Detection of low-copy proteins in proteomic studies: issues and solutions.

Detection of low-copy proteins in complex biological samples is one of the most important issues of modern proteomics. The main reason for inefficient detection of low protein concentrations is the insufficient sensitivity of mass spectrometric detectors and the high dynamic range of protein concentrations. In this study we have investigated the possibilities and limitations of a targeted mass spectrometric analysis using the reconstructed system of standard proteins UPS1 (Universal Proteomic Standard 1) as an example. The study has shown that the sensitivity of the method is affected by the concentration of target proteins of the UPS1 system, as well as by a high level of biological noise modelled by proteins of whole E. coli cell lysate. The limitations of the method have been overcome by concentrating and pre-fractionating the sample peptides in a reversed phase chromatographic system under alkaline elution conditions. Proteomic analysis of the biological sample (proteins of the human hepatocellular carcinoma cell line HepG2 encoded by genes of human chromosome 18) showed an increase in the sensitivity of the method as compared to the standard targeted mass spectrometric analysis. This culminated in registration of 94 proteins encoded by genes located on human chromosome18.

Impact of Charge State on Characterization of Large Middle-Down Sized Peptides by Tandem Mass Spectrometry.

Fragmentation trends of large peptides were characterized by five activation methods, including HCD, ETD, EThcD, 213 nm UVPD, and 193 nm UVPD. Sequence coverages and scores were assessed based on charge site, peptide sequence, and peptide size. The effect of charge state and peptide size on sequence coverage was explored for a Glu-C digest of E. coli ribosomal proteins, and linear regression analysis of the collection of peptides indicated that HCD, ETD, and EThcD have a higher dependence charge state than 193 and 213 nm UV. Four model peptides, neuromedin, glucagon, galanin, and amyloid ß, were characterized in greater detail based on charge site analysis and showed a charge state dependence on sequence coverage for collision and electron-based activation methods.

Top-down proteomic identification of plasmid and host proteins produced by pathogenic Escherichia coli using MALDI-TOF-TOF tandem mass spectrometry.

Fourteen proteins produced by three pathogenic Escherichia coli strains were identified using antibiotic induction, MALDI-TOF-TOF tandem mass spectrometry (MS/MS) and top-down proteomic analysis using software developed in-house. Host proteins as well as plasmid proteins were identified. Mature, intact protein ions were fragmented by post-source decay (PSD), and prominent fragment ions resulted from the aspartic acid effect fragmentation mechanism wherein polypeptide backbone cleavage (PBC) occurs on the C-terminal side of aspartic acid (D), glutamic acid (E) and asparagine (N) residues. These highly specific MS/MS-PSD fragment ions were compared to b- and y-type fragment ions on the C-terminal side of D-, E- and N-residues of in silico protein sequences derived from whole genome sequencing. Nine proteins were found to be post-translationally modified with either removal of an N-terminal methionine or a signal peptide. The protein sequence truncation algorithm of our software correctly identified all full and truncated protein sequences. Truncated sequences were compared to those predicted by SignalP. Nearly complete concurrence was obtained except for one protein where SignalP mis-identified the cleavage site by one residue. Two proteins had intramolecular disulfide bonds that were inferred by the absence of PBC on the C-terminal side of a D-residue located within the disulfide loop. These results demonstrate the utility of MALDI-TOF-TOF for identification of full and truncated bacterial proteins.

Tryptophan Ameliorates Barrier Integrity and Alleviates the Inflammatory Response to Enterotoxigenic Escherichia coli K88 Through the CaSR/Rac1/PLC-γ1 Signaling Pathway in Porcine Intestinal Epithelial Cells.

