Integrated top-down and bottom-up mass spectrometry characterization of Escherichia coli ribosomal protein heterogeneity: identification of protein isoforms and post-translational modifications

The bacterial genome exhibits notable plasticity but is relatively static when compared to the proteome. Protein expression can vary significantly depending on environmental factors, growth stage or stochastic processes within cells. This highly variable character, coupled with the large dynamic range of protein expression levels and the complexity achieved through processes such as post-translational modification (PTM), necessitate accurate, sensitive and high-throughput methods of analysis. The primary aim of this research was to develop an integrated experimental and analysis workflow that combines the analytical power of top-down and bottom-up mass spectrometry towards protein isoform and PTM characterization. We apply this approach to a comprehensive characterization of Escherichia coli ribosomal protein isoform heterogeneity. Our findings uncovered a significant level of heterogeneity in the post-translational modification of a number of ribosomal proteins, revealing a possible mechanism for the regulation of ribosomal protein function both within and beyond the ribosome

Fragmentation Characteristics of Collision-Induced Dissociation in MALDI TOF/TOF Mass Spectrometry

The identification of proteins by tandem mass spectrometry relies on knowledge of the products produced by collision-induced dissociation of peptide ions. Most previous work has focused on fragmentation statistics for ion trap systems. We analyzed fragmentation in MALDI TOF/TOF mass spectrometry, collecting statistics using a curated set of 2459 MS/MS spectra, and applying bootstrap resampling to assess confidence intervals. We calculated the frequency of 18 product ion types, the correlation between both mass and intensity with ion type, the dependence of amide bond breakage on the residues surrounding the cleavage site, and the dependence of product ion detection on residues not adjacent to the cleavage site. The most frequently observed were internal ions, followed by y ions. A strong correlation between ion type and the mass and intensity of its peak was observed, with b and y ions producing the most intense and highest-mass peaks. The amino acids P, W, D and R had a strong effect on amide bond cleavage when situated next to the breakage site, whereas residues including I, K and H had a strong effect on product ion observation when located in the peptide but not adjacent to the cleavage site, a novel observation

CYLD mutation characterizes a subset of HPV-positive head and neck squamous cell carcinomas with distinctive genomics and frequent cylindroma-like histologic features

Mutations in the tumor suppressor CYLD, known to be causative of cylindromas, were recently described in a subset of high-risk (hr) HPV-positive head and neck squamous cell carcinomas (HNSCC). Pathologic and genetic characterization of these CYLD-mutant carcinomas, however, remains limited. Here, we investigated whether CYLD mutations characterize a histopathologically and genomically distinct subset of hrHPV-positive HNSCC. Comprehensive genomic profiling via hybrid capture-based DNA sequencing was performed on 703 consecutive head and neck carcinomas with hrHPV sequences, identifying 148 unique cases (21%) harboring CYLD mutations. Clinical data, pathology reports, and histopathology were reviewed. CYLD mutations included homozygous deletions (n = 61/148; 41%), truncations (n = 52; 35%), missense (n = 26; 18%) and splice-site (n = 9; 6%) mutations, and in-frame deletion (n = 1; 1%). Among hrHPV-positive HNSCC, the CYLD-mutant cohort showed substantially lower tumor mutational burden than CYLD-wildtype cases (n = 555) (median 2.6 vs. 4.4 mut/Mb, p \u3c 0.00001) and less frequent alterations in PIK3CA (11% vs. 34%, p \u3c 0.0001), KMT2D (1% vs. 16%, p \u3c 0.0001), and FBXW7 (3% vs. 11%, p = 0.0018). Male predominance (94% vs. 87%), median age (58 vs. 60 years), and detection of HPV16 (95% vs. 89%) were similar. On available histopathology, 70% of CYLD-mutant HNSCC (98/141 cases) contained hyalinized material, consistent with basement membrane inclusions, within crowded aggregates of tumor cells. Only 7% of CYLD-wildtype cases demonstrated this distinctive pattern (p \u3c 0.0001). Histopathologic patterns of CYLD-mutant HNSCC lacking basement membrane inclusions included nonkeratinizing (n = 22, 16%), predominantly nonkeratinizing (nonkeratinizing SCC with focal maturation; n = 10, 7%), and keratinizing (n = 11, 8%) patterns. The latter two groups showed significantly higher frequency of PTEN alterations compared with other CYLD-mutant cases (38% [8/21] vs. 7% [8/120], p = 0.0004). Within our cohort of hrHPV-positive HNSCCs, CYLD mutations were frequent (21%) and demonstrated distinctive clinical, histopathologic, and genomic features that may inform future study of prognosis and treatment

Rethinking place-making: aligning placeness factors with perceived urban design qualities (PUDQs) to improve the built environment in historical district

Understanding the concept of place is critically important for urban design and place-making practice, and this research attempted to investigate the pathways by which perceived urban design qualities (PUDQs) influence placeness factors in the Chinese context. Twelve hypotheses were developed and combined in a structural equation model for validation. The Tanhualin historical district in Wuhan, China was selected for the analysis. As a result, place attachment was verified as a critical bridge factor that mediated the influence of PUDQs on place satisfaction. Among the five selected PUDQs, walkability and space quality were revealed as the most influential factors associated with place attachment and place satisfaction. Accessibility was actually indirectly beneficial to place-making via the mediation of walkability. Corresponding implications and strategies were discussed to maintain the sense of place for historic districts

