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The recent discovery of SPINDLY (SPY)-catalyzed protein O-fucosylation revealed a novel mechanism for regulating nucleocytoplasmic protein functions in plants. Genetic evidence indicates the important roles of SPY in diverse developmental and physiological processes. However, the upstream signal controlling SPY activity and the downstream substrate proteins O-fucosylated by SPY remain largely unknown. Here, we demonstrated that SPY mediates sugar-dependent growth in Arabidopsis (Arabidopsis thaliana). We further identified hundreds of O-fucosylated proteins using lectin affinity chromatography followed by mass spectrometry. All the O-fucosylation events quantified in our proteomic analyses were undetectable or dramatically decreased in the spy mutants, and thus likely catalyzed by SPY. The O-fucosylome includes mostly nuclear and cytosolic proteins. Many O-fucosylated proteins function in essential cellular processes, phytohormone signaling, and developmental programs, consistent with the genetic functions of SPY. The O-fucosylome also includes many proteins modified by O-linked N-acetylglucosamine (O-GlcNAc) and by phosphorylation downstream of the target of rapamycin (TOR) kinase, revealing the convergence of these nutrient signaling pathways on key regulatory functions such as post-transcriptional/translational regulation and phytohormone responses. Our study identified numerous targets of SPY/O-fucosylation and potential nodes of crosstalk among sugar/nutrient signaling pathways, enabling future dissection of the signaling network that mediates sugar regulation of plant growth and development.

Custom pathway and substrate-kinase libraries for tumor-specific pathway enrichment analyses.

Tyrosine phosphorylation (pTyr) plays a pivotal role in signal transduction and is commonly dysregulated in cancer. As a result, profiling tumor pTyr levels may reveal therapeutic insights critical to combating disease. Existing discovery and targeted mass spectrometry–based methods used to monitor pTyr networks involve a tradeoff between broad coverage of the pTyr network, reproducibility in target identification across analyses, and accurate quantification. To address these limitations, we developed a targeted approach, termed “SureQuant pTyr,” coupling low input pTyr enrichment with a panel of isotopically labeled internal standard peptides to guide data acquisition of low-abundance tyrosine phosphopeptides. SureQuant pTyr allowed for reliable quantification of several hundred commonly dysregulated pTyr targets with high quantitative accuracy, improving the robustness and usability of targeted mass spectrometry assays. We established the clinical applicability of SureQuant pTyr by profiling pTyr signaling levels in human colorectal tumors using minimal sample input, characterizing patient-specific oncogenic-driving mechanisms. While in some cases pTyr profiles aligned with previously reported proteomic, genomic, and transcriptomic molecular characterizations, we highlighted instances of new insights gained using pTyr characterization and emphasized the complementary nature of pTyr measurements with traditional biomarkers for improving patient stratification and identifying therapeutic targets. The turn-key nature of this approach opens the door to rapid and reproducible pTyr profiling in research and clinical settings alike and enables pTyr-based measurements for applications in precision medicine.Significance:SureQuant pTyr is a mass spectrometry–based targeted method that enables sensitive and selective targeted quantitation of several hundred low-abundance tyrosine phosphorylated peptides commonly dysregulated in cancer, including oncogenic signaling networks.

Supplementary figures, table legends, and methods.

The rapidly increasing adoption of high-resolution accurate-mass methods in analytical laboratories has fueled demand for instruments that combine high performance and reliability with small size and greater ease-of-use. This paper presents the major design principles that are driving the evolution of the hybrid quadrupole-Orbitrap instrument architecture to enable a greater range of applications and users. These principles may be summarized as follows: better usage of physical space and better access for service by means of size reduction of pumping and ion optics; expanded use of technologies from electronics in ion-optical design; flexibility in performance via modularity of design of the hardware and software components; and, harmonization of interfaces with other instruments to facilitate sharing and transferability of analytical workflows. The design of a novel family of hybrid mass spectrometers is described in detail, and performance evaluation is carried out on a wide variety of samples for its three representatives: the Orbitrap Exploris 120, Orbitrap Exploris 240 and Orbitrap Exploris 480 mass spectrometers. The new instrument family is shown to offer compelling potential not only for high-end proteomics and biopharmaceutical applications, but also for screening, trace, targeted and clinical analysis by liquid chromatography/mass spectrometry methods.

We report on the combination of nanodroplet sample preparation, ultra-low-flow nanoLC, high-field asymmetric ion mobility spectrometry (FAIMS), and the latest-generation Orbitrap Eclipse Tribrid mass spectrometer for greatly improved single-cell proteome profiling. FAIMS effectively filtered out singly charged ions for more effective MS analysis of multiply charged peptides, resulting in an average of 1056 protein groups identified from single HeLa cells without MS1-level feature matching. This is 2.3 times more identifications than without FAIMS and a far greater level of proteome coverage for single mammalian cells than has been previously reported for a label-free study. Differential analysis of single microdissected motor neurons and interneurons from human spinal tissue indicated a similar level of proteome coverage, and the two subpopulations of cells were readily differentiated based on single-cell label-free quantification., The combination of nanodroplet sample preparation, ultra-low-flow nanoLC, high-field asymmetric ion mobility spectrometry (FAIMS) and latest-generation mass spectrometry instrumentation provides dramatically improved single-cell proteome profiling.