Background: Impaired intestinal barrier integrity plays a crucial role in the development of many diseases such as obesity, inflammatory bowel disease, and type 2 diabetes. Thus, protecting the intestinal barrier from pathological disruption is of great significance. Tryptophan can increase gut barrier integrity, enhance intestinal absorption, and decrease intestinal inflammation. However, the mechanism of tryptophan in decreasing intestinal barrier damage and inflammatory response remains largely unknown. The objective of this study was to test the hypothesis that tryptophan can enhance intestinal epithelial barrier integrity and decrease inflammatory response mediated by the calcium-sensing receptor (CaSR)/Ras-related C3 botulinum toxin substrate 1 (Rac1)/phospholipase Cγ1 (PLC-γ1) signaling pathway. Methods: IPEC-J2 cells were treated with or without enterotoxigenic Escherichia coli (ETEC) K88 in the absence or presence of tryptophan, CaSR inhibitor (NPS-2143), wild-type CaSR overexpression (pcDNA3.1-CaSR-WT), Rac1-siRNA, and PLC-γ1-siRNA. Results: The results showed that ETEC K88 decreased the protein concentration of occludin, zonula occludens-1 (ZO-1), claudin-1, CaSR, total Rac1, Rho family member 1 of porcine GTP-binding protein (GTP-rac1), phosphorylated phospholipase Cγ1 (p-PLC-γ1), and inositol triphosphate (IP3); suppressed the transepithelial electrical resistance (TEER); and enhanced the permeability of FITC-dextran compared with the control group. Compared with the control group, 0.7 mM tryptophan increased the protein concentration of CaSR, total Rac1, GTP-rac1, p-PLC-γ1, ZO-1, claudin-1, occludin, and IP3; elevated the TEER; and decreased the permeability of FITC-dextran and contents of interleukin-8 (IL-8) and TNF-α. However, 0.7 mM tryptophan+ETEC K88 reversed the effects induced by 0.7 mM tryptophan alone. Rac1-siRNA+tryptophan+ETEC K88 or PLC-γ1-siRNA+tryptophan+ETEC K88 reduced the TEER, increased the permeability of FITC-dextran, and improved the contents of IL-8 and TNF-α compared with tryptophan+ETEC K88. NPS2143+tryptophan+ETEC K88 decreased the TEER and the protein concentration of CaSR, total Rac1, GTP-rac1, p-PLC-γ1, ZO-1, claudin-1, occludin, and IP3; increased the permeability of FITC-dextran; and improved the contents of IL-8 and TNF-α compared with tryptophan+ETEC K88. pcDNA3.1-CaSR-WT+Rac1-siRNA+ETEC K88 and pcDNA3.1-CaSR-WT+PLC-γ1-siRNA+ETEC K88 decreased the TEER and enhanced the permeability in porcine intestine epithelial cells compared with pcDNA3.1-CaSR-WT+ETEC K88. Conclusion: Tryptophan can improve intestinal epithelial barrier integrity and decrease inflammatory response through the CaSR/Rac1/PLC-γ1 signaling pathway.

A comparative study of evaluating missing value imputation methods in label-free proteomics.

The presence of missing values (MVs) in label-free quantitative proteomics greatly reduces the completeness of data. Imputation has been widely utilized to handle MVs, and selection of the proper method is critical for the accuracy and reliability of imputation. Here we present a comparative study that evaluates the performance of seven popular imputation methods with a large-scale benchmark dataset and an immune cell dataset. Simulated MVs were incorporated into the complete part of each dataset with different combinations of MV rates and missing not at random (MNAR) rates. Normalized root mean square error (NRMSE) was applied to evaluate the accuracy of protein abundances and intergroup protein ratios after imputation. Detection of true positives (TPs) and false altered-protein discovery rate (FADR) between groups were also compared using the benchmark dataset. Furthermore, the accuracy of handling real MVs was assessed by comparing enriched pathways and signature genes of cell activation after imputing the immune cell dataset. We observed that the accuracy of imputation is primarily affected by the MNAR rate rather than the MV rate, and downstream analysis can be largely impacted by the selection of imputation methods. A random forest-based imputation method consistently outperformed other popular methods by achieving the lowest NRMSE, high amount of TPs with the average FADR < 5%, and the best detection of relevant pathways and signature genes, highlighting it as the most suitable method for label-free proteomics.

Bis(zinc(II)-dipicolylamine)-functionalized sub-2 µm core-shell microspheres for the analysis of N-phosphoproteome.

