Interleukin-13 Treatment of Living Lung Tissue Model Alters the Metabolome and Proteome-A Nano-DESI MS Metabolomics and Shotgun Proteomics Study.

Asthma is a chronic respiratory disease with one of the largest numbers of cases in the world; thus, constant investigation and technical development are needed to unravel the underlying biochemical mechanisms. In this study, we aimed to develop a nano-DESI MS method for the in vivo characterization of the cellular metabolome. Using air-liquid interface (ALI) cell layers, we studied the role of Interleukin-13 (IL-13) on differentiated lung epithelial cells acting as a lung tissue model. We demonstrate the feasibility of nano-DESI MS for the in vivo monitoring of basal-apical molecular transport, and the subsequent endogenous metabolic response, for the first time. Conserving the integrity of the ALI lung-cell layer enabled us to perform temporally resolved metabolomic characterization followed by "bottom-up" proteomics on the same population of cells. Metabolic remodeling was observed upon histamine and corticosteroid treatment of the IL-13-exposed lung cell monolayers, in correlation with alterations in the proteomic profile. This proof of principle study demonstrates the utility of in vivo nano-DESI MS for characterizing ALI tissue layers, and the new markers identified in our study provide a good starting point for future, larger-scale studies.

Potential Use of Supercharging Agents for Improved Mass Spectrometric Analysis of Monoclonal Antibodies and Antibody-Drug Conjugates.

The addition of supercharging (SC) reagents in electrospray ionization coupled mass spectrometry (ESI-MS) has demonstrated several advantages for protein analysis such as reduced required mass range of the instrument, narrowed charge-state distribution, increased sensitivity, and adduct suppression. The potential use of SC reagents to improve analyses of larger and complex protein molecules such as monoclonal antibodies and antibody-drug conjugates (ADCs) has not been previously reported. In this study, the effect of seven SC reagents (meta-nitrobenzyl alcohol (m-NBA), dimethyl sulfoxide (DMSO), ortho-nitroanisole (o-NA), propane sultone (PS), ethylene carbonate (EC), propylene carbonate (PC), and sulfolane) on ESI-MS acquired spectra of deglycosylated, intact, and reduced trastuzumab and a vcMMAE-trastuzumab ADC was investigated under denaturing conditions. The addition of any of the SC reagents resulted in a higher average charge state observed for all tested reagents for both trastuzumab and the ADC and a narrower charge-state envelope for o-NA and 1% sulfolane for trastuzumab. However, improved peak shapes or increased sensitivity was observed for several reagents, overall increasing the spectra quality. Finally, it was shown that SC reagents can be safely used for ADC analysis without impacting the obtained drug-to-antibody (DAR) values, as all DAR values were within 0.1 from the control sample.

Plasma-derived extracellular vesicles from myocardial infarction patients inhibits tumor necrosis factor-alpha induced cardiac cell death.

RATIONALE: Extracellular vesicles (EVs) derived exogenously from pluripotent stem cells or endogenously from healthy human serum exert cardioprotective effects after injury. However role of endogenous EVs from myocardial infarction (MI) patients not well understood in this settings. METHODS AND RESULTS: The EVs from plasma of MI patients with preserved or reduced left ventricular ejection fraction (LVEF) and healthy controls (HC) were purified and characterized by flow cytometry, mass spectrometry (MS) and transmission electron microscopy (TEM). HCM and human cardiac microvascular endothelial cells (hCMVECs), under individual culture or co-culture, were used to study functional effects of EVs upon TNFα stimulation. These effects of EVs on HCM and hCMVECs were observed using cell death assays, western blots and confocal microscopy. Higher concentrations of platelet-, leukocyte-, endothelial- and erythrocyte-derived EVs were found in MI patients, both with preserved and reduced LVEF, compared to HC, and MS data on MI EVs proteome displayed alteration in several proteins. MI EVs protected HCM and hCMVECs against staurosporine-induced apoptosis. Furthermore, MI EVs were observed to abrogate TNFα-triggered HCM and hCMVECs death under both individually cultured and co-cultured conditions. MI EVs failed to inhibit TNFα induced hCMVECs and HCM activation when cultured individually, however co-cultured hCMVECs with HCM supported MI EVs capacity to attenuate TNFα induced cells activation. MI CD41+ EVs but not HC EVs were found to be internalized by HCM directly or migrated through hCMVECs to HCM. MI EVs indirectly restores TNFα mediated drop in mitochondrial membrane potential. CONCLUSIONS: Endogenous EVs from MI patients, regardless of severity of the MI exert cardioprotective potential upon TNFα-induced cell death. Patient-derived EVs needs to be further explored to elucidate their potential cardioprotective role during MI.

