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Ion mobility-mass spectrometry (IM-MS) has emerged as a powerful separation and identification tool to characterize synthetic polymer mixtures and topologies (linear, cyclic, star-shaped,…). Electrospray coupled to IM-MS already revealed the coexistence of several charge state-dependent conformations for a single charge state of biomolecules with strong intramolecular interactions, even when limited resolving power IM-MS instruments were used. For synthetic polymers, the sample’s polydispersity allows the observation of several chain lengths. A unique collision cross-section (CCS) trend is usually observed when increasing the degree of polymerization (DP) at constant charge state, allowing the deciphering of different polymer topologies. In this paper, we report multiple coexisting CCS trends when increasing the DP at constant charge state for linear poly(acrylamide) PAAm in the gas phase. This is similar to observations on peptides and proteins. Biomolecules show in addition population changes when collisionally heating the ions. In the case of synthetic PAAm, fragmentation occurred before reaching the energy for conformation conversion. These observations, which were made on two different IM-MS instruments (SYNAPT G2 HDMS and high resolution multi-pass cyclic T-Wave prototype from Waters), limit the use of ion mobility for synthetic polymer topology interpretations to polymers where unique CCS values are observed for each DP at constant charge state. Graphical Abstract ᅟ

Data-independent mass spectral acquisition is particularly powerful when combined with ultra-performance liquid chromatography (LC) that provides excellent separation of most components present in a given sample. Data-independent analysis (DIA) consists of alternating full MS scans and scans with fragmentation of all ions within a selected m/z range, providing precursor masses and structure information, respectively. Fragmentation spectra are acquired either by sequential isolation and fragmentation of sliding m/z ranges or fragmenting all ions entering the MS instrument with no ion isolation, termed broadband DIA. Previously, broadband DIA has only been possible using collision induced dissociation (CID). Here, we report the use of electron transfer dissociation (ETD) as the fragmentation technique in broadband DIA instead of traditional collision induced dissociation (CID) during MSsup E. In this approach, which we refer to as MSsup ETD, we implement the inherent benefits provided by ETD, such as discrimination of leucine and isoleucine, in a DIA setup. The combination of DIA analysis and ETD fragmentation with supplemental CID energy provides a powerful platform to obtain information on all precursors and their sequence from a single experiment. .

Data-independent mass spectral acquisition is particularly powerful when combined with ultra-performance liquid chromatography (LC) that provides excellent separation of most components present in a given sample. Data-independent analysis (DIA) consists of alternating full MS scans and scans with fragmentation of all ions within a selected m/z range, providing precursor masses and structure information, respectively. Fragmentation spectra are acquired either by sequential isolation and fragmentation of sliding m/z ranges or fragmenting all ions entering the MS instrument with no ion isolation, termed broadband DIA. Previously, broadband DIA has only been possible using collision induced dissociation (CID). Here, we report the use of electron transfer dissociation (ETD) as the fragmentation technique in broadband DIA instead of traditional collision induced dissociation (CID) during MS E . In this approach, which we refer to as MS ETD , we implement the inherent benefits provided by ETD, such as discrimination of leucine and isoleucine, in a DIA setup. The combination of DIA analysis and ETD fragmentation with supplemental CID energy provides a powerful platform to obtain information on all precursors and their sequence from a single experiment. Graphical Abstract ᅟ