Protein N-phosphorylation plays a critical role in central metabolism and two/multicomponent signaling of prokaryotes. However, the current enrichment methods for O-phosphopeptides are not preferred for N-phosphopeptides due to the intrinsic lability of P-N bond under acidic conditions. Therefore, the effective N-phosphoproteome analysis remains challenging. Herein, bis(zinc(II)-dipicolylamine)-functionalized sub-2 µm core-shell silica microspheres (SiO2@DpaZn) are tailored for rapid and effective N-phosphopeptides enrichment. Due to the coordination of phosphate groups to Zn(II), N-phosphopeptides can be effectively captured under neutral conditions. Moreover, the method is successfully applied to an E.coli and HeLa N-phosphoproteome study. These results further broaden the range of methods for the discovery of N-phosphoproteins with significant biological functions.

A comprehensive spectral assay library to quantify the Escherichia coli proteome by DIA/SWATH-MS.

Data-Independent Acquisition (DIA) is a method to improve consistent identification and precise quantitation of peptides and proteins by mass spectrometry (MS). The targeted data analysis strategy in DIA relies on spectral assay libraries that are generally derived from a priori measurements of peptides for each species. Although Escherichia coli (E. coli) is among the best studied model organisms, so far there is no spectral assay library for the bacterium publicly available. Here, we generated a spectral assay library for 4,014 of the 4,389 annotated E. coli proteins using one- and two-dimensional fractionated samples, and ion mobility separation enabling deep proteome coverage. We demonstrate the utility of this high-quality library with robustness in quantitation of the E. coli proteome and with rapid-chromatography to enhance throughput by targeted DIA-MS. The spectral assay library supports the detection and quantification of 91.5% of all E. coli proteins at high-confidence with 56,182 proteotypic peptides, making it a valuable resource for the scientific community. Data and spectral libraries are available via ProteomeXchange (PXD020761, PXD020785) and SWATHAtlas (SAL00222-28).

Escherichia coli and Sf9 Contaminant Databases to Increase Efficiency of Tandem Mass Spectrometry Peptide Identification in Structural Mass Spectrometry Experiments.

Filtering of nonspecifically binding contaminant proteins from affinity purification mass spectrometry (AP-MS) data is a well-established strategy to improve statistical confidence in identified proteins. The CRAPome (contaminant repository for affinity purification) describes the contaminating background content present in many purification strategies. However, full contaminant lists for nickel-nitrilotriacetic acid (NiNTA) and glutathione S-transferase (GST) affinity matrices are lacking. Similarly, no Spodoptera frugiperda (Sf9) contaminants are available, and only the FLAG-purified contaminants are described for Escherichia coli. For MS experiments that use recombinant protein, such as structural mass spectrometry experiments (hydrogen-deuterium exchange mass spectrometry (HDX-MS), chemical cross-linking, and radical foot-printing), failing to include these contaminants in the search database during the initial tandem MS (MS/MS) identification stage can result in complications in peptide identification. We have created contaminant FASTA databases for Sf9 and E. coli NiNTA or GST purification strategies and show that the use of these databases can effectively improve HDX-MS protein coverage, fragment count, and confidence in peptide identification. This approach provides a robust strategy toward the design of contaminant databases for any purification approach that will expand the complexity of systems able to be interrogated by HDX-MS.

Probiotic Escherichia coli Nissle 1917-derived outer membrane vesicles enhance immunomodulation and antimicrobial activity in RAW264.7 macrophages.

