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The dynamics of the collision-induced dissociation of CS2+ molecular ions to CS and S+ fragment ions has been investigated using a crossed-beam tandem hybrid mass spectrometer at collision energies ranging from near threshold to 34.5 eV in the center-of-mass (CM) reference frame. The results show that small impact parameter impulsive collisions are involved in the collisional activation process, resulting in extensive scattering of fragment ions with intensity maxima at non-zero angles. The dissociation of CS2+ to S+ proceeds via a mechanism mainly involving the transfer of the minimum amount of energy required to dissociate the parent ion into S+ (4S) and CS (X1 Σ+) at all collision energies. However, the mechanism for the formation of CS+ depends strongly on the collision energy. Energy transfer is very efficient for this process and a large fraction of the CM collision energy is converted to internal excitation of the colliding CS2+ ions up to a maximum of nearly 16 eV (at 34.5 eV collision energy). At the lowest collision energy we have investigated, 6.5 eV, the lowest energy reaction path to form CS+ (X2 Σ+) and S (3P) is followed exclusively. However, as the collision energy is increased, several additional dissociation channels contribute towards the total dissociation. These results are rationalized by the efficient electronic excitation of the colliding CS2+ ions at these collision energies.

Liquid chromatography coupled with mass spectrometry (LC-MS) is widely used to identify and quantify peptides in complex biological samples. In particular, label-free shotgun proteomics is highly effective for the identification of peptides and subsequently obtaining a global protein profile of a sample. As a result, this approach is widely used for discovery studies. Typically, the objective of these discovery studies is to identify proteins that are affected by some condition of interest (e.g., disease, exposure). However, for complex biological samples, label-free LC-MS proteomics experiments measure peptides and do not directly yield protein quantities. Thus, protein quantification must be inferred from one or more measured peptides. In recent years many computational approaches to compute relative protein quantification of label-free LC-MS data have been published. In this review, we examine the most commonly employed quantification approaches to compute relative protein abundance from peak intensity values, evaluate their individual merits, and discuss challenges in the use of the various computational approaches.

The periplasm of Gram-negative bacteria is a dynamic and physiologically important subcellular compartment where the constant exposure to potential environmental insults amplifies the need for proper protein folding and modifications. Top-down proteomics analysis of the periplasmic fraction at the intact protein level provides unrestricted characterization and annotation of the periplasmic proteome, including the post-translational modifications (PTMs) on these proteins. Here, we used single-dimension ultra-high pressure liquid chromatography coupled with the Fourier transform mass spectrometry (FTMS) to investigate the intact periplasmic proteome of Novosphingobium aromaticivorans. Our top-down analysis provided the confident identification of 55 proteins in the periplasm and characterized their PTMs including signal peptide removal, N-terminal methionine excision, acetylation, glutathionylation, pyroglutamate, and disulfide bond formation. This study provides the first experimental evidence for the expression and periplasmic localization of many hypothetical and uncharacterized proteins and the first unrestrictive, large-scale data on PTMs in the bacterial periplasm.

During acute Lyme disease, bacteria can disseminate to the central nervous system (CNS), leading to the development of meningitis and other neurologic symptoms. Here we have analyzed pooled cerebrospinal fluid (CSF) allowing a deep view into the proteome for patients diagnosed with early disseminated Lyme disease and CSF inflammation. Additionally, we analyzed individual patient samples and quantified differences in protein abundance employing label-free quantitative mass spectrometry-based methods. We identified 108 proteins that differ significantly in abundance in patients with acute Lyme disease from controls. Comparison between infected patients and control subjects revealed differences in proteins in the CSF associated with cell death localized to brain synapses and others that likely originate from brain parenchyma.

