Triclinic Labs can provide complete biophysical analysis of your large molecule samples.
Information about the regulation of protein expression, protein modification, protein:protein interactions and protein function during different stages of drug discovery and development is needed to understand the development and physiology of organisms. This complex analysis of protein function is a major task facing scientists today. Although the field of proteomics was first described only as the study of proteins encoded by the genome, it has now expanded to include the function of all expressed proteins. Thus, it is not just the study of all proteins expressed in a cell but also all protein isoforms and modifications, interactions, structure and high-order complexes (Tyers and Mann, 2003). Triclinic's Protein Characterization practice is headed by Drs. Lake Paul and John Burgner.
Lake Paul, Ph.D. received his doctorate in Structural Biology and Biophysics from Purdue University and most recently has held the position of Biophysics Research Specialist at Purdue University in West Lafayette, IN. During Dr. Paul’s tenure at the National Institute of Environmental Health Sciences and Purdue University, he applied LC/MS techniques to compliment traditional structural biology and demonstrated that protein analyses under Matrix Assisted Laser Desorption Ionization MS conditions coincide with the solution data obtained from analytical ultracentrifuge.
John W. Burgner, Ph.D. received his doctorate in Biochemistry and was most recently Director, Biophysical Analysis Core, Bindley Bioscience Center at Purdue University until retiring in 2009. Dr. Burgner is a recognized authority on hydrodynamic and thermodynamic analysis of macromolecules and has an extensive publication history. Dr. Paul and Dr. Burgner will bring their extensive knowledge in the use of Analytical Ultra Centrifugation (AUC), Surface Plasmon Resonance (SPR), Liquid Chromatography coupled with Mass Spectrometry (LC/MS), as well as other Structural and Kinetic Characterization techniques to bear on client research needs.
Both Dr. Paul and Dr. Burgner have extensive experience in the biophysical characterization of large molecules used in drug discovery and development.
We offer the following services:
Structural Characterization
Triclinic Labs utilizes two analytical ultracentrifuges: Beckman-Coulter XLI and XLA, and a Biacore 3000 for analytical ultracentrifugation and kinetic analysis of biological samples.
These techniques allow one to determine:
- kinetic parameters
- stoichiometry of binding
- size distribution
- shape
- oligomerization state and
- other biophysical measurements
Analytical Ultracentrifugation (AUC)
AUC, including both sedimentation velocity and sedimentation equilibrium, provides a complete analysis for both low concentration (~0.1 mg/ml) as well as samples without chromophores. The XLA AUC is equipped with only absorbance optics and is mainly used for protein/peptide analyses.
Routinely, sedimentation velocity experiments are employed. This type of experiment can be used to evaluate the stoichiometry and kinetics of interactions along with hydrodynamic and thermodynamic properties of the reacting systems. This technique can provide information on the sedimentation coefficient distributions, macromolecular shape distributions, equilibrium/rate constants and molecular weight distributions. Sedimentation equilibrium experiments can also be performed to determine kinetic constants for self associating systems. These experiments are conducted in the native buffer and do not rely on either immobilization or labeling of the samples. AUC is
- the "gold standard" for measuring molecular mass in solution and studying protein self-association and protein-protein interactions (sedimentation equilibrium)
also studies interactions with small molecules when binding is linked to protein association - an excellent method for studying and quantifying protein aggregation (sedimentation velocity)
- often used for cross-validation of SEC methods
- usually allows measurements directly under formulation conditions
- good for demonstrating equivalence of material from different manufacturing processes (comparability protocols)
Surface Plasmon Resonance (SPR)
Determine the kinetic parameters (kon/koff) of biomolecular interactions
The Biacore 3000 is built on the platform that utilizes the Surface Plasmon Resonance (SPR) phenomena, which can be used to monitor the refractive index of a given medium near the surface of a chip. The Biacore 3000 uses a label free system to determine the kinetic parameters (kon/koff) of biomolecular interactions. Evaluation of the kinetic parameters (kon/koff) of a protein-ligand system will require the immobilization of either protein or ligand to the surface of the chip. The binding of analytes flowing of over the surface of the chip can be uses to describe kon (association) and koff (disassociation). The chips are re-useable and have 4 binding surfaces which can be evaluated simultaneously. The Biacore Evaluation software can be used to derive the kinetic parameters for several models.
