Comparative analyses of proteins from Haemophilus influenzae biofilm and planktonic populations using metabolic labeling and mass spectrometry

BACKGROUND: Non-typeable H. influenzae (NTHi) is a nasopharyngeal commensal that can become an opportunistic pathogen causing infections such as otitis media, pneumonia, and bronchitis. NTHi is known to form biofilms. Resistance of bacterial biofilms to clearance by host defense mechanisms and antibiotic treatments is well-established. In the current study, we used stable isotope labeling by amino acids in cell culture (SILAC) to compare the proteomic profiles of NTHi biofilm and planktonic organisms. Duplicate continuous-flow growth chambers containing defined media with either “light” (L) isoleucine or “heavy” (H) (13)C(6)-labeled isoleucine were used to grow planktonic (L) and biofilm (H) samples, respectively. Bacteria were removed from the chambers, mixed based on weight, and protein extracts were generated. Liquid chromatography-mass spectrometry (LC-MS) was performed on the tryptic peptides and 814 unique proteins were identified with 99% confidence. RESULTS: Comparisons of the NTHi biofilm to planktonic samples demonstrated that 127 proteins showed differential expression with p-values ≤0.05. Pathway analysis demonstrated that proteins involved in energy metabolism, protein synthesis, and purine, pyrimidine, nucleoside, and nucleotide processes showed a general trend of downregulation in the biofilm compared to planktonic organisms. Conversely, proteins involved in transcription, DNA metabolism, and fatty acid and phospholipid metabolism showed a general trend of upregulation under biofilm conditions. Selected reaction monitoring (SRM)-MS was used to validate a subset of these proteins; among these were aerobic respiration control protein ArcA, NAD nucleotidase and heme-binding protein A. CONCLUSIONS: The present proteomic study indicates that the NTHi biofilm exists in a semi-dormant state with decreased energy metabolism and protein synthesis yet is still capable of managing oxidative stress and in acquiring necessary cofactors important for biofilm survival. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12866-014-0329-9) contains supplementary material, which is available to authorized users

Phosphoprotein secretome of tumor cells as a source of candidates for breast cancer biomarkers in plasma.

Breast cancer is a heterogeneous disease whose molecular diversity is not well reflected in clinical and pathological markers used for prognosis and treatment selection. As tumor cells secrete proteins into the extracellular environment, some of these proteins reach circulation and could become suitable biomarkers for improving diagnosis or monitoring response to treatment. As many signaling pathways and interaction networks are altered in cancerous tissues by protein phosphorylation, changes in the secretory phosphoproteome of cancer tissues could reflect both disease progression and subtype. To test this hypothesis, we compared the phosphopeptide-enriched fractions obtained from proteins secreted into conditioned media (CM) derived from five luminal and five basal type breast cancer cell lines using label-free quantitative mass spectrometry. Altogether over 5000 phosphosites derived from 1756 phosphoproteins were identified, several of which have the potential to qualify as phosphopeptide plasma biomarker candidates for the more aggressive basal and also the luminal-type breast cancers. The analysis of phosphopeptides from breast cancer patient plasma and controls allowed us to construct a discovery list of phosphosites under rigorous collection conditions, and second to qualify discovery candidates generated from the CM studies. Indeed, a set of basal-specific phosphorylation CM site candidates derived from IBP3, CD44, OPN, FSTL3, LAMB1, and STC2, and luminal-specific candidates derived from CYTC and IBP5 were selected and, based on their presence in plasma, quantified across all cell line CM samples using Skyline MS1 intensity data. Together, this approach allowed us to assemble a set of novel cancer subtype specific phosphopeptide candidates for subsequent biomarker verification and clinical validation

Structural, Kinetic and Proteomic Characterization of Acetyl Phosphate-Dependent Bacterial Protein Acetylation