BACKGROUND: Probiotic Escherichia coli Nissle 1917 (EcN) has been widely studied for the treatment of intestinal inflammatory diseases and infectious diarrhea, but the mechanisms by which they communicate with the host are not well-known. Outer membrane vesicles (OMVs) are produced by Gram-negative bacteria and deliver microbial molecules to distant target cells in the host, which play a very important role in mediating bacteria-host communication. Here, we aimed to investigate whether EcN-derived OMVs (EcN_OMVs) could mediate immune regulation in macrophages. RESULTS: In this study, after the characterization of EcN_OMVs using electron microscopy, nanoparticle tracking and proteomic analyses, we demonstrated by confocal fluorescence microscopy that EcN_OMVs could be internalized by RAW 264.7 macrophages. Stimulation with EcN_OMVs at appropriate concentrations promoted proliferation, immune-related enzymatic activities and phagocytic functions of RAW264.7 cells. Moreover, EcN_OMVs induced more anti-inflammatory responses (IL-10) than pro-inflammatory responses (IL-6 and TNF-α) in vitro, and also modulated the production of Th1-polarizing cytokine (IL-12) and Th2-polarizing cytokine (IL-4). Treatments with EcN_OMVs effectively improved the antibacterial activity of RAW 264.7 macrophages. CONCLUSIONS: These findings indicated that EcN_OMVs could modulate the functions of the host immune cells, which will enrich the existing body of knowledge of EVs as an important mechanism for the communication of probiotics with their hosts.

Extending the Separation Space with Trapped Ion Mobility Spectrometry Improves the Accuracy of Isobaric Tag-Based Quantitation in Proteomic LC/MS/MS.

Two-dimensional separation by nano-LC and trapped ion mobility spectrometry (TIMS) prior to Q/TOF tandem mass spectrometry significantly improves the accuracy of isobaric tag-based quantitation in proteome analysis without the need for additional measurement time for TIMS insertion between LC and Q/TOF MS. The obtained peak capacity of up to 3300 h-1 in LC/TIMS reduced the coisolation of precursor ions at the quadrupole analyzer, resulting in more accurate ratios of reporter ions derived from isobaric tags in product ion spectra obtained at the TOF analyzer. We also found that TIMS with a narrower quadrupole isolation window could reduce the ratio compression effect at least as effectively as the synchronous precursor selection method using MS3 scans without compromising sensitivity or coverage. Our results suggest that the 65 min gradient LC/TIMS/Q/TOF system is an excellent platform for high-throughput proteomics studies.

Enhancing protein discoverability by data independent acquisition assisted by ion mobility mass spectrometry.

Ion mobility (IM) mass spectrometry allows conducting data independent acquisition (DIA) where all ions entering the instrument are fragmented based on their drift time. In this work, DIA operational parameters were first optimized using a design of experiments. The optimization of data treatment involved a smoothing algorithm of the IM dimension, which increased the number of identified peptides. Then, classical DDA and IM-based DIA were compared injecting increasing amounts of a complex proteome digest (E. coli). Results revealed that compared to DDA, DIA allowed to identify from 2 to 3.3 times more proteins, depending on the injected quantity. To evaluate proteome coverage, endogenous proteins in E. coli cells were sorted by abundance deciles. A large majority of the proteins uniquely observed in DDA were part of the 10% most abundant protein groups. Interestingly, owing to the absence of ion-picking algorithm, DIA allowed to identify proteins coming from a broader concentration range therefore greatly improving proteome coverage. Furthermore, ion mobility separation improved coverage by separating co-eluting peptides. Physicochemical properties of peptides uniquely detected by DIA or DDA were also compared using supervised and unsupervised multivariate analysis. As a result, peptides having a higher mass and being relatively hydrophobic were significantly more identified in DIA. Finally, semi-quantitative performance of both methods was investigated and proved to be comparable, except that DIA demonstrated a better sensitivity than DDA. As a conclusion, we demonstrated in this study that both acquisition modes provide complementary information about the proteome under investigation.

Evaluation and minimization of nonspecific tryptic cleavages in proteomic sample preparation.