Human leukocyte antigen class I (HLA-I) molecules bind and present peptides at the cell surface to facilitate the induction of appropriate CD8+ T cell-mediated immune responses to pathogen- and self-derived proteins. The HLA-I peptide-binding cleft contains dominant anchor sites in the B and F pockets that interact primarily with amino acids at peptide position 2 and the C-terminus, respectively. Nonpocket peptide–HLA interactions also contribute to peptide binding and stability, but these secondary interactions are thought to be unique to individual HLA allotypes or to specific peptide antigens. Here, we show that two positively charged residues located near the top of peptide-binding cleft facilitate interactions with negatively charged residues at position 4 of presented peptides, which occur at elevated frequencies across most HLA-I allotypes. Loss of these interactions was shown to impair HLA-I/peptide binding and complex stability, as demonstrated by both in vitro and in silico experiments. Furthermore, mutation of these Arginine-65 (R65) and/or Lysine-66 (K66) residues in HLA-A*02:01 and A*24:02 significantly reduced HLA-I cell surface expression while also reducing the diversity of the presented peptide repertoire by up to 5-fold. The impact of the R65 mutation demonstrates that nonpocket HLA-I/peptide interactions can constitute anchor motifs that exert an unexpectedly broad influence on HLA-I-mediated antigen presentation. These findings provide fundamental insights into peptide antigen binding that could broadly inform epitope discovery in the context of viral vaccine development and cancer immunotherapy.

In the young field of single-cell proteomics (scMS), there is a great need for improved global proteome characterization, both in terms of proteins quantified per cell and quantitative performance thereof. The recently introduced real-time search (RTS) on the Orbitrap Eclipse Tribrid mass spectrometer in combination with SPS-MS3 acquisition has been shown to be beneficial for the measurement of samples that are multiplexed using isobaric tags. Multiplexed single-cell proteomics requires high ion injection times and high-resolution spectra to quantify the single-cell signal, however the carrier channel facilitates peptide identification and thus offers the opportunity for fast on-the-fly precursor filtering before committing to the time intensive quantification scan. Here, we compared classical MS2 acquisition against RTS-SPS-MS3, both using the Orbitrap Eclipse Tribrid MS with the FAIMS Pro ion mobility interface and present a new acquisition strategy termed RETICLE (RTS Enhanced Quant of Single Cell Spectra) that makes use of fast real-time searched linear ion trap scans to preselect MS1 peptide precursors for quantitative MS2 Orbitrap acquisition. We show that classical MS2 acquisition is outperformed by both RTS-SPS-MS3 through increased quantitative accuracy at similar proteome coverage, and RETICLE through higher proteome coverage, with the latter enabling the quantification of over 1000 proteins per cell at a MS2 injection time of 750ms using a 2h gradient.

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 the accurate prediction of peptide fragment ion intensities. Using these predictions to calculate additional intensity-based scores helps to overcome this drawback. Here, we describe a processing workflow termed INFERYS™ rescoring for the intensity-based rescoring of Sequest HT search engine results in Thermo Scientific™ Proteome Discoverer™ 2.5 software. The workflow is based on the deep learning platform INFERYS capable of predicting fragment ion intensities, which runs on personal computers without the need for graphics processing units. This workflow calculates intensity-based scores comparing peptide spectrum matches from Sequest HT and predicted spectra. Resulting scores are combined with classical search engine scores for input to the false discovery rate estimation tool Percolator. We demonstrate the merits of this approach by analyzing a classical HeLa standard sample and exemplify how this workflow leads to a better separation of target and decoy identifications, in turn resulting in increased peptide spectrum match, peptide and protein identification numbers. On an immunopeptidome dataset, this workflow leads to a 50% increase in identified peptides, emphasizing the advantage of intensity-based scores when analyzing low-intensity spectra or analytes with very similar physicochemical properties that require vast search spaces. Overall, the end-to-end integration of INFERYS rescoring enables simple and easy access to a powerful enhancement to classical database search engines, promising a deeper, more confident and more comprehensive analysis of proteomic data from any organism by unlocking the intensity dimension of tandem mass spectra for identification and more confident scoring.

In this chapter, we describe a rapid workflow for the shotgun global phosphoproteomics analysis. The strategy is based on the use of accelerated in-solution trypsin digestion under an ultrasonic field by high-intensity focused ultrasound (HIFU) coupled to titanium dioxide (TiO

In this chapter, we describe a rapid workflow for the shotgun global phosphoproteomics analysis. The strategy is based on the use of accelerated in-solution trypsin digestion under an ultrasonic field by high-intensity focused ultrasound (HIFU) coupled to titanium dioxide (TiO2) selective phosphopeptide enrichment, fractionation by strong cation exchange chromatography (SCX), and analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a high-resolution mass spectrometer (LTQ-Orbitrap XL). The strategy was optimized for the global phosphoproteome analysis of Jurkat T-cells. Using this accelerated workflow, HIFU-TiO2-SCX-LC-MS/MS, 15,367 phosphorylation sites from 13,029 different phosphopeptides belonging to 3,163 different phosphoproteins can be efficiently identified in less than 15 h.

Tyrosine phosphorylation (pTyr) plays a pivotal role in signal transduction and is commonly dysregulated in cancer. As a result, profiling tumor pTyr levels may reveal therapeutic insights critical to combating disease. Existing discovery and targeted mass spectrometry-based methods used to monitor pTyr networks involve a tradeoff between broad coverage of the pTyr network, reproducibility in target identification across analyses, and accurate quantification. To address these limitations, we developed a targeted approach, termed “SureQuant pTyr,” coupling low input pTyr enrichment with a panel of isotopically labeled, tyrosine phosphorylated internal standard (IS) peptides. Using internal standard guided acquisition, the real-time detection of IS peptides during the analysis initiates the sensitive and selective quantitation of endogenous pTyr targets. This framework allows for reliable quantification of several hundred commonly dysregulated pTyr targets with high quantitative accuracy, enhances target detection success rates, and improves the robustness and usability of targeted acquisition. We establish the clinical applicability of SureQuant pTyr by profiling pTyr signaling levels in human colorectal tumors using minimal sample input, characterizing patient specific oncogenic driving mechanisms. While in some cases pTyr profiles align with previously reported proteomic, genomic, and transcriptomic molecular characterizations, we highlight instances of new insights gained using pTyr characterization and emphasize the complementary nature of pTyr measurements with traditional biomarkers for improving patient stratification and identifying therapeutic targets. The turn-key nature of this approach opens the door to rapid and reproducible pTyr profiling in research and clinical settings alike and enable pTyr-based measurements for applications in precision medicine.SummaryA targeted, mass spectrometry-based method, termed “SureQuant pTyr,” enables highly sensitive and reproducible profiling of tyrosine phosphorylation levels in human colorectal tumors and reveals dysregulated signaling networks for enhanced tumor characterization and biomarker identification.

