European Journal of Mass Spectrometry最新文献

Irreversible protein footprinting is a mass spectrometry-based approach in which solvent-accessible sites of a protein are modified to assess high-order protein structure. Structural insights can be gained by determining the position and extents of modification. The usual approach to obtain the "footprint" is to analyze the protein through bottom-up LC-MS/MS. In this approach, the proteins are digested to yield a mixture of peptides that are then separated by LC before locating the modification sites by MS/MS. This process consumes substantial amounts of time and is difficult to accelerate for applications that require quick and high-throughput analysis. Here, we describe employing matrix-assisted laser desorption/ionization (MALDI) in-source decay (ISD) to analyze a footprinted small test protein (ubiquitin) via a top-down approach. Matrix-assisted laser desorption/ionization is easily adapted for high-throughput analysis, and top-down strategies can avoid lengthy proteolysis and LC separation. We optimized the method with model peptides and then demonstrated its feasibility on ubiquitin submitted to two types of footprinting. We found that MALDI ISD can produce a comprehensive set of fragment ions for small proteins, affording footprinting information in a fast manner and giving results that agree with the established methods, and serve as a rough measure of protein solvent accessibility. To assist in the implementation of the MALDI approach, we developed a method of processing top-down ISD data.

Hexahydrocannabinol (HHC) is a cannabinoid that has been known since 1940 but has only recently found its way into recreational use as a psychoactive drug. HHC has been used as a legal alternative to tetrahydrocannabinol (THC) in many countries, but first countries already placed it under their narcotic substances law. Our aim was to evaluate a reliable analytical method for the proof of HHC consumption by LC-MS/MS and GC-MS. We identified the two epimers of HHC and metabolites after HHC consumption by two volunteers (inhalation by use of a vaporizer and oral intake). LC-HR-MS/MS, LC-MS/MS and GC-MS with literature data (EI-MS spectra of derivatives) and reference compounds - as far as commercially available - were used for metabolite identification. Phase-II-metabolites (glucuronides) of HHC and OH-HHC were found in urine samples with LC-HR-MS/MS and LC-MS/MS. The main metabolite was tentatively identified with GC-MS as 4'OH-HHC (stereochemistry on C9 and C4' unknown). Another major side-chain hydroxylated metabolite found by LC-MS/MS could not be unambiguously identified. Both epimers of 11-OH-HHC were found in considerable amounts in urine. (8R, 9R)-8-OH-HHC was identified as a minor metabolite with GC-MS and LC-MS/MS. While (9S)-HHC was found in urine after oral intake and inhalation of HHC, the more psychoactive epimer (9R)-HHC was only found in urine after inhalation. Several other minor metabolites were detected but not structurally identified. We found that after oral or inhalative consumption the urinary main metabolites of a diastereomeric mixture of HHC are different from the respective, major Δ⁹-THC metabolites (11-OH-Δ⁹-THC and 11-nor-9-carboxy-Δ⁹-THC). Although a sensitive LC-MS/MS and GC-SIM-MS method were set-up for the reference compounds (9R)-11-nor-9-carboxy-HHC and (9S)-11-nor-9-carboxy-HHC, these oxidation products were not detected in urine with these techniques. To further increase sensitivity, a GC-MS/MS method was developed, and the 11-nor-9-carboxy metabolites of HHC were confirmed to be present as minor metabolites.