Complementing these traditional biophysical methods, Triclinic also provides mass spectrometric analyses at both the protein and peptide levels using an ABI 4800 mass spectrometer. The 4800 uses matrix assisted laser desorption/ionization (MALDI) to analyze biomolecules such as proteins, peptides and sugars.
With each analytical ultracentrifugation analysis, a complementary mass spectrometric whole protein analysis is conducted. There are matrices which preserve the non-covalent interactions of proteins using ionic liquid matrices (ILM). We can also look at membrane proteins using the Ultra Thin Layer Method. The data gathered from these mass spectrometric analyses agree with the solution data from the analytical ultracentrifuge. Combined, both techniques provide a robust and complete evaluation of your samples.
Circular dichroism (CD) Spectroscopy
CD is an excellent tool for characterizing thermal stability of proteins and the effects of solution conditions and excipients on stability. It can also be used to analyze the reversibility of protein unfolding.The technique is used to provide basic characterization of protein secondary structure (e.g. percentage alpha helix and beta sheet). Additionally, CD can be used for:
- assesment of equivalence of material from different manufacturing processes (comparability and sameness)
- determining if expressed proteins are properly folded
- identifying changes in the tertiary structure around aromatic residues
Laser light scattering
Classical light scattering (CLS) is available.
CLS, when used with size-exclusion chromatography (SEC), measures the true mass of each chromatographic peak, (independent of the molecular shape or elution position), making it an absolute method.The CLS process also confirms quaternary structure for proteins that elute abnormally on SEC and provides excellent sensitivity for detecting small amounts of aggregates. CLS/SEC may also be used to distinguish dimers from unfolded monomers.
Peptide Mapping (by LC-MS) & LC/MS/MS
Used to confirm identity/primary structure of proteins in mixtures. Mass spectrometry is an important method for characterization and sequencing of proteins. The two primary methods for ionization of whole proteins are electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI).
We utilize two approaches for characterizing proteins. In the first, intact proteins are ionized by either ESI or MALDI and then introduced to a mass analyzer. This approach is referred to as "top-down" protein analysis. In the second approach, proteins are enzymatically digested into smaller peptides using proteases such as trypsin or pepsin (either in solution or in gel after electrophoretic separation). The collection of peptide products are then introduced to the mass analyzer.
When the characteristic pattern of peptides is used for the identification of the protein the method is called peptide mass fingerprinting (PMF), if the identification is performed using the sequence data determined in tandem MS analysis it is called de novo sequencing. These approaches are also referred to as the "bottom-up" strategy..
Amino Acid Composition Analysis (AAA)
Determination of amino acid composition of a protein. Used as confirmation of primary structure.
N-terminal Sequencing
Determination of amino acid sequence from the N-terminus of protein. Used as a confirmation of primary structure.
HPLC
SEC-HPLC
Separation of protein based on size. Determination of degree of aggregation.
RP and IEX-HPLC
Identity, content and impurity determination.
Method Development, Validation, and Transfer
Post-Translational Modification (PTM) Analysis
Detection of oxidation and deamidation products
Enzymatic digestion of protein to monitor lot-to-lot variability/post-translational modifications.
Monosaccharide Composition
Quantification of neutral amino sugars: glucosamine, galactosamine, mannose, galactose and fucose.
Salicylic Acid Determination
Quantitation of acidic sugars NANA and NGNA.
Oligosaccharide Profiling
N-linked oligosaccharide profiling by determination of the sialylated and desialylated carbohydrate structures.
Glycosylation/Sulfation/Phosphorylation
Site Identification Using enzymes to determine the peptide location of post-translational modifications.
Disulfide Bond Pattern Determination
Determination of secondary structure. Cystine linkage sites are identified.
UV Analysis
Determination of extinction coefficient and content of protein.
FTIR spectroscopy
Triclinic utilizes a ThermoFinnegan FTIR. With this instrument we are able to provide secondary structure information for lyophilized drug product, spray-dried powders or precipitates, as well as liquid-state drug product. This thechnique, when combined with other spectroscopic approaches, provids an excellent prediction of performance and stability. FTIR is more sensitive than CD when details of beta-sheet structures are required. FTIR also can detect aggregation arising from formation of intermolecular beta sheets.
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