Environmental adaptability is critical for survival of the fungal human pathogen Aspergillus fumigatus in the immunocompromised host lung. We hypothesized that exposure of the fungal pathogen to human serum would lead to significant alterations to the organism's physiology, including metabolic activity and stress response. Shifts in functional pathway and corresponding enzyme reactivity of A. fumigatus upon exposure to the human host may represent much needed prognostic indicators of fungal infection. To address this, we employed a multiplexed activity-based protein profiling (ABPP) approach coupled to quantitative mass spectrometry-based proteomics to measure broad enzyme reactivity of the fungus cultured with and without human serum. ABPP showed a shift from aerobic respiration to ethanol fermentation and utilization over time in the presence of human serum, which was not observed in serum-free culture. Our approach provides direct insight into this pathogen's ability to survive, adapt, and proliferate. Additionally, our multiplexed ABPP approach captured a broad swath of enzyme reactivity and functional pathways and provides a method for rapid assessment of the A. fumigatus response to external stimuli.

Morphine has long been known to have immunosuppressive properties in vivo, but the molecular and immunologic changes induced by it are incompletely understood. To explore how these changes interact with lentiviral infections in vivo, animals from two nonhuman primate species (African green monkeys and pigtailed macaques) were provided morphine and studied using a systems biology approach. Biological specimens were obtained from multiple sources (e.g. lymph node, colon, cerebrospinal fluid, and peripheral blood) before and after the administration of morphine (titrated up to a maximum dose of 5 mg/kg over a period of 20 days). Cellular immune, plasma cytokine, and proteome changes were measured and morphine-induced changes in these parameters were assessed on an interorgan, interindividual, and interspecies basis. In both species, morphine was associated with decreased levels of Ki-67(+) T-cell activation but with only minimal changes in overall T-cell counts, neutrophil counts, and NK cell counts. Although changes in T-cell maturation were observed, these varied across the various tissue/fluid compartments studied. Proteomic analysis revealed a morphine-induced suppressive effect in lymph nodes, with decreased abundance of protein mediators involved in the functional categories of energy metabolism, signaling, and maintenance of cell structure. These findings have direct relevance for understanding the impact of heroin addiction and the opioids used to treat addiction as well as on the potential interplay between opioid abuse and the immunological response to an infective agent.

Sodium dodecyl sulfate (SDS) is one of the most popular laboratory reagents used for biological sample extraction; however, the presence of this reagent in samples challenges LC-MS-based proteomics analyses because it can interfere with reversed-phase LC separations and electrospray ionization. This study reports a simple SDS-assisted proteomics sample preparation method facilitated by a novel peptide-level SDS removal step. In an initial demonstration, SDS was effectively (99.9%) removed from peptide samples through ion substitution-mediated DS(-) precipitation using potassium chloride (KCl), and excellent peptide recovery (95%) was observed for20 μg of peptides. Further experiments demonstrated the compatibility of this protocol with LC-MS/MS analyses. The resulting proteome coverage obtained for both mammalian tissues and bacterial samples was comparable to or better than that obtained for the same sample types prepared using standard proteomics preparation methods and analyzed using LC-MS/MS. These results suggest the SDS-assisted protocol is a practical, simple, and broadly applicable proteomics sample processing method, which can be particularly useful when dealing with samples difficult to solubilize by other methods.

A hemispherical energy analyzer was constructed by using a novel approach to control the fringing electrostatic field. It provides several properties useful in ion spectrometers: namely, rather simple fabrication and compact size, high transmission efficiency at moderate resolution, and the capability to adjust resolution by changing the intersphere potentials. A computer program was developed to evaluate ion trajectories through the hemispherical analyzer. Data obtained from the trajectories were used to predict the characteristics of the analyzer. Experiments performed to determine the kinetic energy dependence of the absolute transmission and the resolution functions are in accord with theoretical calculations.

A crossed-beam tandem hybrid mass spectrometer equipped with a supersonic neutral beam source was used to investigate collision-induced dissociation (CID) of the propane molecular ion at a series of relative collision energies from 1.7–449 eV. The competing processes of elimination of methane and loss of methyl radical were studied using beams of helium and argon neutrals. The dynamics of both dissociation processes change relatively little with increasing collision energy, demonstrating that the CID mechanism does not change with collision energy over a very broad range. The most probable energy transfer into the ion increased only slightly with collision energy. The dynamics are consistent with previous conclusions that internal energy is randomized amongst internl degrees of freedom before dissociation. The intensity maxima at all energies occurred at non-zero scattering angles in the center-of-mass reference frame; scattering angles decrease with increasing collision energy but never decreased to zero. This implies that the CID mechanism for propane ion is dominated by small impact parameter collisions with large angular momentum exchange at all energies.