Pepsin, because of its optimal activity at low acidic pH, has gained importance in mass spectrometric proteome research as a readily available and easy-to-handle protease. Pepsin has also been study object of protein higher-order structure analyses, but questions about how to best investigate pepsin in-solution conformers still remain. We first determined dependencies of pepsin ion charge structures on solvent pH which indicated the in-solution existence of (a) natively folded pepsin (N) which by nanoESI-MS analysis gave rise to a narrow charge state distribution with an 11-fold protonated most intense ion signal, (b) unfolded pepsin (U) with a rather broad ion charge state distribution whose highest ion signal carried 25 protons, and (c) a compactly folded pepsin conformer (C) with a narrow charge structure and a 12-fold protonated ion signal in the center of its charge state envelope. Because pepsin is a protease, unfolded pepsin became its own substrate in solution at pH 6.6 since at this pH some portion of pepsin maintained a compact/native fold which displayed enzymatic activity. Subsequent mass spectrometric ITEM-TWO analyses of pepstatin A - pepsin complex dissociation reactions in the gas phase confirmed a very strong binding of pepstatin A by natively folded pepsin (N). ITEM-TWO further revealed the existence of two compactly folded in-solution pepsin conformers (C^a and C^b) which also were able to bind pepstatin A. Binding strengths of the respective compactly folded pepsin conformer-containing complexes could be determined and apparent gas phase complex dissociation constants and reaction enthalpies differentiated these from each other and from the pepstatin A - pepsin complex which had been formed from natively folded pepsin. Thus, ITEM-TWO turned out to be well suited to pinpoint in-solution pepsin conformers by interrogating quantitative traits of pepstatin A - pepsin complexes in the gas phase.

Mass spectrometry (MS) has developed over the last decades into the most informative and versatile analytical technology in molecular and structural biology (). The platform enables discovery, identification, and characterisation of non-volatile biomolecules, such as proteins, peptides, DNA, RNA, nutrients, metabolites, and lipids at both speed and scale and can elucidate their interactions and effects. The versatility, robustness, and throughput have rendered MS a major research and development platform in molecular human health and biomedical science. More recently, MS has also been established as the central tool for 'Molecular Nutrition', enabling comprehensive and rapid identification and characterisation of macro- and micronutrients, bioactives, and other food compounds. 'Molecular Nutrition' thereby helps understand bioaccessibility, bioavailability, and bioefficacy of macro- and micronutrients and related health effects. Hence, MS provides a lens through which the fate of nutrients can be monitored along digestion via absorption to metabolism. This in turn provides the bioanalytical foundation for 'Personalised Nutrition' or 'Precision Nutrition' in which design and development of diets and nutritional products is tailored towards consumer and patient groups sharing similar genetic and environmental predisposition, health/disease conditions and lifestyles, and/or objectives of performance and wellbeing. The next level of integrated nutrition science is now being built as 'Systems Nutrition' where public and personal health data are correlated with life condition and lifestyle factors, to establish directional relationships between nutrition, lifestyle, environment, and health, eventually translating into science-based public and personal heath recommendations and actions. This account provides a condensed summary of the contributions of MS to a precise, quantitative, and comprehensive nutrition and health science and sketches an outlook on its future role in this fascinating and relevant field.

Alcohol biomarkers are able to reflect the degree of recent or long-term alcohol consumption, covering different windows of detection. Phosphatidylethanols (PEths) are an emerging group of direct alcohol biomarkers that are widely applied in clinical and forensic applications. Their quantification can provide insight into an individual's drinking behaviour. Here, we present a new sub-class of yet unknown PEth species, LysoPEths, which are structurally related to PEth, but miss one fatty acyl chain. LysoPEths can be either a degradation product of PEth or a product of transesterification of lyso-phosphatidylcholine (LysoPC) with ethanol. To set up an analytical method, LysoPEth 16:0 was synthesised from PC 16:0/18:1 and characterised by LC-MS/MS, using an enzymatic method: phospholipase D (PLD), followed by phospholipase A₂ (PLA₂). Then, an LC-MS/MS method in MRM mode for LysoPEth 16:0 with additional LysoPEth species (LysoPEth 18:1, LysoPEth 18:2, and LysoPEth 20:4) and PEth 16:0/20:4 was developed. By incubation of freshly sampled venous blood of a teetotaller with ethanol at different concentrations, the formation of LysoPEth in parallel to PEth was investigated. With increasing ethanol concentrations, LysoPEth 16:0 was formed besides the known PEth species (PEth 16:0/18:1, PEth 16:0/18:2) for up to 72 h with LysoPEth concentrations being about three times lower than PEth concentrations. Storage of ethanol-free PEth-positive blood of an alcohol consumer at 37 °C showed that LysoPEth 16:0 concentrations increased, while PEth 16:0/18:1 concentrations decreased in the first 24 h for frozen/thawed blood, however not for freshly collected blood. Furthermore, LysoPEth 16:0 was detected in venous as well as lyophilised blood from clinical and forensic case work alongside with PEth 16:0/18:1, 16:0/18:2, and other PEth and LysoPEth species (PEth 16:0/20:4, LysoPEth 18:1, LysoPEth 18:2, and LysoPEth 20:4). LysoPEth 16:0 concentrations were found to be in linear correlation with PEth 16:0/18:1 (r²= 0.75).