10 pages, 6 figures, 1 table, We propose a new workflow for fast phosphoproteome profiling. The workflow is based on the use of accelerated in-solution trypsin digestion under an ultrasonic field provided by high-intensity focused ultrasound (HIFU) combined with an inverse strategy based on TiO2 selective phosphopeptide enrichment, fractionation by strong cation exchange chromatography (SCX) and analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) using a high-resolution mass spectrometer. The performance of the method was established for the global phosphoproteome analysis of un-stimulated human Jurkat leukemia T cells (E6.1). Using this accelerated workflow, 15,367 phosphorylation sites from 13,029 different phosphopeptides belonging to 3,163 different phosphoproteins were efficiently identified with high-throughput and reproducibility in less than 15 h. The functional analysis revealed significant phosphorylation-based networks that are implicated in immune function and tumor development pathways. The present strategy, HIFU-TiO2-SCX-LC-MS/MS, is the fastest analytical method reported to date for generating large-scale phosphoproteomics datasets (, This work was supported by the EU Marie Curie actions (FP7-PEOPLE-2012-IEF, ref. 332274) and by the Ramón Areces Foundation (XVII National grant)

Single-cell proteomics can provide unique insights into biological processes by resolving heterogeneity that is obscured by bulk measurements. Gains in the overall sensitivity and proteome coverage through improvements in sample processing and analysis increase the information content obtained from each cell, particularly for less abundant proteins. Here we report on improved single-cell proteome coverage through the combination of the previously developed nanoPOTS platform with further miniaturization of liquid chromatography (LC) separations and implementation of an ultrasensitive latest generation mass spectrometer. Following nanoPOTS sample preparation, protein digests from single cells were separated using a 20 μm i.d. in-housepacked nanoLC column. Separated peptides were ionized using an etched fused-silica emitter capable of stable operation at the ~20 nL/min flow rate provided by the LC separation. Ultrasensitive LC–MS analysis was achieved using the Orbitrap Eclipse Tribrid mass spectrometer. An average of 362 protein groups were identified by tandem mass spectrometry (MS/MS) from single HeLa cells, and 874 protein groups were identified using the Match Between Runs feature of MaxQuant. This represents an >70% increase in label-free proteome coverage for single cells relative to previous efforts using larger bore (30 μm i.d.) LC columns coupled to a previous-generation Orbitrap Fusion Lumos mass spectrometer.

Infection with the influenza virus triggers an innate immune response aimed at initiating the adaptive response to halt viral replication and spread. However, the metabolic response fueling the molecular mechanisms underlying changes in innate immune cell homeostasis remain undefined. Thus, we compared the metabolic response of dendritic cells to that of those infected with active and inactive influenza A virus or treated with toll like receptor agonists. While influenza infects dendritic cells, it does not productively replicate in these cells, and therefore metabolic changes upon infection may represent an adaptive response on the part of the host cells. Using quantitative mass spectrometry along with pulse chase substrate utilization assays and metabolic flux measurements, we found global metabolic changes 17 hours post infection, including significant changes in carbon commitment via glycolysis and glutaminolysis, as well as ATP production via TCA cycle and oxidative phosphorylation. Influenza infection of dendritic cells led to a metabolic phenotype, distinct from that induced by TLR agonists, with significant resilience in terms of metabolic plasticity. We identified Myc as one transcription factor modulating this response. Restriction of either Myc activity or mitochondrial substrates resulted in significant changes in the innate immune functions of dendritic cells, including reduced motility and T cell activation. Transcriptome analysis of inflammatory dendritic cells isolated following influenza infection showed similar metabolic reprogramming occurs in vivo. Thus, early in the infection process dendritic cells respond with global metabolic restructuring that is present in lung DC 9 days following infection and impacts their effector function, suggesting that metabolic switching in dendritic cells plays a vital role in initiating the immune response to influenza infection. Author Summary In response to influenza infection we found that dendritic cells, cells that are critical in mounting an effective immune response, undergo a profound metabolic shift. They alter the concentration and location of hundreds of proteins, including c-MYC, mediating a shift to a highly glycolytic phenotype that is also flexible in terms of fueling respiration. Dendritic cells initiate the immune response to influenza and activate the adaptive response allowing viral clearance and manifesting immune memory for protection against subsequent infections. We found that limiting access to specific metabolic pathways or substrates diminished key immune functions. Previously we described an immediate, fixed, hypermetabolic state in infected respiratory epithelial cells. We now show the metabolic responses of epithelial and dendritic cells are distinct. Here, we also demonstrate that dendritic cells tailor their metabolic response to the pathogen or TLR stimulus. This metabolic reprogramming occurs rapidly in vitro and it is sustained in inflammatory dendritic cells in vivo for at least 9 days following influenza infection. Thus, drugs targeting metabolism are likely to have cell- and pathogen-specific activities in the context of infection. These studies open the possibility of modulating the immune response to viral infection via customizing metabolic therapy to enhance or diminish the function of specific cells. Graphical abstract. Schematic of global metabolic changes in IAV infected DC. Arrows indicate flow of metabolic products. Bar graph represents the percent difference between control uninfected and infected DC for experiments detailed in Figs 2 & 3 +/− SD.