Collisional activation and dissociation of low-energy acetone ions have been studied using a crossed-beam tandem mass spectrometer. A small but significant fraction of acetone ions from 70 eV electron ionization are formed in long-lived excited A state. Ground state acetone ions are also collisionally excited to the A state via conversion of translational into internal energy. Two populations of collisionally excited ions are formed, one which is stable on the time scale of our experiments and one which dissociates into an acetyl ion and a methyl radical. These microscopically related reaction pathways demonstrate the unexpectedly facile interconversion of translational and electronic energy in low-energy collisions.

Two crossed‐beam instruments with different geometry and with low pressure and differentially pumped ion sources were used to investigate collision‐induced dissociation (CID) of acetone molecular ions activated by He and Ar collisions over the kinetic energy range 0.45–10 eV (center of mass). Long‐lived electronically excited state(s) which back‐scatter superelastically with conversion of 2.2±0.3 eV of internal energy into translational energy dominate the low collision energy (Erel

Dissociative scattering of CF3+ ions in collision with a self-assembled monolayer surface of fluorinated alkyl thiol on a gold 111 crystal has been studied at low ion kinetic energies (from 29 to 159 eV) using a custom built tandem mass spectrometer with a rotatable second stage energy analyzer and mass spectrometer detectors. Energy and intensity distributions of the scattered fragment ions were measured as a function of the fragment ion mass and scattering angle. Inelastically scattered CF3+ ions were not observed even at the lowest energy studied here. All fragment ions, CF2+, CF+, F+, and C+, were observed at all energies studied with the relative intensity of the highest energy pathway, C+, increasing and that of the lowest energy pathway, CF2+, decreasing with collision energy. Also, the dissociation efficiency of CF3+ decreased significantly as the collision energy was increased to 159 eV. Energy distributions of all fragment ions from the alkyl thiol surface showed two distinct components, one corresponding to the loss of nearly all of the kinetic energy and scattered over a broad angular range while the other corresponding to smaller kinetic energy losses and scattered closer to the surface parallel. The latter process is due to delayed dissociation of collisionally excited ions after they have passed the collision region as excited parent ions. A similar study performed at 74 eV using a LiF coated surface on a titanium substrate resulted only in one process for all fragment ions; corresponding to the delayed dissociation process. The intensity maxima for these fragmentation processes were shifted farther away from the surface parallel compared to the thiol surface. A new mechanism is proposed for the delayed dissociation process as proceeding via projectile ions' neutralization to long-lived highly excited Rydberg state(s), reionization by the potential field between the collision region and entrance to the energy analyzer, and subsequent dissociation several microseconds after collisional excitation. A kinematic analysis of experimental data plotted as velocity Newton diagrams demonstrates that the delayed dissociation process results from the collisions of the ion with the bulk surface; i.e., the self-assembled monolayer surface acts as a bulk surface. A similar analysis for the highly inelastic collision processes shows that these are due to stronger collisions with a fraction of the thiol molecular chain, varying in length (mass) with the ion energy.

A 22-year-old primiparous woman was referred for a routine antenatal fetal scan. During the scan multiple intracardiac echogenic tumours were detected at 24 weeks gestation. A smaller mass was visible in the left ventricle while a larger one was seen in the right atrium (figure 1). Figure 1 Antenatal ultrasonographic transverse view at the level of the cardia showing echogenic foci within the right atrium (RA) and the left ventricle (LV). Subsequent antenatal MRI showed considerably enlarged well-defined masses isointense to the adjoining myocardium …