<div><p>The emerging view of N^ε-lysine acetylation in eukaryotes is of a relatively abundant post-translational modification (PTM) that has a major impact on the function, structure, stability and/or location of thousands of proteins involved in diverse cellular processes. This PTM is typically considered to arise by the donation of the acetyl group from acetyl-coenzyme A (acCoA) to the ε-amino group of a lysine residue that is reversibly catalyzed by lysine acetyltransferases and deacetylases. Here, we provide genetic, mass spectrometric, biochemical and structural evidence that N^ε-lysine acetylation is an equally abundant and important PTM in bacteria. Applying a recently developed, label-free and global mass spectrometric approach to an isogenic set of mutants, we detected acetylation of thousands of lysine residues on hundreds of <i>Escherichia coli</i> proteins that participate in diverse and often essential cellular processes, including translation, transcription and central metabolism. Many of these acetylations were regulated in an acetyl phosphate (acP)-dependent manner, providing compelling evidence for a recently reported mechanism of bacterial N^ε-lysine acetylation. These mass spectrometric data, coupled with observations made by crystallography, biochemistry, and additional mass spectrometry showed that this acP-dependent acetylation is both non-enzymatic and specific, with specificity determined by the accessibility, reactivity and three-dimensional microenvironment of the target lysine. Crystallographic evidence shows acP can bind to proteins in active sites and cofactor binding sites, but also potentially anywhere molecules with a phosphate moiety could bind. Finally, we provide evidence that acP-dependent acetylation can impact the function of critical enzymes, including glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, and RNA polymerase.</p></div

Multiplexed, Scheduled, High-Resolution Parallel Reaction Monitoring on a Full Scan QqTOF Instrument with Integrated Data-Dependent and Targeted Mass Spectrometric Workflows

Recent advances in commercial mass spectrometers with higher resolving power and faster scanning capabilities have expanded their functionality beyond traditional data-dependent acquisition (DDA) to targeted proteomics with higher precision and multiplexing. Using an orthogonal quadrupole time-of flight (QqTOF) LC-MS system, we investigated the feasibility of implementing large-scale targeted quantitative assays using scheduled, high resolution multiple reaction monitoring (sMRM-HR), also referred to as parallel reaction monitoring (sPRM). We assessed the selectivity and reproducibility of PRM, also referred to as parallel reaction monitoring, by measuring standard peptide concentration curves and system suitability assays. By evaluating up to 500 peptides in a single assay, the robustness and accuracy of PRM assays were compared to traditional SRM workflows on triple quadrupole instruments. The high resolution and high mass accuracy of the full scan MS/MS spectra resulted in sufficient selectivity to monitor 6–10 MS/MS fragment ions per target precursor, providing flexibility in postacquisition assay refinement and optimization. The general applicability of the sPRM workflow was assessed in complex biological samples by first targeting 532 peptide precursor ions in a yeast lysate, and then 466 peptide precursors from a previously generated candidate list of differentially expressed proteins in whole cell lysates from <i>E. coli</i>. Lastly, we found that sPRM assays could be rapidly and efficiently developed in Skyline from DDA libraries when acquired on the same QqTOF platform, greatly facilitating their successful implementation. These results establish a robust sPRM workflow on a QqTOF platform to rapidly transition from discovery analysis to highly multiplexed, targeted peptide quantitation

Crystal structure of glyceraldehyde-3-phosphate dehydrogenase (GapA) from <i>E. coli</i> in native (PDB ID: 1S7C) and acP-modified (PDB ID: 4MVJ) forms.

<p><b>A</b>) Surface representation of GapA with the locations highlighted in red of up-regulated acetylated lysine residues in the <i>ackA</i> mutant as determined by mass spectrometry. Front and back views of the protein are shown and NAD⁺ (shown with sticks) is bound at the active site pocket. <b>B</b>) Electron density map surrounding acetylated K46 in the GapA crystal structure. The F_o-F_c omit map in blue mesh is contoured at the 3 sigma level. Residues are shown as sticks and water is represented as a sphere. <b>C</b>) Electron density map surrounding acetylated K249 in the GapA structure. The F_o-F_c omit map in blue mesh is contoured at the 2 sigma level. <b>D</b>) Electron density map surrounding acetylated K257 in the GapA structure. The F_o-F_c omit map in blue mesh is contoured at the 2 sigma level. Oxygens, nitrogens, and carbons are shown in red, blue and light blue, respectively. <b>E, F</b>) Overlay of the K249 and K257 residue of GapA (PDB ID: 4MVJ) in its acetylated and non-acetylated form, respectively. Phosphate is present in the non-acetylated form and binds in the same location as the acetyl group of the acetylated residue. The protein with the non-acetylated lysine and phosphate bound is shown in orange, the protein with the acetylated lysine is in teal, oxygen atoms are in red and nitrogens are in blue.</p

Mass spectrometric workflow.