Infection with the influenza virus triggers an innate immune response that initiates the adaptive response to halt viral replication and spread. However, the metabolic response fueling the molecular mechanisms underlying changes in innate immune cell homeostasis remain undefined. Although influenza increases parasitized cell metabolism, it does not productively replicate in dendritic cells. To dissect these mechanisms, we compared the metabolism of dendritic cells to that of those infected with active and inactive influenza A virus and those treated with toll-like receptor agonists. Using quantitative mass spectrometry, pulse chase substrate utilization assays and metabolic flux measurements, we found global metabolic changes in dendritic cells 17 hours post infection, including significant changes in carbon commitment via glycolysis and glutaminolysis, as well as mitochondrial respiration. Influenza infection of dendritic cells led to a metabolic phenotype distinct from that induced by TLR agonists, with significant resilience in terms of metabolic plasticity. We identified c-Myc as one transcription factor modulating this response. Restriction of c-Myc activity or mitochondrial substrates significantly changed the immune functions of dendritic cells, such as reducing motility and T cell activation. Transcriptome analysis of inflammatory dendritic cells isolated following influenza infection showed similar metabolic reprogramming occurs in vivo. Thus, early in the infection process, dendritic cells respond with global metabolic restructuring, that is present in inflammatory lung dendritic cells after infection, and this is important for effector function. These findings suggest metabolic switching in dendritic cells plays a vital role in initiating the immune response to influenza infection., Author summary Dendritic cells are critical in mounting an effective immune response to influenza infection by initiating the immune response to influenza and activating the adaptive response to mediate viral clearance and manifest immune memory for protection against subsequent infections. We found dendritic cells undergo a profound metabolic shift after infection. They alter the concentration and location of hundreds of proteins, including c-Myc, facilitating a shift to a highly glycolytic phenotype that is also flexible in terms of fueling respiration. Nonetheless, we found limiting access to specific metabolic pathways or substrates diminished key immune functions. We previously described an immediate, fixed hypermetabolic state in infected respiratory epithelial cells. Here we present data indicating the metabolic response of dendritic cells is increased yet flexible, distinct from what we previously showed for epithelial cells. Additionally, we demonstrate dendritic cells tailor their metabolic response to the pathogen or TLR stimulus. This metabolic reprogramming occurs rapidly in vitro and is sustained in inflammatory dendritic cells in vivo for at least 9 days following influenza infection. These studies introduce the possibility of modulating the immune response to viral infection using customized metabolic therapy to enhance or diminish the function of specific cells.

Determining and linking the structural heterogeneity of recombinant antibodies to function is critical in the biopharmaceutical industry. We introduce a new microfluidic capillary electrophoresis—mass spectrometry (μCE-MS) approach to characterize intact monoclonal antibody (mAb) and simultaneously quantifying distinct variants. Our MS analysis of intact NIST mAb (RM8671) shows 18 variants identified amongst proteolytic and glycolytic modifications with a range of relative abundances between 0.1% and 100%. In order to verify our quantitative MS results, we used an established system based on capillary electrophoresis—with laser induced fluorescence (CE-LIF) for profiling the N-glycans. All major glycans were identified and further substantiated by exoglycosidase digestion. Interestingly, the µCE-MS analysis of intact NIST mAb consistently yielded higher amounts of G2FG2F-Hex glycoform (~3.4%), whereas the CE-LIF analysis indicates that only 1.4% of G2F-Gal is present. Therefore, the additional hexose adduct observed in µCE-MS may have been the glycation product of the mAb. Further analysis of deglycosylated mAb with µCE-MS made it possible to reveal the glycation with 10.5% of one hexose product and 4.9% of two hexose product in the intact deglycosylated antibody. An integrated solution using two orthogonal and complementary techniques for characterizing antibody glycosylation is provided here.

We propose a new workflow for fast phosphoproteome profiling. The workflow is based on the use of accelerated in-solution trypsin digestion under an ultrasonic field provided by high-intensity focused ultrasound (HIFU) combined with an inverse strategy based on TiO

Ubiquitination is an abundant post-translational modification that consists of covalent attachment of ubiquitin to lysine residues or the N-terminus of proteins. Mono and polyubiquitination have been shown to be involved in many critical eukaryotic cellular functions and are often disrupted by intracellular bacterial pathogens. Affinity enrichment of ubiquitinated proteins enables global analysis of this key modification. In this context, the use of ubiquitin-binding domains is a promising, but relatively unexplored alternative to more broadly used immunoaffinity or tagged affinity enrichment methods. In this study, we evaluated the application of eight ubiquitin-binding domains that have differing affinities for ubiquitination states. Small-scale proteomics analysis identified ∼200 ubiquitinated protein candidates per ubiquitin-binding domain pull-down experiment. Results from subsequent Western blot analyses that employed anti-ubiquitin or monoclonal antibodies against polyubiquitination at lysine 48 and 63 suggest that ubiquitin-binding domains from Dsk2 and ubiquilin-1 have the broadest specificity in that they captured most types of ubiquitination, whereas the binding domain from NBR1 was more selective to polyubiquitination. These data demonstrate that with optimized purification conditions, ubiquitin-binding domains can be an alternative tool for proteomic applications. This approach is especially promising for the analysis of tissues or cells resistant to transfection, of which the overexpression of tagged ubiquitin is a major hurdle.

