Plant Protein Innovation Center

Protein Structural & Functional Characterization and Flavor Analysis Capabilities

Instrument-Based Analytical Tools for Structural Characterization

Test Category	Analysis	Purpose
Structural Deviation	Differential Scanning Calorimetry (DSC)	DSC determines the onset temperature of denaturation and the degree of unfolding (estimates protein denaturation state) as affected by processing or modification
	Protein secondary configuration by FTIR	Fourier transform infrared spectroscopy (FTIR) measures the distribution of secondary protein structures as affected by processing conditions or modification
	Protein tertiary structures by surface enhanced Raman spectroscopy (SERS)	Raman spectroscopy determines the distribution of tertiary protein structures and intramolecular interactions as affected by processing conditions or modification
Aggregation	Apparent protein aggregation state by size exclusion HPLC	Size exclusion HPLC identifies degree of polymerization and estimates the extent of intermolecular aggregation with covalent and non-covalent linkages and association of subunits by determining molecular weight of polymers
	Transmission-Scanning Electron Microscopy	Enables visual association of protein and particle dynamics (aggregation morphology)
Chemical Deviation	MALDI-TOF	Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) determines the individual protein subunit/peptide molecular weights, which can be used for identification of different protein subunits. MALDI-TOP can also be used to identify changes in proteins and peptides profile/distributinon as affected by processing and/or enzyme modification. Another application of this technique is to monitor site of modification.
	LC/MS/MS	HPLC with tandem mass spectrometry can be used for multiple identification purposes. For example, it can be used to determine the selectivity of enzymes to specific protein subunits within a mixture of proteins. It can be used to determine the protein that is digested, the most or the least. On the other hand, identifying peptides is essential in studies investigating bioactivity potency of peptides.
Structural Visualization	Confocal Laser Scanning Microscopy (CLSM)	CLSM utilizes fluorescent probes to visualize protein-protein and protein-lipid interactions in dispersed systems. For example, it can be used to visualize the distribution of protein and phospholipids at the interface and in the continuous phase of an oil-in-water emulsion.

Chemical Assays for Structural Characterization

Test Category	Analysis	Purpose
Aggregation/ Molecular Aggregation	One or Two-Dimensional Gel Electrophoresis	Gel electrophoresis determines protein profile under reducing and non-reducing conditions. This analysis will reveal polymerization and/or hydrolysis patterns and potential bonding mechanisms: non-covalent, covalent S-S linkages, or other covalent bonding. It will also reveal the number and changes in prevalence of individual protein subunits.
Surface Properties	Surface Hydrophobicity	This assay provides an indication of protein unfolding and subsequent polymerization driven by hydrophobic interactions. The test estimates the hydrophobic surface area and changes throughout processing.
	Surface Charge (Zeta-Potential)	Zeta Potential estimates net charge density on the surface. It is an indication to changes in surface hydrophilicity, protein unfolding, and possible interactions with macromolecules such as carbohydrates.
	Free Sulfhydryl Concentration	This assay provides an indication of unfolding and revealing of free SH previously entrapped in the three dimensional structure of the protein, in addition to the extent of potential formation of disulfide linkages. This test determines the redox state of the protein.
Chemical Deviation	Alpha-Amine Prevalence (OPA)	The o-phthaladehyde spectrophotometric assay (OPA) determines the extent of amine blocked by the Maillard reaction and and is also used to determine the degree of hydrolysis.

Chemical Assays for Functional Characterization

Analysis	Purpose
Solubility	This assay determines the soluble protein in a protein-water dispersion by measuring the protein concentration of the dispersion before and after centrifugation to sediment insoluble proteins. The inherent solubility of proteins greatly impacts other aspects of their functionality and performance in various applications.
Oil-Binding Capacity	Oil-binding capacity provides an indication of the amount of oil bound to the protein structure. Oil-binding capacity is related to a protein's surface hydrophobicity and influences its emulsification properties.
Water-Binding Capacity	Water-binding capacity provides an indication of the amount of water bound to protein via hydrogen or ionic bonds. Together with the water-holding capacity of a protein, the water-binding capacity influences many other functional properties of proteins, such as dispersibility, wettability, swelling, solubility, thickening/viscosity, gelation, coagulation, emulsification, and foaming.
Water-Holding Capacity	Water-holding capacity reflects the ability of a protein to imbibe and retain water (via hydrogen bonds, ionic bonds, and physical entrapment) against gravity within a protein matrix, i.e. gels.
Gelation	Gelation measures the ability of proteins to form a three-dimensional network that traps water. Gelation depends on partially unfolded proteins forming protein-water-protein interactions via hydrophobic interactions and disulfide linkages. Gel strength is a measure of the force required to rupture the gel. Prior to measuring gel strength, least gelation concentration is determined as a measure of the lowest protein concentration at which a protein solution forms a heat-induced gel and holds its shape.
Foaming Capacity & Stability	Foaming capacity is a measurement of the ability of a protein solution to produce a foam after agitation. Foaming stability, on the other hand, refers to the ability of a protein foam to remain stable over time, and against gravitational or mechanical stresses.
Emulsification Capacity	Emulsification capacity determines the quantity of oil a certain amount of protein can emulsify. Oil is titrated into a protein solution and emulsion failure is measured by a decrease in viscosity.
Emulsification Stability	This assay provides an indication of the stability of an emulsion again chemicophysical forces over time. The stability of an emulsion is measured spectrophotometrically by comparing the change in absorbance of the emulsion over a set period due to destabilization by creaming, brownian flocculation, and coalescence.
Particle Size in Emulsions	The particle size of an emulsion is an indicator of emulsion type (i.e. micro vs nanoemulsion). It can also be used to monitor the physical stability of emulsions by tracking changes in particle size over time.
Color Analysis by Chroma Meter	Color analysis using the Chroma Meter enables the analysis of Lab values of protein ingredients as affected by extraction conditions, glycation, processing conditions, etc.