The Subcellular Proteome of a Planctomycetes Bacterium Shows That Newly Evolved Proteins Have Distinct Fractionation Patterns.

The Planctomycetes bacteria have unique cell architectures with heavily invaginated membranes as confirmed by three-dimensional models reconstructed from FIB-SEM images of Tuwongella immobilis and Gemmata obscuriglobus. The subcellular proteome of T. immobilis was examined by differential solubilization followed by LC-MS/MS analysis, which identified 1569 proteins in total. The Tris-soluble fraction contained mostly cytoplasmic proteins, while inner and outer membrane proteins were found in the Triton X-100 and SDS-soluble fractions, respectively. For comparisons, the subcellular proteome of Escherichia coli was also examined using the same methodology. A notable difference in the overall fractionation pattern of the two species was a fivefold higher number of predicted cytoplasmic proteins in the SDS-soluble fraction in T. immobilis. One category of such proteins is represented by innovations in the Planctomycetes lineage, including unique sets of serine/threonine kinases and extracytoplasmic sigma factors with WD40 repeat domains for which no homologs are present in E. coli. Other such proteins are members of recently expanded protein families in which the newly evolved paralog with a new domain structure is recovered from the SDS-soluble fraction, while other paralogs may have similar domain structures and fractionation patterns as the single homolog in E. coli. The expanded protein families in T. immobilis include enzymes involved in replication-repair processes as well as in rRNA and tRNA modification and degradation. These results show that paralogization and domain shuffling have yielded new proteins with distinct fractionation characteristics. Understanding the molecular intricacies of these adaptive changes might aid in the development of a model for the evolution of cellular complexity.

Proteomic comparison between different tissue preservation methods for identification of promising biomarkers of urothelial bladder cancer.

Samples in biobanks are generally preserved by formalin-fixation and paraffin-embedding (FFPE) and/or optimal cutting temperature compound (OCT)-embedding and subsequently frozen. Mass spectrometry (MS)-based analysis of these samples is now available via developed protocols, however, the differences in results with respect to preservation methods needs further investigation. Here we use bladder urothelial carcinoma tissue of two different tumor stages (Ta/T1-non-muscle invasive bladder cancer (NMIBC), and T2/T3-muscle invasive bladder cancer (MIBC)) which, upon sampling, were divided and preserved by FFPE and OCT. Samples were parallel processed from the two methods and proteins were analyzed with label-free quantitative MS. Over 700 and 1200 proteins were quantified in FFPE and OCT samples, respectively. Multivariate analysis indicates that the preservation method is the main source of variation, but also tumors of different stages could be differentiated. Proteins involved in mitochondrial function were overrepresented in OCT data but missing in the FFPE data, indicating that these proteins are not well preserved by FFPE. Concordant results for proteins such as HMGCS2 (uniquely quantified in Ta/T1 tumors), and LGALS1, ANXA5 and plastin (upregulated in T2/T3 tumors) were observed in both FFPE and OCT data, which supports the use of MS technology for biobank samples and encourages the further evaluation of these proteins as biomarkers.

The Human Adenovirus Type 2 Transcriptome: An Amazing Complexity of Alternatively Spliced mRNAs.