<p><b>A</b>) <i>E. coli</i> strains MG1655 (WT), AJW5052 (<i>ackA</i>), AJW2785 (<i>pta ackA</i>), AJW5037 (<i>cobB</i>), and AJW5164 (<i>yfiQ</i>) were aerated at 37°C in TB7 (3 independent biological replicates) or TB7 supplemented with 0.4% glucose (4 independent biological replicates) and harvested when the OD₆₁₀ reached 1.0. For mass spectrometric analysis, the harvested cells were lysed and the protein lysates were proteolytically digested with trypsin, followed by affinity enrichment for acetyllysine (Ac-Lys)-containing peptides using a polyclonal anti-acetyllysine antibody. Enriched Ac-Lys peptides for each strain/condition were analyzed by high-resolution label-free LC-MS/MS (3 technical replicates each) for acetyl site identification, and these Ac-Lys sites were subsequently subjected to MS1- and MS2-based quantification methods. <b>B</b>) Skyline MS1 Filtering for acetylated peptide NLDAG<b>Kac</b>AGVEVDDR (^AcK284) obtained from dihydrolipoyl dehydrogenase (LpdA) to determine quantitative differences between the <i>ackA</i> mutant (strain AJW5052) and its WT parent (strain MG1655). MS1 ion chromatograms and corresponding peak areas demonstrating one of four biological replicates is shown for WT and <i>ackA</i> mutant (3 technical MS replicates acquired); precursor ions were extracted for M at <i>m/z</i> 750.87⁺⁺, M+1 at <i>m/z</i> 751.37⁺⁺, M+2 at <i>m/z</i> 751.87⁺⁺. <b>C</b>) Independent confirmation and validation of potential candidates and sites for acetyllysine regulation using SWATH MS2 Filtering (SWATH MS2): exemplified for NLDAG<b>Kac</b>AGVEVDDR (^AcK284): 1 biological replicate (3 technical replicates acquired) shown with extracted ion chromatograms and corresponding peak areas for fragment ions y₁₂ at <i>m/z</i> 1273.60⁺, y₁₁ at <i>m/z</i> 1158.57⁺, y₁₀ at <i>m/z</i> 1087.54⁺, y₈ at <i>m/z</i> 860.41⁺, y₇ at <i>m/z</i> 789.37⁺, and y₅ at <i>m/z</i> 633.28⁺.</p

<i>In vitro</i> acetylation of LpdA using acP as the acetyl group donor is sensitive to acP concentration and time of incubation.

<p>Coomassie stain of SDS-polyacrylamide gel and anti-acetyllysine Western immunoblot analysis of acP (5, 10, 15, 20 mM) incubated with 1.25 µM LpdA for various lengths of time (5, 10, 15, and 30 min, 1, 2, 3, 4, 5, and 7 hours) at 37°C. Acetylation signals were quantified using AlphaView and normalized to the signal in the absence of acP.</p

Distribution of acetylation sites in <i>E. coli</i> proteins.

<p>Mass spectrometric analysis of the strains grown in the conditions shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094816#pone-0094816-g002" target="_blank">Figure 2</a> confidently identified 2730 unique lysine acetylation sites across 806 unique acetylated <i>E. coli</i> proteins. <b>A</b>) The frequency of individual proteins relative to the number of acetyllysines per protein. <b>B</b>) The estimated protein copy number per <i>E. coli</i> cell <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094816#pone.0094816-Ishihama1" target="_blank">[39]</a> relative to the number of acetylation sites per protein. The exponential regression trendline is indicated (R²  =  0.13), the y-axis is presented as a logarithmic scale.</p

Crystal structure of <i>E. coli</i> triose phosphate isomerase (TpiA) determined in the presence and absence of acP.

<p><b>A</b>) Cartoon and stick representation of acP bound in the active site of TpiA. The acP ligand is surrounded by the F_o-F_c omit map that was contoured at the 3 sigma level. Side-chain and main-chain interactions with acP are shown as gray dashed lines. Oxygens are shown in red, nitrogens in blue, phosphate in orange, carbons of acP in yellow, and carbons of the protein in light blue. <b>B</b>) Overlay of the crystal structure of the <i>E. coli</i> acP-bound TpiA protein (PDB ID: 4MVA, cyan) with the crystal structure of TpiA from <i>Saccharomyces cerevisiae</i> (PDB ID: 1NEY, gray). The <i>S. cerevisiae</i> structure has the substrate 1,3-dihydroxyacetone phosphate (13P) bound in its active site. K11 is shown in each structure. Nitrogen atoms are blue, oxygens are red, the carbon atoms of acP are cyan and carbon atoms of 13P are gray. An arrow indicates the movement for loop closure between open (cyan) and closed (gray) forms of the protein. <b>C</b>) Surface representation of TpiA (PDB ID: 4MVA) with the locations of up-regulated acetylated lysine residues in the <i>ackA</i> mutant highlighted in red. AcP is bound in the active site.</p