Journal: Nature Communications

Article Title: Reliable identification of protein-protein interactions by crosslinking mass spectrometry

doi: 10.1038/s41467-021-23666-z

Figure Lengend Snippet: a For matches within the same protein sequence (with a non-directional crosslinker ), a crosslink from A1 to A2 is indistinguishable from A2 to A1 (theoretically possible search space shown as purple triangles). In contrast, heteromeric matches are not symmetrical, and therefore occupy a larger random space (green squares). b Fraction of decoys in random picks of 100 self and 100 heteromeric CSMs from the search output before any FDR filtering (random picks, n = 20, i.e. ten per crosslinker dataset). Error bars show standard deviation from the mean. Source data are provided as a Source Data file. c Schematic showing error increase when merging crosslinked residue pairs to PPIs. Proteins are indicated as circles; blue and red lines represent true and false linkages, respectively. d Experimental workflow. E. coli lysate was separated and crosslinked in individual high molecular weight fractions, pooled again to simulate a complex mixture and analyzed by mass spectrometry. Quantitative proteomics of uncrosslinked fractions provided protein coelution data.

Article Snippet: A single clone of Escherichia coli K12 strain (BW25113 purchased from DSMZ, Germany; https://www.dsmz.de/ ) grown on Agar plates was selected for inoculation of lysogeny broth (LB)-media.

Techniques: Sequencing, Standard Deviation, Residue, High Molecular Weight, Mass Spectrometry, Quantitative Proteomics

Journal: Nature Communications

Article Title: Reliable identification of protein-protein interactions by crosslinking mass spectrometry

doi: 10.1038/s41467-021-23666-z

Figure Lengend Snippet: a , b False identifications as a function of merging heteromeric CSMs passing a naïve decoy-based CSM-FDR of 5% for a DSSO and b BS3, respectively. When merging crosslink data from CSMs to PPIs, the number of identifications decreases and the fraction of false identifications increases. CSMs rarely corroborated each other in false PPIs while plausible PPIs were supported by multiple CSMs. Heteromeric CSMs are indicted by circles connected with a straight line; self-CSMs by a circle with curved line. c Proportion of proteins involved in false PPIs with self-links or with only heteromeric crosslinks, of non-crosslinkable E. coli proteins and the entrapment database. d Proteins found exclusively in heteromeric PPIs had a lower abundance than all identified proteins and thus a low chance to be detected. Boxplots depict the median (middle line), upper and lower quartiles (boxes), 1.5 times of the interquartile range (whiskers) as well as outliers (single points). e PPI error resulting from a 5% FDR threshold of FDR approaches performed in other studies (Supplementary Table ). Each bar is from a separate FDR calculation. Diamond denotes the method leading to the PPI error closest to 5%. f Fraction of protein pairs with similar elution profiles (correlation coefficient > 0.5) among the PPIs passing a given FDR threshold, applying different published FDR methods (Supplementary Table ). Averages of BS3 and DSSO data are shown (Also presented sepearately in Supplementary Fig. ). Source data for panels a , b and d are provided as a Source Data file.

Article Snippet: A single clone of Escherichia coli K12 strain (BW25113 purchased from DSMZ, Germany; https://www.dsmz.de/ ) grown on Agar plates was selected for inoculation of lysogeny broth (LB)-media.

Techniques:

Journal: Nature Communications

Article Title: Reliable identification of protein-protein interactions by crosslinking mass spectrometry

doi: 10.1038/s41467-021-23666-z

Figure Lengend Snippet: a Crosslinking MS-derived PPI network of soluble high molecular weight E. coli proteome. Selected proteins (circles) and protein complexes are highlighted. The proteins AceA and TnaA were removed for clarity. b Characterization of the obtained PPI network in comparison to random PPIs from proteins identified in coelution data. Shown are the overlaps with STRING database and coelution data (correlation coefficient > 0.5).