Maintenance of macrophages in their basal state and their rapid activation in response to pathogen detection are central to the innate immune system, acting to limit nonspecific oxidative damage and promote pathogen killing following infection. To identify possible age-related alterations in macrophage function, we have assayed the function of peritoneal macrophages from young (3-4 months) and aged (14-15 months) Balb/c mice. In agreement with prior suggestions, we observe age-dependent increases in the extent of recruitment of macrophages into the peritoneum, as well as ex vivo functional changes involving enhanced nitric oxide production under resting conditions that contribute to a reduction in the time needed for full activation of senescent macrophages following exposure to lipopolysaccharides (LPS). Further, we observe enhanced bactericidal activity following Salmonella uptake by macrophages isolated from aged Balb/c mice in comparison with those isolated from young animals. Pathways responsible for observed phenotypic changes were interrogated using tandem mass spectrometry, which identified age-dependent increases in levels of proteins linked to immune cell pathways under basal conditions and following LPS activation. Immune pathways upregulated in macrophages isolated from aged mice include proteins critical to the formation of the immunoproteasome. Detection of these latter proteins is dramatically enhanced following LPS exposure for macrophages isolated from aged animals; in comparison, the identification of immunoproteasome subunits is insensitive to LPS exposure for macrophages isolated from young animals. Consistent with observed global changes in the proteome, quantitative proteomic measurements indicate that there are age-dependent abundance changes involving specific proteins linked to immune cell function under basal conditions. LPS exposure selectively increases the levels of many proteins involved in immune cell function in aged Balb/c mice. Collectively, these results indicate that macrophages isolated from old mice are in a preactivated state that enhances their sensitivities to LPS exposure. The hyper-responsive activation of macrophages in aged animals may act to minimize infection by general bacterial threats that arise due to age-dependent declines in adaptive immunity. However, this hypersensitivity and the associated increase in the level of formation of reactive oxygen species are likely to contribute to observed age-dependent increases in the level of oxidative damage that underlie many diseases of the elderly.

In this study, we evaluated a concatenated low pH (pH 3) and high pH (pH 10) reversed-phase liquid chromatography strategy as a first dimension for two-dimensional liquid chromatography tandem mass spectrometry ("shotgun") proteomic analysis of trypsin-digested human MCF10A cell sample. Compared with the more traditional strong cation exchange method, the use of concatenated high pH reversed-phase liquid chromatography as a first-dimension fractionation strategy resulted in 1.8- and 1.6-fold increases in the number of peptide and protein identifications (with two or more unique peptides), respectively. In addition to broader identifications, advantages of the concatenated high pH fractionation approach include improved protein sequence coverage, simplified sample processing, and reduced sample losses. The results demonstrate that the concatenated high pH reversed-phased strategy is an attractive alternative to strong cation exchange for two-dimensional shotgun proteomic analysis.

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 to high throughput proteomics experiments. Bottom-up proteomics ultimately provides the peptide sequences derived from the tandem MS analyses of peptides after the proteome has been digested. Top-down proteomics conversely entails the MS analyses of intact proteins for more effective characterization of genetic variations and/or post-translational modifications. Herein, we describe recent efforts toward efficient integration of bottom-up and top-down LC-MS-based proteomics strategies. Since most proteomics separations utilize acidic conditions, we exploited the compatibility of pepsin (where the optimal digestion conditions are at low pH) for integration into bottom-up and top-down proteomics work flows. Pressure-enhanced pepsin digestions were successfully performed and characterized with several standard proteins in either an off-line mode using a Barocycler or an on-line mode using a modified high pressure LC system referred to as a fast on-line digestion system (FOLDS). FOLDS was tested using pepsin and a whole microbial proteome, and the results were compared against traditional trypsin digestions on the same platform. Additionally, FOLDS was integrated with a RePlay configuration to demonstrate an ultrarapid integrated bottom-up top-down proteomics strategy using a standard mixture of proteins and a monkey pox virus proteome.

Trypsin-coated magnetic nanoparticles (EC-TR/NPs), prepared via a simple multilayer random crosslinking of the trypsin molecules onto magnetic nanoparticles, were highly stable and could be easily captured using a magnet after the digestion was complete. EC-TR/NPs showed a negligible loss of trypsin activity after multiple uses and continuous shaking, whereas the conventional immobilization of covalently attached trypsin on NPs resulted in a rapid inactivation under the same conditions due to the denaturation and autolysis of trypsin. A single model protein, a five-protein mixture, and a whole mouse brain proteome were digested at atmospheric pressure and 37°C for 12 h or in combination with pressure cycling technology at room temperature for 1 min. In all cases, EC-TR/NPs performed equally to or better than free trypsin in terms of both the identified peptide/protein number and the digestion reproducibility. In addition, the concomitant use of EC-TR/NPs and pressure cycling technology resulted in very rapid (∼1 min) and efficient digestions with more reproducible digestion results.

Understanding viral pathogenesis is challenging because of confounding factors, including nonabrasive access to infected tissues and high abundance of inflammatory mediators that may mask mechanistic details. In diseases such as influenza and smallpox where the primary cause of mortality results from complications in the lung, the characterization of lung fluid offers a unique opportunity to study host-pathogen interactions with minimal effect on infected animals. This investigation characterizes the global proteome response in the pulmonary fluid, bronchoalveolar lavage fluid, of macaques during upper respiratory infection by monkeypox virus (MPXV), a close relative of the causative agent of smallpox, variola virus. These results are compared and contrasted against infections by vaccinia virus (VV), a low pathogenic relative of MPXV, and with extracellular fluid from MPXV-infected HeLa cells. To identify changes in the pulmonary protein compartment, macaque lung fluid was sampled twice prior to infection, serving as base line, and up to six times following intrabronchial infection with either MPXV or VV. Increased expression of inflammatory proteins was observed in response to both viruses. Although the increased expression resolved for a subset of proteins, such as C-reactive protein, S100A8, and S100A9, high expression levels persisted for other proteins, including vitamin D-binding protein and fibrinogen γ. Structural and metabolic proteins were substantially decreased in lung fluid exclusively during MPXV and not VV infection. Decreases in structural and metabolic proteins were similarly observed in the extracellular fluid of MPXV-infected HeLa cells. Results from this study suggest that the host inflammatory response may not be the only facilitator of viral pathogenesis, but rather maintaining pulmonary structural integrity could be a key factor influencing disease progression and mortality.