Formation and accumulation of fibrillar plaques and aggregates of beta-amyloid peptide (Abeta) in brain have been recognized as characteristics of Alzheimer's disease (AD). Oligomeric aggregates of Ass are considered critical intermediates leading to progressive neurodegeneration; however, molecular details of the oligomerization and aggregation pathway and the structures of Abeta-oligomers are hitherto unclear. Using an in vitro fibril formation procedure of Abeta(1-40), beta-amyloid aggregates were prepared and insoluble aggregates separated from soluble products by centrifugation. In this study, ion mobility mass spectrometry (IM-MS) was applied in combination with electron paramagnetic resonance spectroscopy (EPR) to the identification of the components of Abeta-oligomers, and to their structural and topographical characterization. The formation of Abeta-oligomers and aggregates was monitored by gel electrophoresis, and Abeta-oligomer bands were identified by in-gel tryptic digestion and matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) to consist predominantly of Abeta(1-40) peptide. First, ion mobility-MS studies of soluble Abeta-aggregates prepared by incubation for 5 days were performed on a quadrupole time-of-flight mass spectrometer and revealed (1) the presence of at least two different conformational states, and (2), the formation of Met-35 oxidized products. For estimation of the size of Abeta-aggregates using EPR spectroscopy, a modified Abeta(1-40) peptide containing an additional N-terminal cysteine residue was prepared, and a 3-(2-iodoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy radical spin label derivative (IPSL) was coupled by S-alkylation. The EPR spectra of the spin-labeled Cys-Abeta(1-40) oligomers were matched with spectra simulations using a multi-component simulation strategy, resulting in complete agreement with the gel electrophoresis results.

Data-independent mass spectral acquisition is particularly powerful when combined with ultra-performance liquid chromatography (LC) that provides excellent separation of most components present in a given sample. Data-independent analysis (DIA) consists of alternating full MS scans and scans with fragmentation of all ions within a selected m/z range, providing precursor masses and structure information, respectively. Fragmentation spectra are acquired either by sequential isolation and fragmentation of sliding m/z ranges or fragmenting all ions entering the MS instrument with no ion isolation, termed broadband DIA. Previously, broadband DIA has only been possible using collision induced dissociation (CID). Here, we report the use of electron transfer dissociation (ETD) as the fragmentation technique in broadband DIA instead of traditional collision induced dissociation (CID) during MS . In this approach, which we refer to as MS , we implement the inherent benefits provided by ETD, such as discrimination of leucine and isoleucine, in a DIA setup. The combination of DIA analysis and ETD fragmentation with supplemental CID energy provides a powerful platform to obtain information on all precursors and their sequence from a single experiment. Graphical Abstract ᅟ.

Formation and accumulation of fibrillar plaques and aggregates of ß-amyloid peptide (Aß) in brain have been recognized as characteristics of Alzheimer's disease (AD). Oligomeric aggregates of Aß are considered critical intermediates leading to progressive neurodegeneration; however, molecular details of the oligomerization and aggregation pathway and the structures of Aß-oligomers are hitherto unclear. Using an in vitro fibril formation procedure of Aß(1-40), ß-amyloid aggregates were prepared and insoluble aggregates separated from soluble products by centrifugation. In this study, ion mobility mass spectrometry (IM-MS) was applied in combination with electron paramagnetic resonance spectroscopy (EPR) to the identification of the components of Aß-oligomers, and to their structural and topographical characterization. The formation of Aß-oligomers and aggregates was monitored by gel electrophoresis, and Aß-oligomer bands were identified by in-gel tryptic digestion and matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) to consist predominantly of Aß(1-40) peptide. First, ion mobility-MS studies of soluble Aß-aggregates prepared by incubation for 5 days were performed on a quadrupole time-of-flight mass spectrometer and revealed (1) the presence of at least two different conformational states, and (2), the formation of Met-35 oxidized products. For estimation of the size of Aß-aggregates using EPR spectroscopy, a modified Aß(1-40) peptide containing an additional N-terminal cysteine residue was prepared, and a 3-(2-iodoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy radical spin label derivative (IPSL) was coupled by S-alkylation. The EPR spectra of the spin-labeled Cys-Aß(1-40) oligomers were matched with spectra simulations using a multi-component simulation strategy, resulting in complete agreement with the gel electrophoresis results.