Article Snippet: A single clone of Escherichia coli K12 strain (BW25113 purchased from DSMZ, Germany; https://www.dsmz.de/ ) grown on Agar plates was selected for inoculation of lysogeny broth (LB)-media.

Techniques: Derivative Assay, High Molecular Weight, Comparison

Journal: bioRxiv

Article Title: Molecular and cellular insight into Escherichia coli SslE and its role during biofilm maturation

doi: 10.1101/2021.02.07.430137

Figure Lengend Snippet: a, SAXS bead model of rSslE at pH 7.4. b, Fit of the SAXS bead model (teal line) and negative-stain TEM map (black line) of rSslE to the rSslE SAXS data (black open circles) with χ of 1.2 and 113.7, respectively. c, Representative negative-stain TEM micrograph of rSslE (pH 7.4) at 50,000× nominal magnification with representative 2D classifications. Scale bar represents 20 nm. d, Overlay of the 22 Å resolution TEM map (grey) and SAXS bead model (teal) with the three defined regions in SslE highlighted. e, Docking of an SslE M60 domain homology model (orange) into the TEM map. f, TEM map of rSslE colored based on domain organization. The NT3-M60 interdomain channel is highlighted with an arrow. g, TROSY 1 H 15 N-HSQC spectrum of rSslE (black) overlaid with 1 H 15 N-HSQC spectra of the SslE NT1 (light green) and NT2 (olive green) domains.

Article Snippet: Likewise, SslE NT1 (residues 67-211), NT2 (residues 230-425), NT1-NT2 (residues 67-425), NT3-M60 (residues 426-1497) and RgpB-CTD were transformed into E. coli SHuffle cells (SslE; New England Biolabs) or BL21 (DE3) (RgpB-CTD; New England Biolabs), grown at 37°C in LB media (rSslE, RgpB-CTD: 100 μg/ml ampicillin; NT1, NT2, NT1-NT2, NT3-M60: 100 μg/ml kanamycin) and expression induced with 0.5 mM isopropyl-d-1-thiogalactopyranoside (IPTG) at an OD 600nm of 0.6, followed by growth overnight at 18°C.

Techniques: Staining

Journal: bioRxiv

Article Title: Molecular and cellular insight into Escherichia coli SslE and its role during biofilm maturation

doi: 10.1101/2021.02.07.430137

Figure Lengend Snippet: a, TEM analysis of negatively stained SslE fibres. Two major species are observed. b, Smaller filaments have an average width of ~4.5 nm and are coated with globular structures of ~4.5 nm in diameter. Scale bar, 20 nm. c, Schematic describing the overall dimensions of the smaller SslE fibre species. d, Larger species of SslE fibres appear as ~20 to 40 nm wide structures with variable lengths. Scale bar, 20 nm. e, f, DARR ssNMR spectrum of SslE fibres focussed on the carbonyl ( e, left), aliphatic ( e, right) and aromatic ( f ) regions. Likely amino acid type assignments are highlighted with red dashed lines and annotated in teal. g, S AXS bead model for the repeating unit of the SslE fibre core with overall dimensions given. h, Three units of the SslE fibre core translated along the fibre axis (left). One unit is zoomed in and overlaid with a bead model created of the rSslE negative-stain TEM map with the NT1 domain removed (NT2-3-M60; right). i, Proposed model for SslE fibres. The core of the fibre contains the NT2, NT3 and M60 domains and is decorated with flexible NT1. The core fibre is formed via interactions between the NT2 and NT3 domains of adjacent SslE molecules.

Article Snippet: Likewise, SslE NT1 (residues 67-211), NT2 (residues 230-425), NT1-NT2 (residues 67-425), NT3-M60 (residues 426-1497) and RgpB-CTD were transformed into E. coli SHuffle cells (SslE; New England Biolabs) or BL21 (DE3) (RgpB-CTD; New England Biolabs), grown at 37°C in LB media (rSslE, RgpB-CTD: 100 μg/ml ampicillin; NT1, NT2, NT1-NT2, NT3-M60: 100 μg/ml kanamycin) and expression induced with 0.5 mM isopropyl-d-1-thiogalactopyranoside (IPTG) at an OD 600nm of 0.6, followed by growth overnight at 18°C.

