Tandem mass spectrometry of non-enzymatically glycated peptides and proteins

The thesis presents the study of the reaction of glyoxal (ethanedial) with polypeptides. This reaction is important in the food industry as well as during ageing and diabetes mellitus. To study this reaction a Fourier transform ion cyclotron resonance mass spectrometer coupled with electron capture dissociation and collisionally activated dissociation was used. Initially this reaction was carried out in the neuropeptide Substance P to set up the reaction conditions, sample preparation, as well as the instrumental parameters in the mass spectrometer. The results in Substance P revealed two compounds, with mass additions assigned as C2O and C2H2O2 from glyoxal, were formed. MS/MS results showed that the modification site for both species could be located at either the arginine residue or at the N-terminus. Thus, in order to distinguish N-terminus from arginine modification the position of the arginine was varied in four model peptides. The results indicated that both mass additions C2O, C2H2O2 were located at the arginine residue. Interestingly, two of those model peptides showed an unusual mass addition of 21.9843 Da, which was assigned as a new type of glyoxal modification at the arginine residue showing the addition of two carbon atoms from glyoxal and the loss of two hydrogen atoms from the peptide (C2-H2), herein referred to as 2-imino-imidazole. In order to assess the involvement of other residues in the reaction with glyoxal a new set of experiments in acetylated and non-acetylated undecapeptides were carried out. Unexpectedly, these experiments revealed that two species with the same mass (16.01092 Da) were being formed in the non-acetylated peptide. One of the species corresponded to diglycation, where the results suggest that the glyoxal binding at the lysine residue is crosslinked with the N-terminus. The second species showing the addition of 116.01092 Da was formed at the arginine residue forming a species, here called a glyoxal dimer, at the arginine residue. The formation of the glyoxal dimer species was also observed in the acetylated peptide. Although is clear that crosslinking between the lysine residue and the N-terminus is not possible in the acetylated peptide, the results seem to indicate that crosslinking between the amino group of the lysine and the amide group of glutamine could occur. However, a systematic study varying the position of the lysine relative to the glutamine residue and also relative to the N-terminus needs to be addressed in the future in order to determine the extent of the involvement of the N-terminus and amide group in the glyoxal glycation reaction

Electron capture dissociation mass spectrometry of phosphopeptides: Arginine and phosphoserine

AbstractWe have previously shown that the presence of phosphorylation can inhibit detection of electron capture dissociation (ECD) fragments of doubly charged peptide ions. The presence of non-covalent interactions, in the form of salt-bridges or ionic hydrogen bonds, prevents the separation of fragments following backbone cleavage. Here, we show the electron capture dissociation mass spectrometry of a suite of model peptides designed to investigate the relationship between phosphoserine and arginine position, namely AApSAnRAmKA (n=0–6, m=6–0), the presence of lysine residues (AApSAAKAARAKA) and AAApSARAAAAKAAAK, and the presence of proline A(A/P)ApSARAAA(A/P)KAAAK. The latter are analogous to the peptides studied previously. The results show that the presence of phosphoserine and basic amino acid residues alone does not inhibit ECD fragmentation, even when the number of basic amino acid residues is greater than the precursor charge state. Neither did the presence of proline in the peptide sequence suppress ECD backbone cleavage. Nevertheless, the presence and relative position of the phosphorylated residue do alter the observed backbone fragmentation abundance. In addition, the presence of phosphorylation appears to inhibit cleavage within the arginine side-chain regardless of the relative position of the arginine residue. The results suggest that ECD fragmentation behaviour is dependent on the three-dimensional structure of a peptide rather than its sequence

Liquid Extraction Surface Analysis (LESA) Electron-Induced Dissociation and Collision-Induced Dissociation Mass Spectrometry of Small Molecule Drug Compounds.