High specificity of trypsin is a prerequisite for accurate identification and quantification of proteins in shotgun proteomics. It is important to minimize nonspecific enzymatic cleavages during proteomic sample preparation. METHODS: In this study, protein extraction and trypsin digestion conditions were extensively evaluated using the less-complex Escherichia coli lysates to improve the sensitivity of detecting low-abundance nonspecific peptides by liquid chromatography/tandem mass spectrometry. RESULTS: Trypsin digestion buffers and digestion times were proved to have a significant effect on nonspecific cleavages. The triethylammonium bicarbonate buffer induces significantly lower nonspecific cleavages than the other two buffers, but a freshly prepared urea solution does not induce more than sodium dodecyl sulfate. Because prolonged trypsin digestion resulted in a considerable number of nonspecific cleavages, an optimized 2-h protocol was developed with 45.2% less semispecific tryptic peptides but 18.5% more unmodified peptides identified than the commonly used 16-h protocol. CONCLUSIONS: The significant decrease in nonspecific cleavages and artificial modifications improves the accuracy of protein quantification and the identification of low-abundance proteins, and it is especially useful for studying protein posttranslational modifications. For trypsin digestion, the proposed 2-h protocol can potentially be a replacement for the traditional 16-h protocol.

Transcriptional analysis of RstA/RstB in avian pathogenic Escherichia coli identifies its role in the regulation of hdeD-mediated virulence and survival in chicken macrophages.

Avian pathogenic Escherichia coli (APEC) causes avian colibacillosis in poultry, which is characterized by systemic infections such as septicemia, air sacculitis, and pericarditis. APEC uses two-component regulatory systems (TCSs) to handle the stressful environments present in infected hosts. While many TCSs in E. coli have been well characterized, the RstA/RstB system in APEC has not been thoroughly investigated. The involvement of the RstA regulator in APEC pathogenesis was demonstrated during previous studies investigating its role in APEC persistence in chicken macrophages and respiratory infections. However, the mechanism underlying this phenomenon has not been clarified. Transcriptional analysis of the effect of rstAB deletion was therefore performed to improve the understanding of the RstA/RstB regulatory mechanism, and particularly its role in virulence. The transcriptomes of the rstAB mutant and the wild-type strain E058 were compared during their growth in the bloodstreams of challenged chickens. Overall, 198 differentially expressed (DE) genes were identified, and these indicated that RstA/RstB mainly regulates systems involved in nitrogen metabolism, iron acquisition, and acid resistance. Phenotypic assays indicated that the rstAB mutant responded more to an acidic pH than the wild-type strain did, possibly because of the repression of the acid-resistance operons hdeABD and gadABE by the deletion of rstAB. Based on the reported RstA box motif TACATNTNGTTACA, we identified four possible RstA target genes (hdeD, fadE, narG, and metE) among the DE genes. An electrophoretic mobility shift assay confirmed that RstA binds directly to the promoter of hdeD, and ß-galactosidase assays showed that hdeD expression was reduced by rstAB deletion, indicating that RstA directly regulates hdeD expression. The hdeD mutation resulted in virulence attenuation in both cultured chicken macrophages and experimentally infected chickens. In conclusion, our data suggest that RstA affects APEC E058 virulence partly by directly regulating the acidic resistance gene hdeD.

Kinetics of the thermal inactivation and the refolding of bacterial luciferases in Bacillus subtilis and in Escherichia coli differ.

Here we present a study of the thermal inactivation and the refolding of the proteins in Gram positive Bacillus subtilis. To enable use of bacterial luciferases as the models for protein thermal inactivation and refolding in B. subtilis cells, we developed a variety of bright luminescent B. subtilis strains which express luxAB genes encoding luciferases of differing thermolability. The kinetics of the thermal inactivation and the refolding of luciferases from Photorhabdus luminescens and Photobacterium leiognathi were compared in Gram negative and Gram positive bacteria. In B. subtilis cells, these luciferases are substantially more thermostable than in Escherichia coli. Thermal inactivation of the thermostable luciferase P. luminescens in B. subtilis at 48.5°Ð¡ behaves as a first-order reaction. In E.coli, the first order rate constant (Kt) of the thermal inactivation of luciferase in E. coli exceeds that observed in B. subtilis cells 2.9 times. Incubation time dependence curves for the thermal inactivation of the thermolabile luciferase of P. leiognathi luciferase in the cells of E. coli and B. subtilis may be described by first and third order kinetics, respectively. Here we shown that the levels and the rates of refolding of thermally inactivated luciferases in B. subtilis cells are substantially lower that that observed in E. coli. In dnaK-negative strains of B. subtilis, both the rates of thermal inactivation and the efficiency of refolding are similar to that observed in wild-type strains. These experiments point that the role that DnaKJE plays in thermostability of luciferases may be limited to bacterial species resembling E. coli.

