After hundreds of generations adaptive evolution at exponential growth, Escherichia coli grows as predicted using flux balance analysis (FBA) on genome‐scale metabolic models (GEMs). However, it is not known whether the pathway usage in FBA solutions consistent with gene and protein expression wild‐type evolved strains. Here, we report that >98% active reactions from optimal growth are supported by transcriptomic proteomic data. Moreover, when E. adapts to rate selective pressure, strains...

In this study, we evaluated a concatenated low pH (pH 3) and high 10) reversed-phase liquid chromatography strategy as first dimension for two-dimensional tandem mass spectrometry ("shotgun") proteomic analysis of trypsin-digested human MCF10A cell sample. Compared with the more traditional strong cation exchange method, use first-dimension fractionation resulted in 1.8- 1.6-fold increases number peptide protein identifications (with two or unique peptides), respectively. addition to broader...

The combination of nanodroplet sample preparation, ultra-low-flow nanoLC, high-field asymmetric ion mobility spectrometry (FAIMS) and latest-generation mass instrumentation provides dramatically improved single-cell proteome profiling.

Single-cell proteomics can provide unique insights into biological processes by resolving heterogeneity that is obscured bulk measurements. Gains in the overall sensitivity and proteome coverage through improvements sample processing analysis increase information content obtained from each cell, particularly for less abundant proteins. Here we report on improved single-cell combination of previously developed nanoPOTS platform with further miniaturization liquid chromatography (LC)...

In the young field of single-cell proteomics (scMS), there is a great need for improved global proteome characterization, both in terms proteins quantified per cell and quantitative performance thereof. The recently introduced real-time search (RTS) on Orbitrap Eclipse Tribrid mass spectrometer combination with SPS-MS3 acquisition has been shown to be beneficial measurement samples that are multiplexed using isobaric tags. Multiplexed scMS requires high ion injection times high-resolution...

Nitric oxide is implicated in a variety of signaling pathways different systems, notably endothelial cells. Some its effects can be exerted through covalent modifications proteins and, among these modifications, increasing attention being paid to S-nitrosylation as mechanism. In this work, we show by methods (ozone chemiluminescence, biotin switch, and mass spectrometry) that the molecular chaperone Hsp90 target identify susceptible cysteine residue region C-terminal domain interacts with...

Tyrosine phosphorylation (pTyr) plays a pivotal role in signal transduction and is commonly dysregulated cancer. As result, profiling tumor pTyr levels may reveal therapeutic insights critical to combating disease. Existing discovery targeted mass spectrometry-based methods used monitor networks involve tradeoff between broad coverage of the network, reproducibility target identification across analyses, accurate quantification. To address these limitations, we developed approach, termed...

Database search engines for bottom‐up proteomics largely ignore peptide fragment ion intensities during the automated scoring of tandem mass spectra against protein databases. Recent advances in deep learning allow accurate prediction intensities. Using these predictions to calculate additional intensity‐based scores helps overcome this drawback. Here, we describe a processing workflow termed INFERYS™ rescoring Sequest HT engine results Thermo Scientific™ Proteome Discoverer™ 2.5 software....

Abstract The recent discovery of SPINDLY (SPY)-catalyzed protein O-fucosylation revealed a novel mechanism for regulating nucleocytoplasmic functions in plants. Genetic evidence indicates the important roles SPY diverse developmental and physiological processes. However, upstream signal controlling activity downstream substrate proteins O-fucosylated by remain largely unknown. Here, we demonstrated that mediates sugar-dependent growth Arabidopsis (Arabidopsis thaliana). We further identified...

Proteolytic digestion of proteins in seconds under an ultrasonic field provided by high-intensity focused ultrasound (HIFU) has been achieved. Successful in-solution and in-gel tryptic 60 s or less was demonstrated either MALDI-TOF mass spectrometry liquid chromatography-electrospray ion trap (RP−HPLC−ESI−IT−MS/MS). The efficiency this new procedure for protein compared favorably with those attained using conventional overnight incubation methods. performance the method also specific...

High throughput identification of peptides in databases from tandem mass spectrometry data is a key technique modern proteomics. Common approaches to interpret large scale peptide results are based on the statistical analysis average score distributions, which constructed set best scores produced by collections MS/MS spectra using searching engines such as SEQUEST. Other calculate individual probabilities basis theoretical models or single-spectrum distributions each spectrum. In this work,...