Small cationic and anionic clusters of lithium formate were generated by electrospray ionization and their fragmentations were studied by tandem mass spectrometry (collision-induced dissociation with N2). Singly as well as multiply charged clusters were formed in both positive and negative ion modes with the general formulae, (HCOOLi)nLi(+), (HCOOLi)nLim (m+), (HCOOLi)nHCOO(-), and (HCOOLi)n(HCOO)m (m-). Several magic number cluster (MNC) ions were observed in both the positive and negative ion modes although more predominant in the positive ion mode with (HCOOLi)3Li(+) being the most abundant and stable cluster ion. Fragmentations of singly charged positive clusters proceed first by the loss of a dimer unit ((HCOOLi)2) followed by the loss of monomer units (HCOOLi) although the former remains the dominant dissociation process. In the case of positive cluster ions, all fragmentations lead to the magic cluster (HCOOLi)3Li(+) as the most abundant fragment ion at higher collision energies which then fragments further to dimer and monomer ions at lower abundances. In the negative ion mode, however, singly charged clusters dissociated via sequential loss of monomer units. Multiply charged clusters in both positive and negative ion modes dissociated mainly via Coulomb repulsion. Quantum chemical calculations performed for smaller cluster ions showed that the trimer ion has a closed ring structure similar to the phenalenylium structure with three closed rings connected to the central lithium ion. Further additions of monomer units result in similar symmetric structures for hexamer and nonamer cluster ions. Thermochemical calculations show that trimer cluster ion is relatively more stable than neighboring cluster ions, supporting the experimental observation of a magic number cluster with enhanced stability.

A new Fourier transform ion cyclotron resonance mass spectrometer (FTICR MS) has been constructed in our laboratory. The instrument employs surface-induced dissociation (SID) as an activation method for obtaining structural information on biomolecules in the gas phase. Tandem SID mass spectra can be acquired using either a continuous or a pulsed mode of operation. Collision energy of precursor ion is controlled by a dc offset of the ICR cell. This approach eliminates defocusing of the ion beam by the ion-transfer optics as a function of ion kinetic energy and constitutes a significant improvement over our previous experimental setup. Furthermore, it can be easily implemented on any FTICR mass spectrometer. Very high signal-to-noise ratios of 200-500 were obtained in single-scan SID mass spectra of model peptides with acquisition time less than 1.1 s. Reasonable SID signal was detected in single-scan spectra with total acquisition time of only 0.3 s. The high signal-to-noise ratio and the fast acquisition time point on a potential application of SID for high-throughput studies in FTICR MS.

Collision-induced dissociation of nitromethane molecular ions to NO+ and NO has been studied at 19 eV center-of-mass collision energy using a crossed-beam tandem mass spectrometer. Center-of-mass velocity contour maps for the two dissociation processes are distinctly different; NO has its intensity maximum at 7 degrees with energy transfer endothermicities of 1.8 eV while NO+ has intensity maxima at zero and 8 degrees with energy transfer endothermicities of 0.3 and 5.5 eV, respectively. The observation of two distinct peaks in the NO+ contour map clearly demonstrates that nitromethane ions excited to 5.5 eV internal energy do not dissociate from the ground state potential surface. Dissociation is faster than intramolecular relaxation, in contradiction to the fundamental postulate of statistical theories (RRKM, QET) for unimolecular dissociation.

Aims: To assess the reliability of ultrasound imaging coupled with Color Power Doppler for monitoring the healing after nonsurgical endodontic therapy. Materials and Methods: Ten asymptomatic maxillary anterior teeth with approximately similar-sized periapical radiolucent lesions of average dimension 1.3 - 1.9 cm, as observed on an intraoral periapical radiograph, and confirmed by ultrasound was selected for the study. After confirming the diagnosis all the teeth were subjected to nonsurgical endodontic treatment. Postoperative healing was monitored using postoperative subjective feedback, a radiograph, and an ultrasound with a color power doppler, at intervals of six weeks, three months, and six months. Results: Eight among the ten cases studied showed signs of healing, with an ultrasound, as early as six weeks postoperatively, but the radiographs showed a noticeable change only at the end of three months. At the end of the follow-up period of three months and six months, the findings in terms of change in the lesion size were the same with both the radiograph and ultrasound. Ultrasonography was able to predict the healing very consistently at all recall periods from six weeks onward providing vital information such as changes in vascularity and bone formation as compared to radiographs. Conclusions: Ultrasound with Color Power Doppler is an efficient tool for monitoring bone healing as compared to the conventionally employed radiographic method.

