and neck anatomy to improve tumor region identification. Three anatomy-specific binary classification models were developed (i.e., "base of tongue," "palatine tonsil," and "oral tongue"). FLIm data from patients (N = 85) undergoing upper aerodigestive oncologic surgery were used to train and validate the classification models using a leave-one-patient-out cross-validation method. These models were evaluated for two classification tasks: (1) to discriminate between healthy and cancer tissue, and (2) to apply the binary classification model trained on healthy and cancer to discriminate dysplasia through transfer learning. This approach achieved superior classification performance compared to models that are anatomy-agnostic; specifically, a ROC-AUC of 0.94 was for the first task and 0.92 for the second. Furthermore, the model demonstrated detection of dysplasia, highlighting the generalization of the FLIm-based classifier. Current findings demonstrate that a classifier that accounts for tumor location can improve the ability to accurately identify surgical margins and underscore FLIm's potential as a tool for surgical guidance in head and neck cancer patients, including those subjects of robotic surgery.

Spectroscopic and in silico evaluation of hesperetin, aglycone flavanone, as a prospective regulatory ligand for human salivary α-amylase

The insight into the binding mechanism of hesperetin, an aglycone flavanone, with human salivary α-amylase (HSAA), simulated under physiological salivary condition, was explored using various spectroscopic approaches and in silico method. Hesperetin effectively quenched the intrinsic fluorescence of HSAA and the quenching was mixed quenching mechanism. The interaction perturbed the HSAA intrinsic fluorophore microenvironment and the enzyme global surface hydrophobicity. The negative values of ΔG for thermodynamic parameters and in silico study revealed the spontaneity of HSAA-hesperetin complex while the positive values of enthalpy change (ΔH) and entropy change (ΔS) showed noticeable involvement of hydrophobic bonding in the stabilization of the complex. Hesperetin was a mixed inhibitor for HSAA with a K I of 44.60 ± 1.63 μM and having apparent inhibition coefficient (α) of 0.26. Macromolecular crowding, given rise to microviscosity and anomalous diffusion, regulated the interaction. Sodium ion (Na +) created high ionic strength, also, modulated the interaction. The in silico study proposed the preferential binding of hesperetin at the active cleft domain of HSAA with the least energy of −8.0 kcal/mol. This work gives a novel insight on the potentials of hesperetin as a future prospective medicinal candidate in the management of postprandial hyperglycemic condition. Communicated by Ramaswamy H. Sarma

A Workflow to Quantitatively Determine Age-Related Macular Degeneration Lesion-Specific Variations in Fundus Autofluorescence.

Fundus autofluorescence (FAF) imaging allows the noninvasive mapping of intrinsic fluorophores of the ocular fundus, particularly the retinal pigment epithelium (RPE), now quantifiable with the advent of confocal scanning laser ophthalmoscopy-based quantitative autofluorescence (QAF). QAF has been shown to be generally decreased at the posterior pole in age-related macular degeneration (AMD). The relationship between QAF and various AMD lesions (drusen, subretinal drusenoid deposits) is still unclear. This paper describes a workflow to determine lesion-specific QAF in AMD. A multimodal in vivo imaging approach is used, including but not limited to spectral domain optical coherence tomography (SD-OCT) macular volume scanning and QAF. Using customized FIJI plug-ins, the corresponding QAF image is aligned with the near-infrared image from the SD-OCT scan (characteristic landmarks; i.e., vessel bifurcations). The foveola and the edge of the optic nerve head are marked in the OCT images (and transferred to the registered QAF image) for accurate positioning of the analysis grids. AMD-specific lesions can then be marked on individual OCT BScans or the QAF image itself. Normative QAF maps are created to account for the varying mean and standard deviation of QAF values throughout the fundus (QAF images from a representative AMD group were averaged to build normative standard retinal QAF AMD maps). The plug-ins record the X and Y coordinates, z-score (a numerical measurement that describes the QAF value in relation to the mean of AF maps in terms of standard deviation from the mean), mean intensity value, standard deviation, and number of pixels marked. The tools also determine z-scores from the border zone of marked lesions.This workflow and the analysis tools will improve the understanding of the pathophysiology and clinical AF image interpretation in AMD.

Monitoring oligomeric forms of amyloid beta in the early stages of aggregation using hybrid X-ray footprinting mass spectrometry with inline fluorescence.

