Vrije Universiteit Amsterdam
Analytical Chemistry / Mass Spectrometry
QC Manager Biosimilars at Sandoz
Join LinkedIn & access JS's full profile
Analytical Chemistry / Mass Spectrometry
Senior Director Chemical Analytics and QC
Immunoassay method development
A quantitative method for the determination of proteins in complex biological matrices has been developed based on the selectivity of antibodies for sample purification followed by proteolytic digestion and quantitative mass spectrometry. An immunosorbent of polyclonal anti-bovine serum albumin (BSA) antibodies immobilized on CNBR agarose is used in the on-line mode for selective sample pretreatment. Next, the purified sample is trypsin digested to obtain protein specific peptide markers. Subsequent analysis of the peptide mixture using a desalination procedure and a separation step coupled, on-line to an ion-trap mass spectrometer, reveals that this method enables selective determination of proteins in biological matrices like diluted human plasma. This approach enhances substantially the selectivity compared to common quantitative analysis executed with immunoassays and colorimetry, fluorimetry or luminescence detection. Hyphenation of the immunoaffinity chromatography with on-line digestion and chromatography–mass spectrometry is performed and a completely on-line quantification of the model protein BSA in bovine and human urine was established. A detection limit of 170 nmol/l and a quantification limit of 280 nmol/l is obtained using 50 μl of either standard or spiked biological matrix. The model system allows fully automated absolute quantitative mass spectrometric analysis of intact proteins in biological matrices without time-consuming labeling procedures.
Analytical methodologies for the absolute quantitation of proteins typically include a digest step often using trypsin as the proteolytic enzyme. In the majority of cases, off-line and on-line digestion methods are implemented prior to an LC−MS analysis system, requiring a high sequence coverage for unambiguous protein identification. For proteins with a strong overlap in amino acid sequence, e.g., therapeutic proteins and their metabolites, it is essential to separate proteins prior to digestion and the subsequent electrospray mass spectrometry analysis of marker peptides. Here, we present an on-line postcolumn solution-phase digestion methodology that is based on the continuous infusion of the proteolytic enzyme pepsin downstream to the nano C18 reversed-phase column. Proteins are identified based on their retention time in combination with the detection of specific marker peptides formed in the postcolumn digest. The optimization of important parameters such as enzyme concentration, reaction time, and organic modifier concentration is described. We demonstrated that the continuous-flow solution-phase digest method can be coupled on-line to the reversed-phase gradient liquid chromatography separation of proteins. Detection limits obtained for five model proteins, detected as specific marker peptides with m/z values of 300−1000, range from 30 to 90 fmol, with a linear response up to 3 pmol.
A generic method for the detection of covalent adducts to the cysteine-34 residue of human serum albumin (HSA) has been developed, based on an on-line combination of immunoaffinity chromatography for selective sample pre-treatment, solution phase digestion, liquid chromatography and tandem mass spectrometry. Selective anti-HSA antibodies immobilized on agarose were used for sample pre-concentration and purification of albumin from the chemically produced alkylated HSA. After elution, HSA and HSA adducts are mixed with pronase and directed to a reaction capillary kept at a digestion temperature of 70 °C. The digestion products were trapped on-line on a C18 SPE cartridge. The peptides were separated on a reversed-phase column using a gradient of organic modifier and subsequently detected using tandem mass spectrometry. Modified albumin samples consisted of synthetically alkylated HSA by the reactive metabolite of acetaminophen, N-acetyl-p-benzoquinoneimine (NAPQI), and using the alkylating agent 1-chloro-2,4-dinitrobenzene (CDNB) as reference. The resulting mixture of alkylated versus non-modified albumin has been applied to the on-line system, and alkylation of HSA is revealed by the detection of the modified marker tetra-peptide glutamine–cysteine–proline–phenylalanine (QCPF) adducts NAPQI-QCPF and CDNB-QCPF. Detection of alkylated species was enabled by the use of data comparison algorithms to distinguish between unmodified and modified HSA samples. The in-solution digestion proved to be a useful tool for enabling fast (less than 2 min) and reproducible on-line digestion of HSA. A detection limit of 1.5 μmol/L of modified HSA could be obtained by applying 10 μL of NAPQI-HSA sample.
