Advances in drug-protein adduct analysis Using LC-MS based proteomics

Irth, H. [Promotor]Niessen, W.M.A. [Promotor]Lingeman, H. [Copromotor

With bauer in the east

Bandung106 p.; 23 cm

Protein digestion: An overview of the available techniques and recent developments

Several proteomics approaches are available that are defined by the level (protein or peptide) at which analysis takes place. The most widely applied method still is bottom-up proteomics where the protein is digested into peptides that can be efficiently analyzed with a wide range of LC-MS or MALDI-TOF-MS instruments. Sample preparation for bottom-up proteomics experiments requires several treatment steps in order to get from the protein to the peptide level and can be very laborious. The most crucial step in such approaches is the protein digestion, which is often the bottleneck in terms of time consumption. Therefore, a significant gain in throughput may be obtained by speeding up the digestion process. Current techniques allow for reduction of the digestion time from overnight (∼15 h) to minutes or even seconds. This advancement also makes integration into online systems feasible, thereby reducing the number of tedious sample handling steps and the risk of sample loss. In this review, an overview is given of the currently available digestion strategies and recent developments in the acceleration of the digestion process. Additionally, tailored approaches for classes of proteins that pose specific challenges are discussed. © 2013 American Chemical Society

A high-throughput sample preparation method for cellular proteomics using 96-well filter plates.

A high-throughput sample preparation protocol based on the use of 96-well molecular weight cutoff (MWCO) filter plates was developed for shotgun proteomics of cell lysates. All sample preparation steps, including cell lysis, buffer exchange, protein denaturation, reduction, alkylation and proteolytic digestion are performed in a 96-well plate format, making the platform extremely well suited for processing large numbers of samples and directly compatible with functional assays for cellular proteomics. In addition, the usage of a single plate for all sample preparation steps following cell lysis reduces potential samples losses and allows for automation. The MWCO filter also enables sample concentration, thereby increasing the overall sensitivity, and implementation of washing steps involving organic solvents, for example, to remove cell membranes constituents. The optimized protocol allowed for higher throughput with improved sensitivity in terms of the number of identified cellular proteins when compared to an established protocol employing gel-filtration columns. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Protein digestion optimization for characterization of drug-protein adducts using response surface modeling

The formation of drug-protein adducts in vivo may have important clinical and toxicological implications. Consequently, there is a great interest in the detection of these adducts and the elucidation of their role in the processes leading to adverse and idiosyncratic drug reactions. Enzymatic digestion is a crucial step in bottom-up proteomics strategies for the analysis of drug-protein adducts. The chosen proteolytic enzyme and digestion conditions have a large influence on the protein coverage of the modified protein and identification of its modification site. In this work, the enzymatic digestion conditions (pH, temperature and time) of trypsin and thermolysin were optimized specifically for the characterization of Human Serum Albumin (HSA) adducts. Using a Design of Experiments (DOE), it was found that of the three optimized parameters mainly pH and temperature showed strong effects on both responses. The optimized digestion conditions were different from those obtained from the suppliers or literature. Their application to HSA adducts resulted in improved protein coverage and signal intensity regarding the peptide containing the modification site, thereby highlighting the importance of a detailed optimization of digestion conditions. © 2010 Elsevier B.V

Identification and quantification of drug-albumin adducts in serum samples from a drug exposure study in mice

The formation of drug-protein adducts following the bioactivation of drugs to reactive metabolites has been linked to adverse drug reactions (ADRs) and is a major complication in drug discovery and development. Identification and quantification of drug-protein adducts in vivo may lead to a better understanding of drug toxicity, but is challenging due to their low abundance in the complex biological samples. Human serum albumin (HSA) is a well-known target of reactive drug metabolites due to the free cysteine on position 34 and is often the first target to be investigated in covalent drug binding studies. Presented here is an optimized strategy for targeted analysis of low-level drug-albumin adducts in serum. This strategy is based on selective extraction of albumin from serum through affinity chromatography, efficient sample treatment and clean-up using gel filtration chromatography followed by tryptic digestion and LC-MS analysis. Quantification of the level of albumin modification was performed through a comparison of non-modified and drug-modified protein based on the relative peak area of the tryptic peptide containing the free cysteine residue. The analysis strategy was applied to serum samples resulting from a drug exposure experiment in mice, which was designed to study the effects of different acetaminophen (APAP) treatments on drug toxicity. APAP is bioactivated to N-acetyl-p-benzoquinoneimine (NAPQI) in both humans and mice and is known to bind to cysteine 34 (cys34) of HSA. Analysis of the mouse serum samples revealed the presence of extremely low-level NAPQI-albumin adducts of approximately 0.2% of the total mouse serum albumin (MSA), regardless of the length of drug exposure. Due to the targeted nature of the strategy, the NAPQI-adduct formation on cys34 could be confirmed while adducts to the second free cysteine on position 579 of MSA were not detected. © 2013 Elsevier B.V

Top-Down MALDI-In-Source Decay-FTICR Mass Spectrometry of Isotopically Resolved Proteins

Proteomic

In-Depth Characterization of Protein Disulfide Bonds by Online Liquid Chromatography-Electrochemistry-Mass Spectrometry

Genetics of disease, diagnosis and treatmen