High-Accuracy Peptide Mass Fingerprinting Using Peak Intensity Data with Machine Learning

For MALDI-TOF mass spectrometry, we show that the intensity of a peptide–ion peak is directly correlated with its sequence, with the residues M, H, P, R, and L having the most substantial effect on ionization. We developed a machine learning approach that exploits this relationship to significantly improve peptide mass fingerprint (PMF) accuracy based on training data sets from both true-positive and false-positive PMF searches. The model’s cross-validated accuracy in distinguishing real versus false-positive database search results is 91%, rivaling the accuracy of MS/MS-based protein identification

Global discovery of high-NaCl-induced changes of protein phosphorylation

U ovoj su radnji izvršene analize novih pristupa u prenaponskoj zaštiti srednjenaponskih elektroenergetskih mreža. Analizirani su nastanci i djelovanja prenapona, te je dan pregled postojećih metoda prenaponske zaštite u srednjenaponskim distribucijskim mrežama. Kao novi pristupi u prenaponskoj zaštiti, analizirani su dugi preskočni odvodnik prenapona s antenskim modulom, te najnovija saznanja o uzrocima i zaštitnim mjerama na polju prenaponske zaštite uslijed djelovanja induciranih napona. Predstavljen je princip djelovanja LFA-AM (Long Flashover Arrester with Antenna-module) dugog preskočnog odvodnika prenapona s antenskim modulom, te izvršena analiza laboratorijskih testova. Prenaponska zaštita od pojave previsokih induciranih napona je obrađena od analize metoda proračuna induciranih napona, preko utjecaja zaštitnog vodiča na vodu i susjednih objekata na učestalost i veličinu induciranih napona, do prijedloga zaštite i usporedbe s vrijednostima dobivenima u ispitivanju na terenu. Dan je poseban osvrt na stanje prenaponske zaštite u srednjenaponskom elektroenergetskom sustavu EPHZHB.In this master thesis, a new approaches to overvoltage protection of middlevoltage power networks are carried out. Overvoltage generation and its effects are analysed and also an overview of existing methods of overvoltage protection in middle-voltage power networks is presented. As new approaches to overvoltage protection, Long Flashover Arrester with Antenna-module (LFA-AM) and the newest cognitions about causes and protection measures on the area of protection against induced voltages are analysed. Operating principle and design of Long Flashover Arrester with Antenna-module(LFA-AM) are presented and appropriate laboratory tests are performed. Overvoltage protection against induced voltages is done through: analysis of calculation methods of induced voltages, analysis of ground wire and nearby objects effect on occurrence frequency and values of induced voltages, suggestions of overvoltage protection measures and comparison of calculated results to field data. A special overview of overvoltage protection of middle-voltage power networks in EPHZHB power electricity company is given

The Development of Ciprofloxacin Resistance in Pseudomonas aeruginosa Involves Multiple Response Stages and Multiple Proteins ▿ † ‡

Microbes have developed resistance to nearly every antibiotic, yet the steps leading to drug resistance remain unclear. Here we report a multistage process by which Pseudomonas aeruginosa acquires drug resistance following exposure to ciprofloxacin at levels ranging from 0.5× to 8× the initial MIC. In stage I, susceptible cells are killed en masse by the exposure. In stage II, a small, slow to nongrowing population survives antibiotic exposure that does not exhibit significantly increased resistance according to the MIC measure. In stage III, exhibited at 0.5× to 4× the MIC, a growing population emerges to reconstitute the population, and these cells display heritable increases in drug resistance of up to 50 times the original level. We studied the stage III cells by proteomic methods to uncover differences in the regulatory pathways that are involved in this phenotype, revealing upregulation of phosphorylation on two proteins, succinate-semialdehyde dehydrogenase (SSADH) and methylmalonate-semialdehyde dehydrogenase (MMSADH), and also revealing upregulation of a highly conserved protein of unknown function. Transposon disruption in the encoding genes for each of these targets substantially dampened the ability of cells to develop the stage III phenotype. Considering these results in combination with computational models of resistance and genomic sequencing results, we postulate that stage III heritable resistance develops from a combination of both genomic mutations and modulation of one or more preexisting cellular pathways

Rapid identification of sequences for orphan enzymes to power accurate protein annotation.

The power of genome sequencing depends on the ability to understand what those genes and their proteins products actually do. The automated methods used to assign functions to putative proteins in newly sequenced organisms are limited by the size of our library of proteins with both known function and sequence. Unfortunately this library grows slowly, lagging well behind the rapid increase in novel protein sequences produced by modern genome sequencing methods. One potential source for rapidly expanding this functional library is the "back catalog" of enzymology--"orphan enzymes," those enzymes that have been characterized and yet lack any associated sequence. There are hundreds of orphan enzymes in the Enzyme Commission (EC) database alone. In this study, we demonstrate how this orphan enzyme "back catalog" is a fertile source for rapidly advancing the state of protein annotation. Starting from three orphan enzyme samples, we applied mass-spectrometry based analysis and computational methods (including sequence similarity networks, sequence and structural alignments, and operon context analysis) to rapidly identify the specific sequence for each orphan while avoiding the most time- and labor-intensive aspects of typical sequence identifications. We then used these three new sequences to more accurately predict the catalytic function of 385 previously uncharacterized or misannotated proteins. We expect that this kind of rapid sequence identification could be efficiently applied on a larger scale to make enzymology's "back catalog" another powerful tool to drive accurate genome annotation