On-line digestion coupled prior to or after an analytical separation in combination with electrospray ionization mass spectrometric detection offers many possibilities to analyse proteins by means of peptides and can result in selective, efficient, fast and accurate analytical information. However, compatibility issues need to be kept in mind when directly coupling biochemical reactions with chemical separation and detection principles. This chapter deals with different modes of on-line digestion. In-solution digestion can be used in a similar way to the automated approach frequently used in immobilized enzyme reactors, but without the need to immobilize enzymes.
This unit describes the solid-phase synthesis and downstream processing for RNA oligonucleotides with a length of up to 40 to 50 nucleotides on a 1- to 4-mmol scale with subsequent conjugation to PEG using the l-RNA spiegelmer NOX-E36 as an example. Following synthesis and two-step deprotection, the crude oligonucleotide is purified by preparative reversed-phase HPLC and desalted by tangential flow ultrafiltration. The resulting intermediate amino-modified oligonucleotide is reacted with NHS-ester-activated PEG, and the oligonucleotide-PEG conjugate is obtained after preparative AX-HPLC purification, followed by ultrafiltration and lyophilization. Critical process parameters are described, as well as time considerations and examples for analytical methods used as in-process and quality controls.
The ability to verify the sequence of a nucleic acid-based therapeutic is an essential step in the drug development process. The challenge associated with sequence identification increases with the length and nuclease resistance of the nucleic acid molecule, the latter being an important attribute of therapeutic oligonucleotides. We describe methods for the sequence determination of Spiegelmers, which are enantiomers of naturally occurring RNA with high resistance to enzymatic degradation. Spiegelmer sequencing is effected by affixing a label or hapten to the 5′-end of the oligonucleotide and chemically degrading the molecule in a controlled fashion to generate fragments that are then resolved and identified using liquid chromatography-mass spectrometry. The Spiegelmer sequence is then derived from these fragments. Examples are shown for two different Spiegelmers (NOX-E36 and NOX-A12), and the specificity of the method is shown using a NOX-E36 mismatch control.
Many biochemical assays used for the quantification of e.g. proteins and peptides in biological matrices are based on bioaffinity principles in combination with colorimetric or fluorescence detection. Although the detection limits of such methods are frequently very
satisfying, the information provided on the target molecule to be analyzed is very poor. Direct application with more selective detection principles such as mass spectrometric detection appears to be very complex due to the chemical composition of the analytes, such as high molecular weight and limited ionization possibilities. In my thesis, several approaches are presented which directly combine biospecificity of affinity interaction assays with selective mass spectrometric detection.
Activities and Societies: Projects with Toxicology group Projects for TNO (Netherlands) Analysis of proteins (ELISA, LCMS, Biacore)
Activities and Societies: Internship at Roche Pharmaceuticals in Mannheim (Germany) - HPLC method development Diploma work at Roche Diagnostics in Penzberg (Germany) - Hypersensitive ELISA development on PEGylated proteins + Immunoassay development of Synthetic large proteins
Over 300 million professionals are already on LinkedIn. Find who you know.
VP Drug Discovery and Preclinical Research bei NOXXON Pharma AG
VP Chemistry&Manufacturing bei NOXXON Pharma AG
Medical Director bei NOXXON Pharma AG
Chief Business Officer at NOXXON Pharma AG
Director Biophysical Analysis bei NOXXON Pharma AG
Application Development Teamleader Drug Imaging and Structure Analysis Solutions R/D
Senior Clinical Trial Manager bei NOXXON Pharma AG
Chief Scientific Officer at apceth GmbH & Co. KG
VP Project Management at NOXXON Pharma AG