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

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

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

Mapping onto a related organism allows rapid MS-based proteomic identification of fructose 5-dehydrogenase from the unsequenced organism <i>Gluconobacter industrius.</i>

<p>MS-based proteomic identification of fructose 5-dehydrogenase. (A) 1-D SDS PAGE of 140 ug of fructose 5-dehydrogenase (F5D) (Sigma Aldrich: #F4892) yields 7 distinct protein bands after Coomassie staining. A band at ∼75 kDa (boxed) was excised for MS analysis. (B) Zoomed-in mass spectra illustrating the triply charged precursor ion at 710.67 m/z observed in MS scans subsequently identified through MS/MS as the tryptic peptide DALGIPHPEVTYDVGEYVR from fructose dehydrogenase large subunit protein in <i>G. frateurri</i>. (C) Complete amino acid sequence of the <i>G. frateurri</i> fructose dehydrogenase large subunit protein identified in protein database searches. High confidence (>90%) MS-identified peptides are colored green. Additional peptide matches are colored red.</p

Active site residues are well conserved across MPUS homologs.

<p>Sequence alignment was generated using MAFFT alignment program. Residues that are conserved in more than 95% species are highlighted in black, residues that are conserved in more than 80% species are highlighted in dark grey and residues that are conserved in more than 50% species are highlighted in grey. Residues that have been experimentally shown to be functionally important are denoted with asterisks on the top and residues proposed to be important for the function are denoted with asterisks at the bottom.</p

“Filtering” by MW allows rapid MS-based proteomic identification of mannosylphosphorylundecaprenol synthase.

<p>(A) 1-D SDS PAGE of a partially purified <i>M. luteus</i> protein fraction exhibiting MPU synthase activity reveals multiple protein bands ranging from 26–35 kDa. The indicated band (boxed) was excised for MS analysis. (B) MS/MS fragmentation analysis of a doubly charged peptide observed at 598.81 m/z identifies the tryptic peptide DGLGGAYIAGFR from a glycosyl transferase protein in <i>M. luteus</i>. (C) Complete amino acid sequence of a DPM1-like glycosyl transferase identified in queries against a custom <i>M. luteus</i> protein database. High confidence (>90%) MS-identified peptides are colored green. Additional peptide matches are colored red.</p

“Filtering” by MW allows rapid MS-based proteomic identification of maltose epimerase.

<p>(A) 1-D SDS PAGE of maltose epimerase (ME) (Sigma Aldrich #M0902) resolves as a band at ∼40 kDa (boxed), the expected molecular weight for maltose epimerase. (B) Tryptic digest and MS analysis of the ∼40 kDa protein band yields a prominent doubly charged precursor ion peak at 727.84 m/z. (C) MS/MS fragmentation analysis of the 727.84 m/z peptide identified as the tryptic peptide DTPIATIGDTTGHR from the <i>L. brevis</i> protein aldose 1-epimerase. b-ion (green) and y-ion (red) series are shown. (D) Complete amino acid sequence of <i>L. brevis</i> protein aldose 1-epimerase with high confidence (>95%) MS-identified peptides colored green. Additional peptide matches are colored red.</p

Structural superimposition between a model structure of maltose epimerase and structures of its homologous enzymes shows conservation of active site residues.

<p>Red: Galactose 1-epimerase from <i>Lactobacillus acidophilus</i> pdb:3imh; magenta: human galactose mutarotase pdb:1snz; yellow: galactose mutarotase/UDP-galactose 4-epimerase from <i>Saccharomyces cerevisiae</i> pdb:1z45; blue: galactose mutarotase from <i>Lactococcus lactis</i> pdb:1l7j; cayan: model structure of maltose epimerase. The numbering is according to galactose mutarotase from <i>Lactococcus lactis</i>.</p

Identification of MPUS re-annotates approximately 430 proteins.

<p>Each node represents one of the protein sequences identified as homologous to MPUS from <i>Micrococcus luteus</i>; edges between nodes are drawn only if the similarity between a pair of sequences is better than an E-value threshold cutoff of 1e⁻⁵⁰. The network is visualized using the organic layout in Cytoscape. Nodes are colored according to assigned function in UniProtKB database; megenta: DPM1, blue: apolipoprotein N-acyltransferase Lnt, red: Ppm1, cyan: MPUS. Larger nodes are proteins that are used to generate sequence alignment. Large rectangular nodes with green borders are proteins with evidence of existence at the protein level.</p