Identification and characterization of OmpT-like proteases in uropathogenic Escherichia coli clinical isolates.

Bacterial colonization of the urogenital tract is limited by innate defenses, including the production of antimicrobial peptides (AMPs). Uropathogenic Escherichia coli (UPEC) resist AMP-killing to cause a range of urinary tract infections (UTIs) including asymptomatic bacteriuria, cystitis, pyelonephritis, and sepsis. UPEC strains have high genomic diversity and encode numerous virulence factors that differentiate them from non-UTI-causing strains, including ompT. As OmpT homologs cleave and inactivate AMPs, we hypothesized that UPEC strains from patients with symptomatic UTIs have high OmpT protease activity. Therefore, we measured OmpT activity in 58 clinical E. coli isolates. While heterogeneous OmpT activities were observed, OmpT activity was significantly greater in UPEC strains isolated from patients with symptomatic infections. Unexpectedly, UPEC strains exhibiting the greatest protease activities harbored an additional ompT-like gene called arlC (ompTp). The presence of two OmpT-like proteases in some UPEC isolates led us to compare the substrate specificities of OmpT-like proteases found in E. coli. While all three cleaved AMPs, cleavage efficiency varied on the basis of AMP size and secondary structure. Our findings suggest the presence of ArlC and OmpT in the same UPEC isolate may confer a fitness advantage by expanding the range of target substrates.

Sortase-mediated fluorescent labeling of CRISPR complexes.

Fluorescent labeling of proteins is a critical requirement for single-molecule imaging studies. Many protein labeling strategies require harsh conditions or large epitopes that can inactivate the target protein, either by decreasing the protein's enzymatic activity or by blocking protein-protein interactions. Here, we provide a detailed protocol to efficiently label CRISPR-Cas complexes with a small fluorescent peptide via sortase-mediated transpeptidation. The sortase tag consists of just a few amino acids that are specifically recognized at either the N- or the C-terminus, making this strategy advantageous when the protein is part of a larger complex. Sortase is active at high ionic strength, 4°C, and with a broad range of organic fluorophores. We discuss the design, optimization, and single-molecule fluorescent imaging of CRISPR-Cas complexes on DNA curtains. Sortase-mediated transpeptidation is a versatile addition to the protein labeling toolkit.

Online Parallel Accumulation-Serial Fragmentation (PASEF) with a Novel Trapped Ion Mobility Mass Spectrometer.

In bottom-up proteomics, peptides are separated by liquid chromatography with elution peak widths in the range of seconds, whereas mass spectra are acquired in about 100 microseconds with time-of-flight (TOF) instruments. This allows adding ion mobility as a third dimension of separation. Among several formats, trapped ion mobility spectrometry (TIMS) is attractive because of its small size, low voltage requirements and high efficiency of ion utilization. We have recently demonstrated a scan mode termed parallel accumulation - serial fragmentation (PASEF), which multiplies the sequencing speed without any loss in sensitivity (Meier et al., PMID: 26538118). Here we introduce the timsTOF Pro instrument, which optimally implements online PASEF. It features an orthogonal ion path into the ion mobility device, limiting the amount of debris entering the instrument and making it very robust in daily operation. We investigate different precursor selection schemes for shotgun proteomics to optimally allocate in excess of 100 fragmentation events per second. More than 600,000 fragmentation spectra in standard 120 min LC runs are achievable, which can be used for near exhaustive precursor selection in complex mixtures or accumulating the signal of weak precursors. In 120 min single runs of HeLa digest, MaxQuant identified more than 6,000 proteins without matching to a library and with high quantitative reproducibility (R > 0.97). Online PASEF achieves a remarkable sensitivity with more than 2,500 proteins identified in 30 min runs of only 10 ng HeLa digest. We also show that highly reproducible collisional cross sections can be acquired on a large scale (R > 0.99). PASEF on the timsTOF Pro is a valuable addition to the technological toolbox in proteomics, with a number of unique operating modes that are only beginning to be explored.