Collision-induced dissociation (CID) of CS2 + molecular ions to S2 + ions at laboratory collision energies ranging from 1 to 6 keV energy has been studied by mass analyzed ion kinetic energy spectrometry (MIKES) utilizing a reversed geometry tandem mass spectrometer. The dissociation proceeds via four energetically distinct pathways which are strongly dependent upon the kinetic energy of the ion and on the nature of the collision gas. CID with argon neutrals is dominated at all energies by the lowest energy threshold reaction, which is endothermic by about 6 eV, whereas the energetics of the process differ and are strongly dependent on the ion kinetic energy when lighter collision gases (He, H2 and D2) are used for collisional activation. The most probable energy transfer from kinetic to internal modes is 11 ± 2 eV for these collision gases. A highly endothermic channel with kinetic energy loss of ~41 ± 4 eV opens up when the energy of the ions is reduced to 3 keV and less. At 1 keV ion energy, there is a small contribution to the total CID from a highly exothermic channel (kinetic energy release of 47 ± 5 eV) that is observed with all collision gases under low pressure conditions in the collision cell.

A hybrid tandem mass spectrometer has been constructed to study the dynamics of collision-induced dissociation processes in the energy range of less than 1 eV to several keV. A mass- and energy-analyzed high-energy ion beam is decelerated to low energies by a series of cylindrical and rectangular tube lenses. The decelerated ion beam collides with a supersonic neutral beam at right angles. Energy and mass analysis of the fragment ions is performed by a novel hemispherical energy analyzer followed by a quadrupole mass filter. The detector system can be rotated about the collision center to provide angular analysis of scattered fragment ions. Ion beams of moderate intensity have been obtained in the entire energy range, it is especially significant that good intensity is obtained in the lower threshold energy range of 0.2–5 eV. The performance of the instrument has been evaluated by an extensive series of ion transmission, focus, and energy measurements and by comparison of CID results with known mstastable and collision-induced dissociation processes.

A crossed-beam study of the collision-induced dissociation of CH 4 + by Ar was carried out at a center-of-mass (c.m.) collision energy of 5.5 eV. The scattering shows three patterns for the formation of CH 2 + , (1) large-angle scattering at preferred impact parameters with little internal excitation of the products, (2) scattering near the c.m. with nearly all collision energy transferred into products internal energy and (3) superelastic scattering, i.e. conversion of internal energy to translational energy, implying the reaction is initiated by a long-lived excited state of CH 4 + generated by electron impact ionization of methane. No previous evidence exists, to our knowledge, that excited states of CH 4 + thus generated may have microsecond lifetimes.

Investigations of the non-reactive scattering of Ar+ by N2 using a crossed-beam apparatus reveal that the two spin-orbit states of Ar+ are back-scattered into different regions of velocity space (center-of-mass reference frame); this fact permits the ratio of these two states in the primary ion beam to be monitored as a function of ion source conditions. Adjustments of source pressure and ionizing current density are the key parameters which control the extent of quenching of the Ar+ (2P 1 2 ) initially formed by electron impact on Ar. Superelastic scattering of electrons is suggested as a probable mechanism and a rate coefficient, k ≥ 8 × 10−11 cm3 s−1, is estimated for the reaction Ar+2P 1 2 ) + e− → Ar+(2P 3 2 ) + e− + 0.18 eV

The collision-induced dissociation (CID) reaction CH3COCH+3 + Ar → CH3CO+ + CH3 + Ar has been studied with a tandem hybrid mass spectrometer (a high-resolution mass spectrometer coupled with a supersonic neutral beam, which uses post-collision energy and mass analysis) at ion laboratory energies in the range 4.5–300 eV. Kinetic energy and angular distributions of the fragment CH3CO+ ions were measured and scattering contour diagrams for this process were constructed. The results show that the dissociation proceeds via low-impact parameter collisions with extensive momentum transfer. At 4.5 eV, the product is primarily backward scattered with some intensity at the center-of-mass (completely inelastic collision). At higher energies the product ion is progressively more forward scattered but over the energy range investigated the maximum intensity never shifts to 0°. Collectively, these results suggest that impulsive mechanisms dominate CID reactions at all energy ranges for this ion and that the dominant mechanism is kinematically different at low and high energy.