Here, we present liquid extraction surface analysis (LESA) coupled with electron-induced dissociation (EID) mass spectrometry in a Fourier-transform ion cyclotron resonance mass spectrometer for the analysis of small organic pharmaceutical compounds directly from dosed tissue. First, the direct infusion electrospray ionisation EID and collision-induced dissociation (CID) behaviour of erlotinib, moxifloxacin, clozapine and olanzapine standards were compared. EID mass spectra were also compared with experimental or reference electron impact ionisation mass spectra. The results show that (with the exception of erlotinib) EID and CID result in complementary fragment ions. Subsequently, we performed LESA EID MS/MS and LESA CID MS/MS on singly charged ions of moxifloxacin and erlotinib extracted from a thin tissue section of rat kidney from a cassette-dosed animal. Both techniques provided structural information, with the majority of peaks observed for the drug standards also observed for the tissue-extracted species. Overall, these results demonstrate the feasibility of LESA EID MS/MS of drug compounds from dosed tissue and extend the number of molecular structures for which EID behaviour has been determined. Graphical Abstract ᅟ

BioPAN: a web-based tool to explore mammalian lipidome metabolic pathways on LIPID MAPS.

Lipidomics increasingly describes the quantification using mass spectrometry of all lipids present in a biological sample.  As the power of lipidomics protocols increase, thousands of lipid molecular species from multiple categories can now be profiled in a single experiment.  Observed changes due to biological differences often encompass large numbers of structurally-related lipids, with these being regulated by enzymes from well-known metabolic pathways.  As lipidomics datasets increase in complexity, the interpretation of their results becomes more challenging.  BioPAN addresses this by enabling the researcher to visualise quantitative lipidomics data in the context of known biosynthetic pathways.  BioPAN provides a list of genes, which could be involved in the activation or suppression of enzymes catalysing lipid metabolism in mammalian tissues

THP-1 macrophage cholesterol efflux is impaired by palmitoleate through Akt activation.

Lipoprotein lipase (LPL) is upregulated in atherosclerotic lesions and it may promote the progression of atherosclerosis, but the mechanisms behind this process are not completely understood. We previously showed that the phosphorylation of Akt within THP-1 macrophages is increased in response to the lipid hydrolysis products generated by LPL from total lipoproteins. Notably, the free fatty acid (FFA) component was responsible for this effect. In the present study, we aimed to reveal more detail as to how the FFA component may affect Akt signalling. We show that the phosphorylation of Akt within THP-1 macrophages increases with total FFA concentration and that phosphorylation is elevated up to 18 hours. We further show that specifically the palmitoleate component of the total FFA affects Akt phosphorylation. This is tied with changes to the levels of select molecular species of phosphoinositides. We further show that the total FFA component, and specifically palmitoleate, reduces apolipoprotein A-I-mediated cholesterol efflux, and that the reduction can be reversed in the presence of the Akt inhibitor MK-2206. Overall, our data support a negative role for the FFA component of lipoprotein hydrolysis products generated by LPL, by impairing macrophage cholesterol efflux via Akt activation

Integrated lipidomics and proteomics reveal cardiolipin alterations, upregulation of HADHA and long chain fatty acids in pancreatic cancer stem cells.

Pancreatic cancer stem cells (PCSCs) play a key role in the aggressiveness of pancreatic ductal adenocarcinomas (PDAC); however, little is known about their signaling and metabolic pathways. Here we show that PCSCs have specific and common proteome and lipidome modulations. PCSCs displayed downregulation of lactate dehydrogenase A chain, and upregulation of trifunctional enzyme subunit alpha. The upregulated proteins of PCSCs are mainly involved in fatty acid (FA) elongation and biosynthesis of unsaturated FAs. Accordingly, lipidomics reveals an increase in long and very long-chain unsaturated FAs, which are products of fatty acid elongase-5 predicted as a key gene. Moreover, lipidomics showed the induction in PCSCs of molecular species of cardiolipin with mixed incorporation of 16:0, 18:1, and 18:2 acyl chains. Our data indicate a crucial role of FA elongation and alteration in cardiolipin acyl chain composition in PCSCs, representing attractive therapeutic targets in PDAC

CD151 regulates expression of FGFR2 in breast cancer cells via PKC-dependent pathways.