Flavor Methods

Test Category	Analysis	Purpose
Extraction Techniques	Solvent Assisted Flavor Extraction (SAFE)	SAFE is considered one of the best overall methods for "clean" aroma extraction and is capable of extracting compounds from complex matrices. Volatile aroma compounds are isolated from an organic solvent extract under high pressure resulting in high recovery of aroma compounds from non-volatile compounds.
	Static Headspace	The static headspace technique involves the extraction of volatile and semi-volatile compounds in the vapor/headspace above a food sample in a vial that is equilibrated at the desired temperature. An aliquot of the vapor/headspace can be analyzed by gas chromatography to determine the concentration of the compounds of interest.
	Solid Phase Micro Extraction (SPME)	SPME is a solvent-free sample prep technology. SPME uses a fiber coated with a polymer sorbent for extracting the coumpounds from the sample by adsorption. It is then injected directly into the GC-MS for desorption and analysis.
	Stir Bar Sorptive Extraction (SBSE)	Following the SBSE technique, a magnetic stir bar with a polymer coating is placed inside the sample vial. The extraction is a function of an equilibrium between the coating and the analytes in solution. This method is used for a quick evaluation of flavor compounds without much sample preparation.
	Purge and Trap/ Dynamic Headspace	Purge and trap is a dynamic headspace extaction method. Samples are purged with inert gas causing volatile and semi-volatile compounds from the sample to enter the vapor/headspace and become adsorbed onto a trap containing adsorption material.
	Gerstel Thermodesorption System (TDS) and Cooled Injection System (CIS)	After extraction of flavor compounds using Stir Bar Sorptive Extraction or Purge and Trap, compounds are thermally desorbed using the Gerstel TDS and are then cryofixed and injected into a gas chromatograph using a Gerstel CIS. The Gerstel TDS and CIS allows for highly sensitive and accurate determination of vlatile and semi-volatile flavor compounds.
Analytical Measurement of Flavor Compounds	Gas Chromatography (GC)	GC is a common type of chromatography used in separating compounds that can be vaporized (gas). Volatile flavor compounds are carried by a carrier gas and separated through a polymer coated column. Analytes can then be analyzed using various types of detectors.
	Detector: Flame Ionization Detector	FID is the most commonly used detector used to detect ionized compounds formed during combustion via hydrogen flame. The generated ions are proportional to the concentration of the compound.
	Detector: Mass Spectrophotometer	MS detects and measures the mass-to-charge ratio of ions resulting in a mass spectra that can be used to identify compounds. Multiple MS detectors can be used in tandem to further evaluate analytes. Different types of MS detectors are available, such as MS quadrupole and MS time of flight, which have different levels of sensitivity.
	Olfactometry	Olfactometry allows for detection of odor active compounds. Analytes separated by GC can be split for detection (e.g. FID or MS) and evaluation by sniffing at a sniff port to identify the compounds that are odor active and their odor intensity.
	Ultra Performance Liquic Chromatography (UPLC)-MS Quadrapole	UPLC-MS Quadropole is used for isolating and analyzing non-volatile taste compounds that impact taste and flavor using liquid chromatography to separate compounds and MS to subsequently identify them.
	Ultra Performance Liquic Chromatography (UPLC)-MS LTQ Quadrapole	UPLC-MS Quadropole is used for isolating and analyzing non-volatile taste compounds that impact taste and flavor using liquid chromatography to separate compounds and MS to subsequently identify them. The LTQ MS has a higher sensitivity than the quadapole previously mentioned.
	Ultra Performance Liquic Chromatography (UPLC)-MS qTOF	UPLC-MS q-TOF is used for analyzing intact protein flavor interactions. It has an electrospray ionization and time of flight detector. It has a mass accuracy up to four decimal places. Tandem MS can be performed as part of this method.