The process of protein digestion is a critical step for successful protein identification in the bottom-up proteomic analysis. To substitute the present practice of in-solution protein digestion, which is long, tedious, and difficult to automate, a lot of efforts have been dedicated for the development of a rapid, recyclable and automated digestion system. Recent advances of nanobiocatalytic approaches have improved the performance of protein digestion by using various nanomaterials such as nanoporous materials, magnetic nanoparticles, and polymer nanofibers. Especially, the unprecedented success of trypsin stabilization in the form of trypsin-coated nanofibers, showing no activity decrease under repeated uses for one year and retaining good resistance to proteolysis, has demonstrated its great potential to be employed in the development of automated, high-throughput, and on-line digestion systems. This review discusses recent developments of nanobiocatalytic approaches for the improved performance of protein digestion in speed, detection sensitivity, recyclability, and trypsin stability. In addition, we also introduce the protein digestions under unconventional energy inputs for protein denaturation and the development of microfluidic enzyme reactors that can benefit from recent successes of these nanobiocatalytic approaches.

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 sample loop into a liquid chromatography-based separation system, both sample and enzyme (e.g., trypsin) can be simultaneously introduced to produce a complete, yet rapid digestion. Both standard proteins and a complex Shewanella oneidensis global protein extract were digested and analyzed using the automated online pressurized digestion system coupled to an ion mobility time-of-flight mass spectrometer, an ion trap mass spectrometer, or both. The system denatured, digested, and separated product peptides in a manner of minutes, making it amenable to on-line high-throughput applications. In addition to simplifying and expediting sample processing, the system was easy to implement and no cross-contamination was observed among samples. As a result, the online digestion system offers a powerful approach for high-throughput screening of...

A new sample processing workflow that uses high intensity focused ultrasound to rapidly reduce and alkylate cysteines, digest proteins and then label peptides with (18)O was developed for quantitative proteomics applications. Each step was individually refined to minimize reaction times, peptide loses and undesired byproducts or modifications. When this novel workflow was used, mouse plasma proteins were successfully denatured, alkylated, in-solution digested, and (18)O-labeled in10 min for subsequent analysis by liquid chromatography-electrospray ionization high resolution mass spectrometry. Performance was evaluated in terms of the number of mouse plasma peptides and proteins identified in a shotgun approach and the quantitative dynamic range. The results were compared with previously published results obtained using conventional sample preparation methods and were found to be similar. Advantages of the new method include greatly simplified and accelerated sample processing, as well as being readily amenable to automation.

High throughput identification of peptides in databases from tandem mass spectrometry data is a key technique in modern proteomics. Common approaches to interpret large scale peptide identification results are based on the statistical analysis of average score distributions, which are constructed from the set of best scores produced by large collections of MS/MS spectra by using searching engines such as SEQUEST. Other approaches calculate individual peptide identification probabilities on the basis of theoretical models or from single-spectrum score distributions constructed by the set of scores produced by each MS/MS spectrum. In this work, we study the mathematical properties of average SEQUEST score distributions by introducing the concept of spectrum quality and expressing these average distributions as compositions of single-spectrum distributions. We predict and demonstrate in the practice that average score distributions are dominated by the quality distribution in the spectra collection, except in the low probability region, where it is possible to predict the dependence of average probability on database size. Our analysis leads to a novel indicator, the probability ratio, which takes optimally into account the statistical information provided by the first and second best scores. The probability ratio is a non-parametric and robust indicator that makes spectra classification according to parameters such as charge state unnecessary and allows a peptide identification performance, on the basis of false discovery rates, that is better than that obtained by other empirical statistical approaches. The probability ratio also compares favorably with statistical probability indicators obtained by the construction of single-spectrum SEQUEST score distributions. These results make the robustness, conceptual simplicity, and ease of automation of the probability ratio algorithm a very attractive alternative to determine peptide identification confidences and error rates in high throughput experiments.

Rapid identification of proteins is of primary importance for the analytical community. Protein-biomarker discovery for medical diagnostics or pharmacological purposes is becoming one of the hottest research topics. Moreover, rapid identification of proteins can help unambiguous bacterial and virus detection. In addition, the fast identification of bacteria can be used to beat bioterrorism. As a consequence, new analytical methodologies have emerged recently with the aim of making protein analysis as fast and as confident as possible. In this article, we critically review the new trends in sample treatment for protein identification and comment on the prospects for the future in this promising analytical area.

Nitric oxide is implicated in a variety of signaling pathways in different systems, notably in endothelial cells. Some of its effects can be exerted through covalent modifications of proteins and, among these modifications, increasing attention is being paid to S-nitrosylation as a signaling mechanism. In this work, we show by a variety of methods (ozone chemiluminescence, biotin switch, and mass spectrometry) that the molecular chaperone Hsp90 is a target of S-nitrosylation and identify a susceptible cysteine residue in the region of the C-terminal domain that interacts with endothelial nitric oxide synthase (eNOS). We also show that the modification occurs in endothelial cells when they are treated withS-nitroso-l-cysteine and when they are exposed to eNOS activators. Hsp90 ATPase activity and its positive effect on eNOS activity are both inhibited by S-nitrosylation. Together, these data suggest that S-nitrosylation may functionally regulate the general activities of Hsp90 and provide a feedback mechanism for limiting eNOS activation.

Recent technological advances have made multidimensional peptide separation techniques coupled with tandem mass spectrometry the method of choice for high-throughput identification of proteins. Due to these advances, the development of software tools for large-scale, fully automated, unambiguous peptide identification is highly necessary. In this work, we have used as a model the nuclear proteome from Jurkat cells and present a processing algorithm that allows accurate predictions of random matching distributions, based on the two SEQUEST scores Xcorr and DeltaCn. Our method permits a very simple and precise calculation of the probabilities associated with individual peptide assignments, as well as of the false discovery rate among the peptides identified in any experiment. A further mathematical analysis demonstrates that the score distributions are highly dependent on database size and precursor mass window and suggests that the probability associated with SEQUEST scores depends on the number of candidate peptide sequences available for the search. Our results highlight the importance of adjusting the filtering criteria to discriminate between correct and incorrect peptide sequences according to the circumstances of each particular experiment.