A new strategy for the fast monitoring of peptide biomarkers is described. It based on use accelerated in-solution trypsin digestions under an ultrasonic field provided by high-intensity focused ultrasound (HIFU) and several peptides selected MS/MS ion in a linear trap mass spectrometer. The performance method was established unequivocal identification all commercial fish species belonging to Merlucciidae family. Using particular combination only 11 peptides, resulting from HIFU-assisted...

Recent technological advances have made multidimensional peptide separation techniques coupled with tandem mass spectrometry the method of choice for high-throughput identification proteins. Due to these advances, development software tools large-scale, fully automated, unambiguous is highly necessary. In this work, we used as a model nuclear proteome from Jurkat cells and present processing algorithm that allows accurate predictions random matching distributions, based on two SEQUEST scores...

Quantitative strategies relying on stable isotope labeling and dilution mass spectrometry have proven to be a very robust alternative the well established gel-based techniques for study of dynamic proteome. Postdigestion 18O is becoming popular mainly due simplicity enzyme-catalyzed exchange reaction, peptide handling storage procedures, flexibility versatility introduced by decoupling protein digestion from labeling. Despite recent progresses, quantification postdigestion still involves...

A new method for rapid proteolytic digestion of proteins under high pressure that uses cycling technology in the range 5−35 kpsi was demonstrated proteomic analysis. Successful in-solution digestions single and complex protein mixtures were achieved 60 s then analyzed by reversed phase liquid chromatography-electrospray ionization ion trap-mass spectrometry. Method performance terms number Shewanella oneidensis peptides identified a shotgun approach evaluated relative to traditional...

Abstract Mass spectrometry (MS) is a technique of paramount importance in Proteomics, and developments this field have been possible owing to novel MS instrumentation, experimental strategies, bioinformatics tools. Today it identify determine relative expression levels thousands proteins biological system by analysis peptides produced proteolytic digestion. In some situations, however, the precise characterization particular peptide species very complex mixture needed. While single‐fragment...

Abstract A stable and robust trypsin‐based biocatalytic system was developed demonstrated for proteomic applications. The utilizes polymer nanofibers coated with trypsin aggregates immobilized protease digestions. After covalently attaching an initial layer of to the nanofibers, highly concentrated molecules are crosslinked layered by way a glutaraldehyde treatment. This process produced 300‐fold increase in activity compared conventional method covalent immobilization, proved be that it...

Maintenance of macrophages in their basal state and rapid activation response to pathogen detection are central the innate immune system, acting limit nonspecific oxidative damage promote killing following infection. To identify possible age-related alterations macrophage function, we have assayed function peritoneal from young (3-4 months) aged (14-15 Balb/c mice. In agreement with prior suggestions, observe age-dependent increases extent recruitment into peritoneum, as well ex vivo...

Quantitative proteomics using stable isotopic 16O/18O labeling has emerged as a very powerful tool, since it number of advantages over other methods, including the simplicity chemistry, constant mass tag at C termini and its general applicability. However, due to small difference between labeled unlabeled peptide species, this approach usually been restricted high-resolution spectrometers. In study we explored whether scanning mode, together with extremely high speed linear IT allows...

Integrated top-down bottom-up proteomics combined with on-line digestion has great potential to improve the characterization of protein isoforms in biological systems and is amendable high throughput experiments. Bottom-up ultimately provides peptide sequences derived from tandem MS analyses peptides after proteome been digested. Top-down conversely entails intact proteins for more effective genetic variations and/or post-translational modifications. Herein, we describe recent efforts toward...

An efficient on-line digestion system that reduces the number of sample manipulation steps has been demonstrated for high-throughput proteomics. By incorporating a pressurized loop into liquid chromatography-based separation system, both and enzyme (e.g., trypsin) can be simultaneously introduced to produce complete, yet rapid digestion. Both standard proteins complex Shewanella oneidensis global protein extract were digested analyzed using automated online coupled an ion mobility...

A new sample processing workflow that uses high intensity focused ultrasound to rapidly reduce and alkylate cysteines, digest proteins then label peptides with 18O was developed for quantitative proteomics applications. Each step individually refined minimize reaction times, peptide loses undesired byproducts or modifications. When this novel used, mouse plasma were successfully denatured, alkylated, in-solution digested, 18O-labeled in <10 min subsequent analysis by liquid...