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Amino Acid Analysis
Protein quantification and amino acid analysis
Purified or total protein quantification
Method, where the protein mixture or purified protein sample is subject to hydrolysis, creating free amino acids. These are then analyzed by IEX-HPLC with post-column ninhydrin derivatization. Up to 20 amino acid are accurately identified and quantified, along with calculation of the total protein content from summed amino acid amounts.
An amino acid standard mixture is utilized for calibration of the instrument and a NIST BSA standard is applied for precision and accuracy assessment of each batch run. Finally, a standard amino acid solution is analyzed in between every 5 samples to correct for ninhydrin activity.
N-terminal sequencing by Edman degradation
N-terminal Edman sequencing (Edman degradation)
Technique where one residue at a time is removed from the N-terminal/amino end of a peptide or protein. The residue is derivatized to a PTH-amino acid and identified by chromatography. The sequencing is a cyclic approach, that cleaves one residue off at a time and analyzes it.
Edman sequencing of mAbs
The method for monoclonal antibodies includes separation of the heavy and light chains by 1D SDS-PAGE, blotting onto a PVDF membrane, staining and cutting off the bands, and finally sequencing of up to 30 amino acids by Edman degradation. The n-terminus of many mAbs are blocked for the Edman chemistry by pyro-glutamic acid that has to be enzymatically removed before analysis.
1D SDS-PAGE & 2D SDS-PAGE
Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS-PAGE)
Separation method for proteins with a polyacrylamide gel as the medium and SDS for denaturation of the proteins. After denaturation, the proteins have a net negative charge that allows them to migrate towards an anode (+). Larger molecules are restrained in the gel more than smaller molecules, thus separating the molecules based on their size.
SDS-PAGE separation can be performed as either One Dimensional (1D) or Two Dimensional (2D):
- 1D SDS-PAGE
Separates protein by molecular weight.
- 2D SDS-PAGE
Separates protein by molecular weight and isoelectric point.
Enzyme-Linked Immunosorbent Assay (ELISA)
Immunological, plate-based assay used for measurement and quantification of molecules, such as antibodies, peptides and proteins.
In a “sandwich ELISA”, the 96-well microtiter plate is coated with a capture antibody which binds the sample antigen (e.g. protein). Hereafter, the antigen is bound by a detecting antibody. A secondary antibody binds and cleaves an added substrate through its linked enzyme, leading to a detectable signal. The amount of signal depend on the antigen amount.
Electrospray Ionization - Mass Spectrometry
Electrospray Ionization (ESI)
Ionization technique that transforms ions of proteins and peptides from a liquid solution to a gaseous state before mass spectrometric detection. ESI Mass Spectrometry (ESI-MS) has a high sensitivity and robustness, thus making it well-suited for non-volatile molecules and complex sample solutions.
Liquid Chromatography Mass Spectrometry (LC-MS & LC-MS/MS)
A technique, where peptides and proteins in a liquid solution are physically separated in the HPLC due to their various chemical properties (hydrophobicity, hydrophilicity, size, charge). The components are released from the stationary phase into the mobile phase and subsequently detected and analyzed in the mass spectrometer (MS). If the mixture is subject to tandem mass spectrometry (MS/MS), the first MS selects precursor ions by their mass (m/z), which are then selected, fragmented and detected as product ions in the second MS.
It is also possible to combine LC-MS with UV detection, thereby achieving both UV trace chromatogram, MS ion current chromatogram and MS spectra. This method is known as UV-LC-MS analysis.
Variant of LC-MS/MS with flow rates in the nanoliter/min range. The system can run smaller sample amounts and has higher sensitivity than the conventional LC-MS/MS setup. NanoLC-MS/MS has very long analysis time due to low flow-rates.
SWATH is a data independent acquisition (DIA) technique for detection and quantitation of all detectable compounds in a complex sample. The analysis can only be performed with a SCIEX TripleTOF instrument.
Multiple Reaction Monitoring (MRM LC-MS/MS)
Technique especially suited for quantification of specific, single proteins of particular interest (HCPs). MRM has a high sensitivity and specificity, thus making it perfect for targeted analysis of a set of low-level proteins, even if they are only ppm-level abundant.
High-Performance Liquid Chromatography
High-Performance Liquid Chromatography (HPLC)
Form of column chromatography, where the sample solution in solvent (mobile phase) is pumped through the column (stationary phase) at high pressure. The technique separates and identifies molecules based on their chemical properties in a liquid solution. A UV or fluorescent detector is often used for detection, if the HPLC is not directly coupled to a mass spectrometer.
Most commonly used HPLC type with a non-polar stationary phase and an aqueous, polar mobile phase. In RP-HPLC, hydrophobic molecules tend to interact with the stationary phase, whereas hydrophilic molecules elute first.
Size Exclusion (SEC-HPLC)
Type of HPLC that separates molecules based on their molecular size. The column packing with fine, porous beads allows for separation of small and big molecules, since the smaller molecules can enter the pores. Thus, they move slower than bigger molecules that cannot interact and enter the pores.
Ion Exchange (IEX-HPLC)
Type of HPLC that separates ionizable molecules based on their net charge. After loading the protein sample on the column, a washing step removes impurities, before proteins elute from the column with a change in pH or a salt gradient.
Hydrophilic Interaction Liquid Chromatography (HILIC)
HILIC is an alternative HPLC mode with a polar stationary phase and an aqueous mobile phase, often as a gradient from high percentage of organic solvent to high percentage of aqueous solvent. HILIC is especially useful for separation of polar compounds, such as glycosylated proteins and released glycans.
Matrix-Assisted Laser Desorption/Ionization
Matrix-Assisted Laser Desorption/Ionization (MALDI)
Ionization technique that takes advantage of a laser absorbing matrix. Large molecules are transformed into ions with low fragmentation. MALDI coupled Mass Spectrometry (MALDI MS) is a crucial technique in proteomics for protein MW-profiling.
Tandem Mass Spectrometry (MALDI MS/MS)
MALDI MS fragmentation technique. In MS/MS mode, a LIFT cell selects a specific parent ion and its fragments formed by post-source decay (PSD). The MALDI reflector separates and detects the product ions. MALDI MS/MS is typically used for peptide fragmentation in protein identification analysis.
In Source Decay (MALDI ISD)
MALDI technique that can determine both N- and C-terminal sequences of 20-50 residues. The intact protein is fragmented by in-source-decay and the fragment masses determined. Also known as top-down sequencing.
Sample Prep Robotics
Automated robotics for multi-step sample handling
Automated robotics for multi-step sample handling
Robust and reproducible sample preparation is a key element in high quality analysis of biopharmaceutical products. Top-of-the-line pipetting robotics are applied together with in-house developed procedures to secure fast and consistent sample preparation.