Biotin synthase (BS) catalyses the final sulfur incorporation step in the biosynthesis of biotin. Its chemical mechanism, primary amino acid sequence and tertiary place it in the Radical-SAM-dependent superfamily of >600 proteins. The gene encoding E. coli biotin synthase (bioB) has been expressed as a histidine-fusion protein (6HisBS) and the recombinant protein purified in a single step using immobilised metal-affinity chromatography (IMAC). In addition, a number of single and double amino acid mutations of the conserved cysteine residues within the ‘cys box’ were constructed and purified using similar methodology. Wild-type and mutant 6HisBS proteins were compared using mass spectrometry (LC-ESI-MS) and UV-visible spectroscopy to probe characteristics altered by the mutation. Biotin synthase was analysed for its ability to bind the cofactor pyridoxal 5’-phosphate (PLP). Two single point mutations (K490Q and K49R) of a putative PLP-binding residue, Lys 49, were isolated and compared with wild-type BS using a combination of biochemical and mass spectrometry techniques. Each protein was subjected to enzymatic proteolysis and peptide modification analysed by means of MALDI-ToF MS, ESI-QTOF MS and MS/MS. We identified a unique Lys49-containing fragment of the 6HisBS which corresponds to the PLP modified peptide. In contrast, this modified peptide is absent in the mutant proteins. This observation is further confirmed by MS/MS sequencing of this MS observed species which shows CID of both the peptide backbone and covalently attached PLP moiety. Biotin synthase was prepared in the presence of excess PLP and its interaction with cysteine monitored with UV-visible spectroscopy. We observed marked changes in the UV-vis profile of biotin synthase under these conditions which is consistent observations of PLP-dependent cysteine desulferase enzymes.

Male breast cancer accounts for approximately 1% of all breast cancer. To date, risk factors for male breast cancer are poorly defined, but certain risk factors and genetic features appear common to both male and female breast cancer. Genome-wide association studies (GWAS) have recently identified common single nucleotide polymorphisms (SNPs) that influence female breast cancer risk; 12 of these have been independently replicated. To examine if these variants contribute to male breast cancer risk, we genotyped 433 male breast cancer cases and 1,569 controls. Five SNPs showed a statistically significant association with male breast cancer: rs13387042 (2q35) (odds ratio (OR)  = 1.30, p = 7.98×10⁻⁴), rs10941679 (5p12) (OR = 1.26, p = 0.007), rs9383938 (6q25.1) (OR = 1.39, p = 0.004), rs2981579 (FGFR2) (OR = 1.18, p = 0.03), and rs3803662 (TOX3) (OR = 1.48, p = 4.04×10⁻⁶). Comparing the ORs for male breast cancer with the published ORs for female breast cancer, three SNPs--rs13387042 (2q35), rs3803662 (TOX3), and rs6504950 (COX11)--showed significant differences in ORs (p<0.05) between sexes. Breast cancer is a heterogeneous disease; the relative risks associated with loci identified to date show subtype and, based on these data, gender specificity. Additional studies of well-defined patient subgroups could provide further insight into the biological basis of breast cancer development.

Targeting Meis1 and Hoxb13 transcriptional activity could be a viable therapeutic strategy for heart regeneration. In this study, we performd an in silico screening to identify FDA-approved drugs that can inhibit Meis1 and Hoxb13 transcriptional activity based on the resolved crystal structure of Meis1 and Hoxb13 bound to DNA. Paromomycin (Paro) and neomycin (Neo) induced proliferation of neonatal rat ventricular myocytes in vitro and displayed dose-dependent inhibition of Meis1 and Hoxb13 transcriptional activity by luciferase assay and disruption of DNA binding by electromobility shift assay. X-ray crystal structure revealed that both Paro and Neo bind to Meis1 near the Hoxb13-interacting domain. Administration of Paro-Neo combination in adult mice and in pigs after cardiac ischemia/reperfusion injury induced cardiomyocyte proliferation, improved left ventricular systolic function and decreased scar formation. Collectively, we identified FDA-approved drugs with therapeutic potential for induction of heart regeneration in mammals.

Nick Orr and colleagues report a genome-wide association study for male breast cancer. They identify a new susceptibility locus at RAD51B and examine association evidence for known female breast cancer loci in these cohorts. We conducted a genome-wide association study of male breast cancer comprising 823 cases and 2,795 controls of European ancestry, with validation in independent sample sets totaling 438 cases and 474 controls. A SNP in RAD51B at 14q24.1 was significantly associated with male breast cancer risk ( P = 3.02 × 10 −13 ; odds ratio (OR) = 1.57). We also refine association at 16q12.1 to a SNP within TOX3 ( P = 3.87 × 10 −15 ; OR = 1.50).