The aggregation-prone Amyloid Beta (Aβ) peptide has been the target of the majority of efforts to develop Alzheimer's Disease therapeutics, most of which have failed or have shown mixed results. It is no longer widely believed that buildup of amyloid fibrils is a causal factor in cognitive decline associated with the disease, and a growing body of evidence now suggests that low molecular weight oligomeric forms of Aβ may be the culprits in disease progression. However, while numerous Aβ fibril structures have been characterized, primarily by NMR and cryo-EM, obtaining structural information on oligomeric forms of Aβ that presumably precede and/or seed fibril formation has proved challenging. These transient forms are heterogeneous, and depend heavily on experimental conditions such as buffer, temperature, concentration, and degree of quiescence during measurement. Here, we present a new approach to delineating structural features of early-stage low molecular weight Aβ oligomers using a solvent accessibility assay in conjunction with inline fluorescence measurements. In this method, inline fluorescence is collected nearly simultaneously from samples during collection of X-ray footprinting mass spectrometry (XFMS) data. In the XFMS method, X-ray irradiation dissociates solvent water to produce hydroxyl radicals, which covalently modify solvent accessible side chains. Liquid chromatography-mass spectrometry is subsequently used to analyze the covalent modifications produced, and the data provide a “water map” at the single residue level, which is used to determine protein structure. This new hybrid approach circumvents issues in batch-to-batch variability of Aβ aggregation which has made this peptide so difficult to study, and provides a method to combine residue-specific structural information nearly simultaneously with global protein structure readouts from either intrinsic protein fluorescence or fluorophores such as Thioflavin T.

A generic rotamer model to explain the temperature dependence of BSA protein fluorescence

AbstractProtein fluorescence signals essential information about the conformational dynamics of proteins. Different types of intrinsic fluorophores reflect different protein local or global structural changes. Bovine Serum Albumin (BSA) is a transport protein that contains two intrinsic fluorophores: Tryptophan134 (Trp134) and Tryptophan213 (Trp213). This protein displays an interesting temperature dependence of the tryptophan fluorescence. However, the molecular mechanism of the temperature dependence is still unclear. In this work, we propose a generic rotamer model to explain this phenomenon. The model assumes the presence of rotamer‐specific fluorescence lifetimes. The fluorescence temperature dependence is caused by the population shifts between different rotamers due to thermal effects. As a proof of concept, we show that the tryptophan's two fluorescence lifetimes (𝜏1 = 0.4–0.5 ns and 𝜏2 = 2‐4 ns) are sufficient to qualitatively explain the fluorescence intensity change at different temperatures, both in buffer solution (water) and in the protein. To computationally verify our rotamer hypothesis, we use an all‐atom molecular dynamics simulation to study the effects of temperature on the two tryptophans' rotamer dynamics. The simulations show that Trp134 is more sensitive to temperature, consistent with experimental observations. Overall, the results support that the temperature dependence of fluorescence in the protein BSA is due to local conformational changes at the residue level. This work sheds light on the relationship between tryptophan's rotamer dynamics and its ability to fluorescence.

In-vivo Testing of Oral Mucosal Lesions with an In-house Developed Portable Imaging Device and Comparison with Spectroscopy Results.

Progression of oral mucosal lesions is generally marked by changes in the concentration of the intrinsic fluorophores such as collagen, nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD) and porphyrin present in the human oral tissue. In this study, we have probed the changes in FAD and porphyrin by exciting with 405nm laser light on different sites (tongue, buccal mucosa, lip etc.) of the oral cavity. Testing has been done by an in-house developed fluorescence-based portable imaging device on oral squamous cell carcinoma (OSCC) patients, dysplastic patients and control (normal) group. Fluorescence images recorded from OSCC and dysplastic patients have displayed an enhancement in the red band (porphyrin) as compared to those from the normal volunteers. Porphyrin to FAD intensity ratio (IPorphyrin/IFAD), referred to red to green ratio (Ired/Igreen) has been taken as the diagnostic marker for classification among the groups. Receiver operating characteristic (ROC) analysis applied on IPorphyrin/IFAD is able to discriminate OSCC to normal, dysplasia to normal and OSCC to dysplasia with sensitivities of 100%, 81%, 92% and specificities of 100%, 93% and 92% respectively. Fluorescence imaging probe can capture a large area of oral lesions in a single scan and hence would be useful for initial scanning. On comparison with spectroscopy studies performed by our group, it is found that combining both spectroscopy and imaging as a device may be effective for the early detection of oral lesions. This clinical study was registered on the date 13/10/2017 in the clinical trials registry-India (CTRI) with registration number CTRI/2017/10/010102.

