A comparative study of synthetic winged peptides for absolute protein quantification

A proper internal standard choice is critical for accurate, precise, and reproducible mass spectrometry-based proteomics assays. Synthetic isotopically labeled (SIL) proteins are currently considered the gold standard. However, they are costly and challenging to obtain. An alternative approach uses SIL peptides or SIL "winged" peptides extended at C- or/and N-terminus with an amino acid sequence or a tag cleaved during enzymatic proteolysis. However, a consensus on the design of a winged peptide for absolute quantification is missing. In this study, we used human serum albumin as a model system to compare the quantitative performance of reference SIL protein with four different designs of SIL winged peptides: (i) commercially available SIL peptides with a proprietary trypsin cleavable tag at C-terminus, (ii) SIL peptides extended with five amino acid residues at C-terminus, (iii) SIL peptides extended with three and (iv) with five amino acid residues at both C- and N-termini. Our results demonstrate properties of various SIL extended peptides designs, e.g., water solubility and efficiency of trypsin enzymatic cleavage with primary influence on quantitative performance. SIL winged peptides extended with three amino acids at both C- and N-termini demonstrated optimal quantitative performance, equivalent to the SIL protein

On-line electrochemistry–bioaffinity screening with parallel HR-LC-MS for the generation and characterization of modified p38α kinase inhibitors

In this study, an integrated approach is developed for the formation, identification and biological characterization of electrochemical conversion products of p38α mitogen-activated protein kinase inhibitors. This work demonstrates the hyphenation of an electrochemical reaction cell with a continuous-flow bioaffinity assay and parallel LC-HR-MS. Competition of the formed products with a tracer (SKF-86002) that shows fluorescence enhancement in the orthosteric binding site of the p38α kinase is the readout for bioaffinity. Parallel HR-MSn experiments provided information on the identity of binders and non-binders. Finally, the data produced with this on-line system were compared to electrochemical conversion products generated off-line. The electrochemical conversion of 1-{6-chloro-5-[(2R,5S)-4-(4-fluorobenzyl)-2,5-dimethylpiperazine-1-carbonyl]-3aH-indol-3-yl}-2-morpholinoethane-1,2-dione resulted in eight products, three of which showed bioaffinity in the continuous-flow p38α bioaffinity assay used. Electrochemical conversion of BIRB796 resulted, amongst others, in the formation of the reactive quinoneimine structure and its corresponding hydroquinone. Both products were detected in the p38α bioaffinity assay, which indicates binding to the p38α kinase

Online electro-Fenton-mass spectrometry reveals 2,4′,5-trichlorobiphenyl oxidation products and binding to organic matter

Electrochemistry–mass spectrometry is used to simulate redox reactions in many research disciplines because this technique is fast and provides information on compound metabolites. However, the analysis of the degradation of refractory organic pollutants by reactive oxygen species is difficult to achieve by the electrochemistry step. Therefore, here we use online electro-Fenton-mass spectrometry to study for the first time the oxidation of 2,4′,5-trichlorobiphenyl [polychlorinated biphenyl (PCB) 31] by reactive oxygen species and the binding reactions of PCB degradation products with model substances of natural organic matter. The degradation products were identified by coupled Q Trap mass spectrometry. We observed a binding of a degradation product with γ-l-glutamyl-l-cysteinyl-glycine. We propose a transformation pathway. We conclude that online electro-Fenton-mass spectrometry is a promising technique to study the oxidation of refractory organic pollutants and further binding of degradation products with natural organic matter

Quantifying Protein Measurands by Peptide Measurements: Where Do Errors Arise?

Proteomic