The loss of methyl radical in collision-activated dissociation (CAD) of acetone and propane molecular ions has been studied at low energy using a tandem hybrid mass spectrometer. Although the two processes are very similar chemically and energetically, very different dynamical features are observed. Acetyl ions from acetone ion are predominantly backward-scattered, with intensity maxima lying inside and outside the elastic scattering circle, confirming our previous observation that electronically excited states are important in low-energy acetone CAD. Ethyl ions from propane ion show a forward-scattered peak maximum at a nonzero scattering angle, which is consistent with generally accepted models for vibrational excitation and redistribution of energy before dissociation. Both processes demonstrate that CAD at low energy proceeds via small-impact-parameter collisions with momentum transfer. Comparison of the present results with higher energy CAD dynamics studies and earlier work leads to some tentative general conclusions about energy tranfer in these processes.

Systems biology offers considerable promise in uncovering novel pathways by which viruses and other microbial pathogens interact with host signaling and expression networks to mediate disease severity. In this study, we have developed an unbiased modeling approach to identify new pathways and network connections mediating acute lung injury, using severe acute respiratory syndrome coronavirus (SARS-CoV) as a model pathogen. We utilized a time course of matched virologic, pathological, and transcriptomic data within a novel methodological framework that can detect pathway enrichment among key highly connected network genes. This unbiased approach produced a high-priority list of 4 genes in one pathway out of over 3,500 genes that were differentially expressed following SARS-CoV infection. With these data, we predicted that the urokinase and other wound repair pathways would regulate lethal versus sublethal disease following SARS-CoV infection in mice. We validated the importance of the urokinase pathway for SARS-CoV disease severity using genetically defined knockout mice, proteomic correlates of pathway activation, and pathological disease severity. The results of these studies demonstrate that a fine balance exists between host coagulation and fibrinolysin pathways regulating pathological disease outcomes, including diffuse alveolar damage and acute lung injury, following infection with highly pathogenic respiratory viruses, such as SARS-CoV.IMPORTANCESevere acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2002 and 2003, and infected patients developed an atypical pneumonia, acute lung injury (ALI), and acute respiratory distress syndrome (ARDS) leading to pulmonary fibrosis and death. We identified sets of differentially expressed genes that contribute to ALI and ARDS using lethal and sublethal SARS-CoV infection models. Mathematical prioritization of our gene sets identified the urokinase and extracellular matrix remodeling pathways as the most enriched pathways. By infecting Serpine1-knockout mice, we showed that the urokinase pathway had a significant effect on both lung pathology and overall SARS-CoV pathogenesis. These results demonstrate the effective use of unbiased modeling techniques for identification of high-priority host targets that regulate disease outcomes. Similar transcriptional signatures were noted in 1918 and 2009 H1N1 influenza virus-infected mice, suggesting a common, potentially treatable mechanism in development of virus-induced ALI.

The broad range and diversity of interferon-stimulated genes (ISGs) function to induce an antiviral state within the host, impeding viral pathogenesis. While successful respiratory viruses overcome individual ISG effectors, analysis of the global ISG response and subsequent viral antagonism has yet to be examined. Employing models of the human airway, transcriptomics and proteomics datasets were used to compare ISG response patterns following highly pathogenic H5N1 avian influenza (HPAI) A virus, 2009 pandemic H1N1, severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome CoV (MERS-CoV) infection. The results illustrated distinct approaches utilized by each virus to antagonize the global ISG response. In addition, the data revealed that highly virulent HPAI virus and MERS-CoV induce repressive histone modifications, which downregulate expression of ISG subsets. Notably, influenza A virus NS1 appears to play a central role in this histone-mediated downregulation in highly pathogenic influenza strains. Together, the work demonstrates the existence of unique and common viral strategies for controlling the global ISG response and provides a novel avenue for viral antagonism via altered histone modifications.IMPORTANCEThis work combines systems biology and experimental validation to identify and confirm strategies used by viruses to control the immune response. Using a novel screening approach, specific comparison between highly pathogenic influenza viruses and coronaviruses revealed similarities and differences in strategies to control the interferon and innate immune response. These findings were subsequently confirmed and explored, revealing both a common pathway of antagonism via type I interferon (IFN) delay as well as a novel avenue for control by altered histone modification. Together, the data highlight how comparative systems biology analysis can be combined with experimental validation to derive novel insights into viral pathogenesis.