A graph-based filtering method for top-down mass spectral identification.

BACKGROUND: Database search has been the main approach for proteoform identification by top-down tandem mass spectrometry. However, when the target proteoform that produced the spectrum contains post-translational modifications (PTMs) and/or mutations, it is quite time consuming to align a query spectrum against all protein sequences without any PTMs and mutations in a large database. Consequently, it is essential to develop efficient and sensitive filtering algorithms for speeding up database search. RESULTS: In this paper, we propose a spectrum graph matching (SGM) based protein sequence filtering method for top-down mass spectral identification. It uses the subspectra of a query spectrum to generate spectrum graphs and searches them against a protein database to report the best candidates. As the sequence tag and gaped tag approaches need the preprocessing step to extract and select tags, the SGM filtering method circumvents this preprocessing step, thus simplifying data processing. We evaluated the filtration efficiency of the SGM filtering method with various parameter settings on an Escherichia coli top-down mass spectrometry data set and compared the performances of the SGM filtering method and two tag-based filtering methods on a data set of MCF-7 cells. CONCLUSIONS: Experimental results on the data sets show that the SGM filtering method achieves high sensitivity in protein sequence filtration. When coupled with a spectral alignment algorithm, the SGM filtering method significantly increases the number of identified proteoform spectrum-matches compared with the tag-based methods in top-down mass spectrometry data analysis.

Stress response of Escherichia coli to essential oil components - insights on low-molecular-weight proteins from MALDI-TOF.

The antibacterial effects of essential oils and their components (EOCs) are usually attributed to effects on membranes and metabolism. Studies of the effects of EOCs on protein expression have primarily analysed proteins larger than 10 kDa using gel electrophoresis. In the present study, we used MALDI-TOF-MS to investigate the effects of EOCs on low-molecular-weight proteins. From 297 m/z features, we identified 94 proteins with important differences in expression among untreated samples, samples treated with EOCs, and samples treated with antibiotics, peroxide, or chlorine. The targets of these treatments obviously differ, even among EOCs. In addition to ribosomal proteins, stress-, membrane- and biofilm-related proteins were affected. These findings may provide a basis for identifying new targets of essential oils and synergies with other antibiotics.

Proteomic investigation on bio-corona of functionalized multi-walled carbon nanotubes.

BACKGROUND: The formation of bio-corona, due to adsorption of biomolecules onto carbon nanotubes (CNTs) surface in a physiological environment, may lead to a modified biological "identity" of CNTs, contributing to determination of their biocompatibility and toxicity. METHODS: Multi-walled carbon nanotubes surfaces (f-MWCNTs) were modified attaching acid and basic chemical functions such as carboxyl (MWCNTs-COOH) and ammonium (MWCNTs-N) groups respectively. The investigation of interactions between f-MWCNTs and proteins present in biological fluids, like human plasma, was performed by electrophoretic separation (SDS-PAGE) and mass spectrometry analysis (nLC-MS/MS). RESULTS: A total of 52 validated proteins was identified after incubation of f-MWCNTs in human plasma. 86% of them was present in bio-coronas formed on the surface of all f-MWCNTs and 29% has specifically interacted with only one type of f-MWCNTs. CONCLUSIONS: The evaluation of proteins primary structures, present in all bio-coronas, did not highlight any correlation between the chemical functionalization on MWCNTs and the content of acid, basic and hydrophobic amino acids. Despite this, many proteins of bio-corona, formed on all f-MWCNTs, were involved in the inhibitor activity of serine- or cysteine- endopeptidases, a molecular function completely unrevealed in the human plasma as control. Finally, the interaction with immune system's proteins and apolipoproteins has suggested a possible biocompatibility and a favored bio-distribution of tested f-MWCNTs. GENERAL SIGNIFICANCE: Considering the great potential of CNTs in the nanomedicine, a specific chemical functionalization onto MWCNTs surface could control the protein corona formation and the biocompatibility of nanomaterials.