Expression of the tetraspanin CD151 is frequently upregulated in epithelial malignancies and correlates with poor prognosis. Here, we report that CD151 is involved in regulation of the expression of fibroblast growth factor receptor 2 (FGFR2). Depletion of CD151 in breast cancer cells resulted in an increased level of FGFR2. Accordingly, an inverse correlation between CD151 and FGFR2 was observed in breast cancer tissues. CD151-dependent regulation of the FGFR2 expression relies on post-transcriptional mechanisms involving HuR (also known as ELAVL1), a multifunctional RNA-binding protein, and the assembly of processing bodies (P-bodies). Depletion of CD151 correlated with inhibition of PKC, a well-established downstream target of CD151. Accordingly, the levels of dialcylglycerol species were decreased in CD151-negative cells, and inhibition of PKC resulted in the increased expression of FGFR2. Whereas expression of FGFR2 itself did not correlate with any of the clinicopathological data, we found that FGFR2-/CD151+ patients were more likely to have developed lymph node metastasis. Conversely, FGFR2-/CD151- patients demonstrated better overall survival. These results illustrate functional interdependency between CD151 complexes and FGFR2, and suggest a previously unsuspected role of CD151 in breast tumorigenesis

LIPID MAPS: Update to databases and tools for the lipidomics community

LIPID MAPS (LIPID Metabolites and Pathways Strategy), www.lipidmaps.org, provides a systematic and standardized approach to organizing lipid structural and biochemical data. Founded 20 years ago, the LIPID MAPS nomenclature and classification has become the accepted community standard. LIPID MAPS provides databases for cataloging and identifying lipids at varying levels of characterization in addition to numerous software tools and educational resources, and became an ELIXIR-UK data resource in 2020. This paper describes the expansion of existing databases in LIPID MAPS, including richer metadata with literature provenance, taxonomic data and improved interoperability to facilitate FAIR compliance. A joint project funded by ELIXIR-UK, in collaboration with WikiPathways, curates and hosts pathway data, and annotates lipids in the context of their biochemical pathways. Updated features of the search infrastructure are described along with implementation of programmatic access via API and SPARQL. New lipid-specific databases have been developed and provision of lipidomics tools to the community has been updated. Training and engagement have been expanded with webinars, podcasts and an online training school

The circadian clock components BMAL1 and REV-ERBα regulate flavivirus replication.

The circadian clock regulates immune responses to microbes and affects pathogen replication, but the underlying molecular mechanisms are not well understood. Here we demonstrate that the circadian components BMAL1 and REV-ERBα influence several steps in the hepatitis C virus (HCV) life cycle, including particle entry into hepatocytes and RNA genome replication. Genetic knock out of Bmal1 and over-expression or activation of REV-ERB with synthetic agonists inhibits the replication of HCV and the related flaviruses dengue and Zika via perturbation of lipid signaling pathways. This study highlights a role for the circadian clock component REV-ERBα in regulating flavivirus replication

Liquid Extraction Surface Analysis (LESA) Electron-Induced Dissociation and Collision-Induced Dissociation Mass Spectrometry of Small Molecule Drug Compounds.

Here, we present liquid extraction surface analysis (LESA) coupled with electron-induced dissociation (EID) mass spectrometry in a Fourier-transform ion cyclotron resonance mass spectrometer for the analysis of small organic pharmaceutical compounds directly from dosed tissue. First, the direct infusion electrospray ionisation EID and collision-induced dissociation (CID) behaviour of erlotinib, moxifloxacin, clozapine and olanzapine standards were compared. EID mass spectra were also compared with experimental or reference electron impact ionisation mass spectra. The results show that (with the exception of erlotinib) EID and CID result in complementary fragment ions. Subsequently, we performed LESA EID MS/MS and LESA CID MS/MS on singly charged ions of moxifloxacin and erlotinib extracted from a thin tissue section of rat kidney from a cassette-dosed animal. Both techniques provided structural information, with the majority of peaks observed for the drug standards also observed for the tissue-extracted species. Overall, these results demonstrate the feasibility of LESA EID MS/MS of drug compounds from dosed tissue and extend the number of molecular structures for which EID behaviour has been determined. Graphical Abstract ᅟ