Techniques: Staining

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: Comprehensive molecular analyses of cuproptosis-related genes with regard to prognosis, immune landscape, and response to immune checkpoint blockers in lung adenocarcinoma

doi: 10.1007/s00432-024-05774-7

Figure Lengend Snippet: Multiple-omics landscape of cuproptosis-related genes in lung adenocarcinoma (LUAD). A Signaling and mechanism of cuproptosis. B Metascape enrichment analysis of the 13 cuproptosis-related genes. C Mutation frequency of 13 cuproptosis-related genes in 497 patients with LUAD from the TCGA-LUAD cohort. Each column represents an individual patient with LUAD. The upper bar plot represents TMB. The right-hand bar indicates the proportion of each variate type. D The CNV frequency of cuproptosis-related genes in the TCGA-LUAD cohort. The height of the column indicates the alteration frequency. Blue dots represent deletion frequency; red dots represent amplification frequency. E Kaplan–Meier curves of patients with LUAD from different risk groups divided by the expression profile of dihydrolipoamide S-acetyltransferase (DLAT), dihydrolipoamide branched chain transacylase E2 (DBT), and LIPT1. F Expression levels of 13 cuproptosis-related genes between LUAD tissues and normal tissues in the TCGA-LUAD cohort. Tumor, green; normal, red. (G) Correlation analysis of the 13 cuproptosis-related genes in LUAD

Article Snippet: Dihydrolipoamide branched chain transacylase E2 (DBT) polyclonal antibody (12,451-1-AP; Proteintech) and dihydrolipoamide S-acetyltransferase (DLAT) polyclonal antibody (13,426-1-AP; Proteintech) were used as the primary antibodies.

Techniques: Mutagenesis, Amplification, Expressing

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: Comprehensive molecular analyses of cuproptosis-related genes with regard to prognosis, immune landscape, and response to immune checkpoint blockers in lung adenocarcinoma

doi: 10.1007/s00432-024-05774-7

Figure Lengend Snippet: Immunohistochemistry (IHC) for DBT and DLAT was conducted on 165 normal lung and LUAD samples. A Representative IHC images showing the high expression levels of DBT and DLAT in LUAD and their low expression levels in normal lung tissues. B IHC scores of DBT and DLAT were significantly decreased in normal lung tissues compared to LUAD tissues. C IHC scores of DBT and DLAT were significantly decreased in LUAD tissues at early stages compared to LUAD tissues at advanced stages. D Kaplan–Meier survival analysis and log‐rank tests indicate that high levels of DLAT and DBT expression were associated with worse outcomes in LUAD ( P < 0.001)

Article Snippet: Dihydrolipoamide branched chain transacylase E2 (DBT) polyclonal antibody (12,451-1-AP; Proteintech) and dihydrolipoamide S-acetyltransferase (DLAT) polyclonal antibody (13,426-1-AP; Proteintech) were used as the primary antibodies.

Techniques: Immunohistochemistry, Expressing

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: Comprehensive molecular analyses of cuproptosis-related genes with regard to prognosis, immune landscape, and response to immune checkpoint blockers in lung adenocarcinoma

doi: 10.1007/s00432-024-05774-7

Figure Lengend Snippet: Cox regression analysis of overall survival in LUAD patients

Article Snippet: Dihydrolipoamide branched chain transacylase E2 (DBT) polyclonal antibody (12,451-1-AP; Proteintech) and dihydrolipoamide S-acetyltransferase (DLAT) polyclonal antibody (13,426-1-AP; Proteintech) were used as the primary antibodies.

Techniques: Expressing