Cryogenic Fluorescence Spectroscopy of Ionic Fluorones in Gaseous and Condensed Phases: New Light on Their Intrinsic Photophysics.

Fluorescence spectroscopy of gas-phase ions generated through electrospray ionization is an emerging technique able to probe intrinsic molecular photophysics directly without perturbations from solvent interactions. While there is ample scope for the ongoing development of gas-phase fluorescence techniques, the recent expansion into low-temperature operating conditions accesses a wealth of data on intrinsic fluorophore photophysics, offering enhanced spectral resolution compared with room-temperature measurements, without matrix effects hindering the excited-state dynamics. This perspective reviews current progress on understanding the photophysics of anionic fluorone dyes, which exhibit an unusually large Stokes shift in the gas phase, and discusses how comparison of gas- and condensed-phase fluorescence spectra can fingerprint structural dynamics. The capacity for temperature-dependent measurements of both fluorescence emission and excitation spectra helps establish the foundation for the use of fluorone dyes as fluorescent tags in macromolecular structure determination. We suggest ideas for technique development.

Thin-Plate Superstructures of the Immunogenic 33-mer Gliadin Peptide.

Gluten related-disorders have a prevalence of 1-5 % worldwide triggered by the ingestion of gluten proteins in wheat, rye, barley, and some oats. In wheat gluten, the most studied protein is gliadin, whose immunodominant 33-mer amino acid fragment remains after digestive proteolysis and accumulates in the gut mucosa. Here, we report the formation of 33-mer thin-plate superstructures using intrinsic tyrosine (Tyr) steady-state fluorescence anisotropy and cryo-TEM in combination with water tension measurements. Furthermore, we showed that fluorescence decay measurements of 33-mer intrinsic fluorophore Tyr provided information on the early stages of the formation of the thin-plate structures. Finally, conformational analysis of Tyr residues using minimalist models by molecular dynamic simulations (MD) demonstrated that changes in Tyr rotamer states depend on the oligomerization stage. Our findings further advance the understanding of the formation of the 33-mer gliadin peptide superstructures and their relation to health and disease.

Impact of the Flavonoid Quercetin on β-Amyloid Aggregation Revealed by Intrinsic Fluorescence.

We report the effects of quercetin, a flavonoid present in the human diet, on early stage beta-amyloid (Aβ) aggregation, a seminal event in Alzheimer’s disease. Molecular level changes in Aβ arrangements are monitored by time-resolved emission spectral (TRES) measurements of the fluorescence of Aβ’s single tyrosine intrinsic fluorophore (Tyr). The results suggest that quercetin binds β-amyloid oligomers at early stages of their aggregation, which leads to the formation of modified oligomers and hinders the creation of β-sheet structures, potentially preventing the onset of Alzheimer’s disease.

Performance of feature extraction method for classification and identification of proteins based on three-dimensional fluorescence spectrometry

Three-dimensional excitation emission matrix (EEM) fluorescence spectroscopy was employed to discriminate protein samples comprising bovine serum albumin, neurotensin, ovalbumin, ricin, trypsin from bovine pancreas and trypsin from porcine pancreas. Two methods of feature extraction with and without parameterization were applied to the spectral data in order to evaluate their performance of discrimination between protein samples. The discrimination of protein samples was conducted by k-means clustering algorithm and eigenvalue extracting procedure based on principal component analysis (PCA). It was found that the method of feature extraction without parameterization performed best, correctly attributing 100% of the spectral data in the condition of two principal components (PCs) captured. Features extracted with spectral parameterization failed to separate ricin and trypsin from bovine pancreas in same condition. Without spectral parameterization, less dimensionality and unique principal components captured by PCA indicates the spectrally-resolved features of corresponding protein samples. By clustering using each spectrum at fixed excitation wavelength, excitation wavelengths matched with common intrinsic fluorophores were found to be more sensitive to the classification accuracy. Contributions of spectral features extracted from EEM to the principal components were discussed and demonstrated their feature differentiation capabilities among six protein samples. These results reveal that appropriate extraction approach of features in combination with PCA analysis could be used in discrimination of protein samples at species level as a spectroscopic diagnostic tool. Our study provides fundamental references about computational strategies when EEM are used to explore proteins in ambient environment.