We have used the Nanopore long-read sequencing platform to demonstrate how amazingly complex the human adenovirus type 2 (Ad2) transcriptome is with a flexible splicing machinery producing a range of novel mRNAs both from the early and late transcription units. In total we report more than 900 alternatively spliced mRNAs produced from the Ad2 transcriptome whereof more than 850 are novel mRNAs. A surprising finding was that more than 50% of all E1A transcripts extended upstream of the previously defined transcriptional start site. The novel start sites mapped close to the inverted terminal repeat (ITR) and within the E1A enhancer region. We speculate that novel promoters or enhancer driven transcription, so-called eRNA transcription, is responsible for producing these novel mRNAs. Their existence was verified by a peptide in the Ad2 proteome that was unique for the E1A ITR mRNA. Although we show a high complexity of alternative splicing from most early and late regions, the E3 region was by far the most complex when expressed at late times of infection. More than 400 alternatively spliced mRNAs were observed in this region alone. These mRNAs included extended L4 mRNAs containing E3 and L5 sequences and readthrough mRNAs combining E3 and L5 sequences. Our findings demonstrate that the virus has a remarkable capacity to produce novel exon combinations, which will offer the virus an evolutionary advantage to change the gene expression repertoire and protein production in an evolving environment.IMPORTANCE Work in the adenovirus system led to the groundbreaking discovery of RNA splicing and alternative RNA splicing in 1977. These mechanisms are essential in mammalian evolution by increasing the coding capacity of a genome. Here, we have used a long-read sequencing technology to characterize the complexity of human adenovirus pre-mRNA splicing in detail. It is mindboggling that the viral genome, which only houses around 36,000 bp, not being much larger than a single cellular gene, generates more than 900 alternatively spliced mRNAs. Recently, adenoviruses have been used as the backbone in several promising SARS-CoV-2 vaccines. Further improvement of adenovirus-based vaccines demands that the virus can be tamed into an innocent carrier of foreign genes. This requires a full understanding of the components that govern adenovirus replication and gene expression.

Inhibition properties of free and conjugated leupeptin analogues.

Leupeptin is a naturally occurring inhibitor of various proteases, in particular serine proteases. Following its discovery, the inhibitory properties of several other peptidyl argininals have been studied. The specificity of leupeptin is most likely due to the Leu-Leu-Argininal sequence, and its C-terminal aldehyde group has been suggested to enhance the binding efficiency and to be essential for function. The terminal aldehyde group makes the structure less vulnerable to carboxypeptidases. Here, we investigated whether the inhibitory function of leupeptin toward serine proteases is retained after oxidation or reduction of the aldehyde group. The oxidized form, which corresponds to the natural precursor, was shown to be superior to the reduced form in terms of inhibitory properties. However, the original leupeptin possessed enhanced inhibitory properties as compared with the oxidized form. Based on these results, new synthetic leupeptin analogues, 6-aminohexanoic acid (Ahx)-Phe-Leu-Arg-COOH and Ahx-Leu-Leu-Arg-COOH, were prepared by solid-phase peptide synthesis using the Fmoc strategy. In these analogues, the N-terminal capping acetyl group was replaced with a 6-aminohexanoyl group to allow conjugation. The structures of the modified leupeptin and the synthetic peptides were confirmed by mass spectrometry. Determination of the inhibitory properties against trypsin (IEC 3.4.21.4, Chymotrypsin IEC 3.4.21.1) revealed that these further modified tripeptides were tight binding inhibitors to their target enzyme, similar to the naturally occurring leupeptin, with Ki values generally in the micromolar range. The Ahx-Phe-Leu-Arg-COOH analogue was selected for conjugation to inorganic oxide nanoparticles and agarose gel beads. All conjugates exhibited inhibitory activity in the same range as for the free peptides.

Investigating the Impact of Sample Preparation on Mass Spectrometry-Based Drug-To-Antibody Ratio Determination for Cysteine- and Lysine-Linked Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) are heterogeneous biotherapeutics and differ vastly in their physicochemical properties depending on their design. The number of small drug molecules covalently attached to each antibody molecule is commonly referred to as the drug-to-antibody ratio (DAR). Established analytical protocols for mass spectrometry (MS)-investigation of antibodies and ADCs often require sample treatment such as desalting or interchain disulfide bond reduction prior to analysis. Herein, the impact of the desalting and reduction steps-as well as the sample concentration and elapsed time between synthesis and analysis of DAR-values (as acquired by reversed phase liquid chromatography MS (RPLC-MS))-was investigated. It was found that the apparent DAR-values could fluctuate by up to 0.6 DAR units due to changes in the sample preparation workflow. For methods involving disulfide reduction by means of dithiothreitol (DTT), an acidic quench is recommended in order to increase DAR reliability. Furthermore, the addition of a desalting step was shown to benefit the ionization efficiencies in RPLC-MS. Finally, in the case of delayed analyses, samples can be stored at four degrees Celsius for up to one week but are better stored at -20 °C for longer periods of time. In conclusion, the results demonstrate that commonly used sample preparation procedures and storage conditions themselves may impact MS-derived DAR-values, which should be taken into account when evaluating analytical procedures.