In systems biology studies, the integration of multiple omics measurements (i.e., genomics, transcriptomics, proteomics, metabolomics, and lipidomics) has been shown to provide a more complete and informative view of biological pathways. Thus, the prospect of extracting different types of molecules (e.g., DNAs, RNAs, proteins, and metabolites) and performing multiple omics measurements on single samples is very attractive, but such studies are challenging due to the fact that the extraction conditions differ according to the molecule type. Here, we adapted an organic solvent-based extraction method that demonstrated broad applicability and robustness, which enabled comprehensive proteomics, metabolomics, and lipidomics analyses from the same sample., Integrative multi-omics analyses can empower more effective investigation and complete understanding of complex biological systems. Despite recent advances in a range of omics analyses, multi-omic measurements of the same sample are still challenging and current methods have not been well evaluated in terms of reproducibility and broad applicability. Here we adapted a solvent-based method, widely applied for extracting lipids and metabolites, to add proteomics to mass spectrometry-based multi-omics measurements. The metabolite, protein, and lipid extraction (MPLEx) protocol proved to be robust and applicable to a diverse set of sample types, including cell cultures, microbial communities, and tissues. To illustrate the utility of this protocol, an integrative multi-omics analysis was performed using a lung epithelial cell line infected with Middle East respiratory syndrome coronavirus, which showed the impact of this virus on the host glycolytic pathway and also suggested a role for lipids during infection. The MPLEx method is a simple, fast, and robust protocol that can be applied for integrative multi-omic measurements from diverse sample types (e.g., environmental, in vitro, and clinical). IMPORTANCE In systems biology studies, the integration of multiple omics measurements (i.e., genomics, transcriptomics, proteomics, metabolomics, and lipidomics) has been shown to provide a more complete and informative view of biological pathways. Thus, the prospect of extracting different types of molecules (e.g., DNAs, RNAs, proteins, and metabolites) and performing multiple omics measurements on single samples is very attractive, but such studies are challenging due to the fact that the extraction conditions differ according to the molecule type. Here, we adapted an organic solvent-based extraction method that demonstrated broad applicability and robustness, which enabled comprehensive proteomics, metabolomics, and lipidomics analyses from the same sample. Author Video: An author video summary of this article is available.

Background Influenza infection causes respiratory disease that can lead to death. The complex interplay between virus-encoded and host-specific pathogenicity regulators – and the relative contributions of each toward viral pathogenicity – is not well-understood. Results By analyzing a collection of lung samples from mice infected by A/Vietnam/1203/2004 (H5N1; VN1203), we characterized a signature of transcripts and proteins associated with the kinetics of the host response. Using a new geometrical representation method and two criteria, we show that inoculation concentrations and four specific mutations in VN1203 mainly impact the magnitude and velocity of the host response kinetics, rather than specific sets of up- and down- regulated genes. We observed analogous kinetic effects using lung samples from mice infected with A/California/04/2009 (H1N1), and we show that these effects correlate with morbidity and viral titer. Conclusions We have demonstrated the importance of the kinetics of the host response to H5N1 pathogenesis and its relationship with clinical disease severity and virus replication. These kinetic properties imply that time-matched comparisons of ‘omics profiles to viral infections give limited views to differentiate host-responses. Moreover, these results demonstrate that a fast activation of the host-response at the earliest time points post-infection is critical for protective mechanisms against fast replicating viruses.