Sample preparation oftentimes can be the Achilles Heel of any analytical process, and in the field of proteomics, preparing samples for mass spectrometric analysis is no exception. Current goals, concerning proteomic sample preparation on a large scale, include efforts toward improving reproducibility, reducing the time of processing and ultimately the automation of the entire workflow. This chapter reviews an array of recent approaches applied to bottom-up proteomics sample preparation to reduce the processing time down from hours to minutes. The current state-of-the-art approaches in the field use different energy inputs such as microwave, ultrasound or pressure to perform the four basic steps in sample preparation: protein extraction, denaturation, reduction/alkylation, and digestion. No single energy input for enhancement of proteome sample preparation has become the universal gold standard. Instead, a combination of different energy inputs tends to produce the best results. This chapter further describes the future trends in the field such as the hyphenation of sample preparation with downstream detection and analysis systems. Finally, a detailed protocol describing the combined use of both pressure cycling technology and ultrasonic energy inputs to hasten proteomic sample preparation is presented.

2 tablas, 3 figuras, A new strategy for the fast monitoring of peptide biomarkers is described. It is based on the use of accelerated in-solution trypsin digestions under an ultrasonic field provided by High-Intensity Focused Ultrasound (HIFU) and the monitoring of several peptides by Selected MS/MS Ion Monitoring (SMIM) in a linear ion trap (LIT) mass spectrometer. The performance of the method was established for the unequivocal identification of all commercial fish species belonging to the Merlucciidae family. Using a particular combination of only eleven peptides, resulting from the HIFU-assisted tryptic digestion of the thermostable proteins parvalbumins (PRVBs), the workflow allowed the unequivocal identification of these closely related fish species in any seafood product, including processed and precooked products, in less than 2 h. The present strategy constitutes the fastest method for peptide biomarker monitoring. Its application for food quality control provides to the authorities an effective and rapid method of food authentication and traceability to guarantee the quality and safety to the consumers., This work was supported by the Comisión Interministerial de Ciencia y Tecnología (CICyT) (Project AGL2000-0440-P4-02).

Understanding viral pathogenesis is challenging because of confounding factors, including nonabrasive access to infected tissues and high abundance of inflammatory mediators that may mask mechanistic details. In diseases such as influenza and smallpox where the primary cause of mortality results from complications in the lung, the characterization of lung fluid offers a unique opportunity to study host-pathogen interactions with minimal effect on infected animals. This investigation characterizes the global proteome response in the pulmonary fluid, bronchoalveolar lavage fluid, of macaques during upper respiratory infection by monkeypox virus (MPXV), a close relative of the causative agent of smallpox, variola virus. These results are compared and contrasted against infections by vaccinia virus (VV), a low pathogenic relative of MPXV, and with extracellular fluid from MPXV-infected HeLa cells. To identify changes in the pulmonary protein compartment, macaque lung fluid was sampled twice prior to infection, serving as base line, and up to six times following intrabronchial infection with either MPXV or VV. Increased expression of inflammatory proteins was observed in response to both viruses. Although the increased expression resolved for a subset of proteins, such as C-reactive protein, S100A8, and S100A9, high expression levels persisted for other proteins, including vitamin D-binding protein and fibrinogen γ. Structural and metabolic proteins were substantially decreased in lung fluid exclusively during MPXV and not VV infection. Decreases in structural and metabolic proteins were similarly observed in the extracellular fluid of MPXV-infected HeLa cells. Results from this study suggest that the host inflammatory response may not be the only facilitator of viral pathogenesis, but rather maintaining pulmonary structural integrity could be a key factor influencing disease progression and mortality.

The construction of a trypsin column for rapid and efficient protein digestion in proteomics is described. Electrospun and alcohol-dispersed polymer nanofibers were used for the fabrication of highly stable trypsin coatings, which was prepared by a two-step process of covalent attachment and enzyme crosslinking. In a comparative study with the trypsin coatings on as-spun and non-dispersed nanofibers, it has been observed that a simple step of alcohol dispersion improved not only the enzyme loading but also the performance of protein digestion. In-column digestion of enolase was successfully performed in less than twenty minutes. By applying the alcohol dispersion of polymer nanofibers, the bypass of samples was reduced by filling up the column with well-dispersed nanofibers, and subsequently, interactions between the protein and the trypsin coatings were improved, yielding more complete and reproducible digestions. Regardless of alcohol-dispersion or not, trypsin coatings showed better digestion performance and improved performance stability under recycled uses than covalently-attached trypsin, in-solution digestion, and commercial trypsin beads. The combination of highly stable trypsin coatings and alcohol-dispersion of polymer nanofibers has opened up a new potential to develop a trypsin column for on-line and automated protein digestion.

An efficient protein digestion in proteomic analysis requires the stabilization of proteases such as trypsin. In the present work, trypsin was stabilized in the form of enzyme coating on electrospun polymer nanofibers (EC-TR), which crosslinks additional trypsin molecules onto covalently-attached trypsin (CA-TR). EC-TR showed better stability than CA-TR in rigorous conditions, such as at high temperatures of 40 °C and 50 °C, in the presence of organic co-solvents, and at various pH's. For example, the half-lives of CA-TR and EC-TR were 0.24 and 163.20 hours at 40 oC, respectively. The improved stability of EC-TR can be explained by covalent-linkages on the surface of trypsin molecules, which effectively inhibits the denaturation, autolysis, and leaching of trypsin. The protein digestion was performed at 40 °C by using both CA-TR and EC-TR in digesting a model protein, enolase. EC-TR showed better performance and stability than CA-TR by maintaining good performance of enolase digestion under recycled uses for a period of one week. In the same condition, CA-TR showed poor performance from the beginning, and could not be used for digestion at all after a few usages. The enzyme coating approach is anticipated to be successfully employed not only for protein digestion in proteomic more » analysis, but also for various other fields where the poor enzyme stability presently hampers the practical applications of enzymes. « less