A Single Shot Pre-fusion-Stabilized Bovine RSV F Vaccine is Safe and Effective in Newborn Calves with Maternally Derived Antibodies.

Achieving safe and protective vaccination against respiratory syncytial virus (RSV) in infants and in calves has proven a challenging task. The design of recombinant antigens with a conformation close to their native form in virus particles is a major breakthrough. We compared two subunit vaccines, the bovine RSV (BRSV) pre-fusion F (preF) alone or with nanorings formed by the RSV nucleoprotein (preF+N). PreF and N proteins are potent antigenic targets for neutralizing antibodies and T cell responses, respectively. To tackle the challenges of neonatal immunization, three groups of six one-month-old calves with maternally derived serum antibodies (MDA) to BRSV received a single intramuscular injection of PreF, preF+N with MontanideTM ISA61 VG (ISA61) as adjuvant or only ISA61 (control). One month later, all calves were challenged with BRSV and monitored for virus replication in the upper respiratory tract and for clinical signs of disease over one week, and then post-mortem examinations of their lungs were performed. Both preF and preF+N vaccines afforded safe, clinical, and virological protection against BRSV, with little difference between the two subunit vaccines. Analysis of immune parameters pointed to neutralizing antibodies and antibodies to preF as being significant correlates of protection. Thus, a single shot vaccination with preF appears sufficient to reduce the burden of BRSV disease in calves with MDA.

Magnetic Beads for Desalting of Monoclonal Antibodies and Antibody-Drug Conjugates.

Characterization of antibody-drug conjugates (ADCs) using mass spectrometry (MS) is important in drug discovery and formulation development and as part of the quality control processes. To facilitate electrospray ionization (ESI) and produce high-quality mass spectra, common components of storage solutions for monoclonal antibodies (mAbs) and ADCs, such as nonvolatile phosphate-buffered saline (PBS), should be replaced before analysis. Centrifugal spin-type kits are extensively used for the desalting or buffer-exchange of mAbs and ADCs samples. The commercially available kits commonly require at least 100 µL of a sample at 1 mg/mL for optimal recovery. However, most ESI-MS based analyses require no more than 25 µg of protein for triplicate injection. In this study, we present a novel method for desalting of ADCs and mAbs building on the SP3 approach with nonfunctionalized carboxylate coated magnetic beads without affinity ligands. The analytes bind to the hydrophilic beads upon the addition of organic solvent, and various solutions of volatile salts or acids can be used in the elution step. The optimized protocol allowed for 88% recovery of ADC at a 25 µL sample volume and 90% recovery at 100 µL. More than 90% of the salts were removed using a process of 20 min. The intra- and interday precision showed little variation with an RSD of 1% and 2%, respectively. The compatibility of this new workflow with low sample volumes is highly valuable since a smaller fraction of the sample is wasted for analysis of the expensive analytes, without compromising recovery.

Phosphorylation Time-Course Study of the Response during Adenovirus Type 2 Infection.