Ubiquitin, a conserved protein in eukaryotic cells, exists as a monomer or polyubiquitin chains known as isopeptide-linked polymers. These chains are attached to a substrate or other ubiquitin molecules through a covalent bond between the α-amino group of lysine in ubiquitin and glycine in the C-terminal of the subsequent ubiquitin unit. The choice of the specific lysine residue in ubiquitin for forming ubiquitin-ubiquitin chains determines its biochemical and biological function. A detailed chemical structure-function evaluation of the respective polyubiquitin chain is required. Interestingly, specific lysine linkage polyubiquitin chains become covalently bonded to many pathological inclusions seen in serious human disease states which appear to be resistant to normal degradation, so the interaction between polyubiquitin chains and ubiquitin antibodies is very useful. For example, the neurofibrillary tangles of Alzheimer's disease and the Lewy bodies seen in Parkinson's disease are heavily ubiquitinated and can be readily visualized using specific ubiquitin antibodies. This study utilized synthetic ubiquitin building block peptides that contained various lysine residues (K6, K11, K33, K48, and K63) linked to a Gly-Gly dipeptide, with the aim of exploring the recognition specificity of the Lys63-polyubiquitin antibody. The interaction studies between different ubiquitin building blocks and the specific Lys63-ubiquitin (K63-Ub) antibody were performed by affinity-mass spectrometry (Affinity-MS) and immunoblotting which enables direct protein identification from biological material with unprecedented selectivity. Affinity-MS and dot blot data proved the specific binding of the K63-Ub antibody to the ubiquitin peptides containing Lys6 or Lys63 residues. In epitope excision for mass spectrometric epitope identification, the ubiquitin building block with Lys63 residue bound to the immobilized K63-Ub antibody was proteolytically cleaved using pronase. The resulting epitope and non-epitope fractions were subjected to matrix-assisted laser desorption/ionization-time of flight analysis, revealing that the epitope is located within the sequence ubiquitin(60-66). Epitope extraction-MS consistently confirmed these findings.

Osteosarcoma (OS) is the most common primary malignant tumor of bone, which occupying about 20% of all bone cancers. To increase understanding of the biology of OS, we developed and evaluated a top-down mass spectrometry approach to detect, identify and quantify low molecular weight (MW) proteins (i.e., 1 kDa < MW < 30 kDa) in osteosarcoma cells. Top-down proteomic (TDP) data was acquired using reversed phase nano-liquid chromatography in conjunction with high-resolution mass spectrometry and resulted in the assignment of 328 proteins and 820 proteoforms or degradation products with high confidence. Eight post-translational modifications (PTMs) were identified in the present study, including N-terminal acetylation, lysine acetylation, succinylation, malonylation, serine/tyrosine phosphorylation, histidine methylation and N-acetylleucine. We confirmed that a truncated N-terminal proteoform lost 73 Da of mass through removal of the N-terminal Met (-131 Da), acetylation of the second amino acid (+42 Da), and Met oxidation (+16 Da). The results showed that the levels of proteoforms/biodegradable peptides correlated with the metastatic phenotypes of osteosarcoma cell lines. This study demonstrates the benefits of TDP for the characterization and relative quantification of proteoforms with relevance to OS biology and the potential of small molecular weight proteoforms to serve as a still underappreciated source of biomarkers.

The way in which professor Michael Przybylski has combined the spirit of research with entrepreneurship has set an example for any and all scientists. He has made significant achievements in the fields of mass spectrometry, biochemistry and medicine, and has initiated important technological developments in the area of protein analysis. Between 2016 and 2023 professor Przybylski's scientific focus shifted on protein interactions with emphasis on aptamer-protein and antibody-protein analysis. This review focuses on professor Przybylski's achievements in the last few years highlighting his impact on the scientific community, on his students and colleagues.