A new method that uses immobilized trypsin concomitant with ultrasonic irradiation results in ultra-rapid digestion and thorough 18O labeling for quantitative protein comparisons. The reproducible and highly efficient method provided effective digestions in

Understanding the molecular mechanisms that modulate macrophage radioresistance is necessary for the development of effective radiation therapies, as tumor-associated macrophages promote both angiogenesis and matrix remodeling that, in turn, enhance tumor metastasis. In this respect, we have identified a dose-dependent increase in the abundance (i.e., expression level) of the calcium regulatory protein calmodulin (CaM) in RAW 264.7 macrophages upon irradiation. At low doses of irradiation there are minimal changes in the abundance of other cellular proteins detected using mass spectrometry, indicating that increases in CaM levels are part of a specific radiation-dependent cellular response. CaM overexpression results in increased macrophage survival following radiation exposure, acting to diminish the sensitivity to low-dose radiation exposures. Following macrophage irradiation, increases in CaM abundance also result in an increase in the number of phosphorylated histone H2AX foci, associated with DNA repair, with no change in the extent of double-stranded DNA damage. In comparison, when nuclear factor kappaB (NFkappaB)-dependent pathways are inhibited, through the expression of a dominant-negative IkappaB construct, there is no significant increase in phosphorylated histone H2AX foci upon irradiation. These results indicate that the molecular basis for the up-regulation of histone H2AX-mediated DNA repair pathways is not the result of nonspecific NFkappaB-dependent pathways or a specific threshold of DNA damage. Rather, increases in CaM abundance act to minimize the low-dose hypersensitivity to radiation by enhancing macrophage radioresistance through processes that include the up-regulation of DNA repair pathways involving histone H2AX phosphorylation.

A new method for rapid proteolytic digestion of proteins under high pressure that uses pressure cycling technology in the range of 5 to 35 kpsi was demonstrated for proteomic analysis. Successful in-solution digestions of single proteins and complex protein mixtures were achieved in 60 s and then analyzed by reversed phase liquid chromatography-electrospray ionization ion trap-mass spectrometry. Method performance in terms of the number of Shewanella oneidensis peptides and proteins identified in a shotgun approach was evaluated relative to a traditional “overnight” sample preparation method. Advantages of the new method include greatly simplified sample processing, easy implementation, no cross contamination among samples, and cost effectiveness.

13 pages, 4 figures.-- PMID: 17960563 [PubMed].-- Printed version published Nov 2007.-- Inmaculada Jorge ... et al., Mass spectrometry (MS) is a technique of paramount importance in Proteomics, and developments in this field have been possible owing to novel MS instrumentation, experimental strategies, and bioinformatics tools. Today it is possible to identify and determine relative expression levels of thousands of proteins in a biological system by MS analysis of peptides produced by proteolytic digestion. In some situations, however, the precise characterization of a particular peptide species in a very complex peptide mixture is needed. While single-fragment ion-based scanning modes such as selected ion reaction monitoring (SIRM) or consecutive reaction monitoring (CRM) may be highly sensitive, they do not produce MS/MS information and their actual specificity must be determined in advance, a prerequisite that is not usually met in a basic research context. In such cases, the MS detector may be programmed to perform continuous MS/MS spectra on the peptide ion of interest in order to obtain structural information. This selected MS/MS ion monitoring (SMIM) mode has a number of advantages that are fully exploited by MS detectors that, like the linear ion trap, are characterized by high scanning speeds. In this work, we show some applications of this technique in the context of biological studies. These results were obtained by selecting an appropriate combination of scans according to the purpose of each one of these research scenarios. They include highly specific identification of proteins present in low amounts, characterization and relative quantification of post-translational modifications such as phosphorylation and S-nitrosylation and species-specific peptide identification., This work was supported by grants BIO2003-01805, BIO2006-10085, GR/SAL/0141/2004 (CAM), CAM BIO/0194/2006, the Fondo de Investigaciones Sanitarias (Ministerio de Sanidad y Consumo, Instituto Salud Carlos III, RECAVA), and by an institutional grant by Fundación Ramón Areces to CBMSO.

In the late 1980s, the advent of soft ionization techniques capable of generating stable gas phase ions from thermally unstable biomolecules, namely matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI), laid the way for the development of a set of powerful alternatives to the traditional Edman chemistry for the structural characterization of peptides and proteins. The rapid protein identification capabilities that, coupled with two-dimensional gel electrophoresis, provided insights into all sorts of biological systems since the dawn of proteomics and have been exploited in the last few years for the development of more powerful and automatable gel-free strategies, mainly based on multidimensional chromatographic separations of peptides from proteolytic digests. In parallel to the evolution of ion sources, mass analysers and scan modes, the invention of new elegant biochemical strategies to fractionate or simplify highly complex mixtures, or to introduce isotopic labels in peptides in a variety of ways now makes also possible large-scale, high-coverage quantitative studies in a wide dynamic range. In this review, we provide the fundamental concepts of mass spectrometry (MS) and describe the technological progress of MS-based proteomics since its earliest days. Representative literature examples of their true power, either when employed as exploratory or as targeted techniques, is provided as well.

24 páginas, 2 figuras, 3 tablas, Sample preparation is a fundamental step in the proteomics workflow. However, it is not easy to find compiled information updating this subject. In this paper, the strategies and protocols for protein extraction and identification, following either classical or second generation proteomics methodologies, are reviewed. Procedures for: tissue disruption, cell lysis, sample pre-fractionation, protein separation by 2-DE, protein digestion, mass spectrometry analysis, multidimensional peptide separations and quantification of protein expression level are described.