PTMs such as phosphorylations are usually involved in signal transduction pathways. To investigate the temporal dynamics of phosphoproteome changes upon viral infection, a model system of IMR-90 cells infected with human adenovirus type 2 (Ad2) is used in a time-course quantitative analysis combining titanium dioxide (TiO2 ) particle enrichment and SILAC-MS. Quantitative data from 1552 phosphorylated sites clustered the highly altered phosphorylated sites to the signaling by rho family GTPases, the actin cytoskeleton signaling, and the cAMP-dependent protein kinase A signaling pathways. Their activation is especially pronounced at early time post-infection. Changes of several phosphorylated sites involved in the glycolysis pathway, related to the activation of the Warburg effect, point at virus-induced energy production. For Ad2 proteins, 32 novel phosphorylation sites are identified and as many as 52 phosphorylated sites on 17 different Ad2 proteins are quantified, most of them at late time post-infection. Kinase predictions highlighted activation of PKA, CDK1/2, MAPK, and CKII. Overlaps of kinase motif sequences for viral and human proteins are observed, stressing the importance of phosphorylation during Ad2 infection.

Application of triple quadrupole mass spectrometry for the characterization of antibody-drug conjugates.

Antibody-drug conjugates (ADCs) are an inherently heterogeneous class of biotherapeutics, the development of which requires extensive characterization throughout. During the earliest phases of preclinical development, when synthetic routes towards the desired conjugate are being assessed, the main interest lies in the determination of the average drug-to-antibody ratio (DAR) of a given batch as well as information about different conjugation species. There has been a trend in mass spectrometry (MS)-based characterization of ADCs towards the use of high-resolving mass spectrometry for many of these analyses. Considering the high cost for such an instrument, the evaluation of cheaper and more accessible alternatives is highly motivated. We have therefore tested the applicability of a quadrupole mass analyzer for the aforementioned characterizations. Eight ADCs consisting of trastuzumab and varying stoichiometries of Mc-Val-Cit-PABC-monomethyl auristatin E conjugated to native cysteines were synthesized and served as test analytes. The average DAR value and molecular weights (Mw) of all detected chains from the quadrupole mass analyzer showed surprisingly high agreement with results obtained from a time-of-flight (TOF) mass analyzer and hydrophobic interaction chromatography (HIC)-derived values for all investigated ADC batches. Acquired Mw were within 80 ppm of TOF-derived values, and DAR was on average within 0.32 DAR units of HIC-derived values. Quadrupole mass spectrometers therefore represent a viable alternative for the characterization of ADC in early-stage development. Graphical abstract.

Transcriptomic and proteomic analyses reveal new insights into the regulation of immune pathways during adenovirus type 2 infection.

BACKGROUND: Human adenovirus (Ad) infection leads to the changes of host cell gene expression and biosynthetic processes. Transcriptomics in adenovirus type 2 (Ad2)-infected lung fibroblasts (IMR-90) cells has previously been studied using RNA sequencing. However, this study included only two time points (12 and 24 hpi) using constrained 76 bp long sequencing reads. Therefore, a more detailed study of transcription at different phases of infection using an up-graded sequencing technique is recalled. Furthermore, the correlation between transcription and protein expression needs to be addressed. RESULTS: In total, 3556 unique cellular genes were identified as differentially expressed at the transcriptional level with more than 2-fold changes in Ad2-infected cells as compared to non-infected cells by using paired-end sequencing. Based on the kinetics of the gene expression changes at different times after infection, these RNAs fell into 20 clusters. Among them, cellular genes involved in immune response were highly up-regulated in the early phase before becoming down-regulated in the late phase. Comparison of differentially expressed genes at transcriptional and posttranscriptional levels revealed low correlation. Particularly genes involved in cellular immune pathways showed a negative correlation. Here, we highlight the genes which expose inconsistent expression profiles with an emphasis on key factors in cellular immune pathways including NFκB, JAK/STAT, caspases and MAVS. Different from their transcriptional profiles with up- and down-regulation in the early and late phase, respectively, these proteins were up-regulated in the early phase and were sustained in the late phase. A surprising finding was that the target genes of the sustained activators failed to show response. CONCLUSION: There were features common to genes which play important roles in cellular immune pathways. Their expression was stimulated at both RNA and protein levels during the early phase. In the late phase however, their transcription was suppressed while protein levels remained stable. These results indicate that Ad2 and the host cell use different strategies to regulate cellular immune pathways. A control mechanism at the post-translational level must thus exist which is under the control of Ad2.

Qualitative analysis of antibody-drug conjugates (ADCs): an experimental comparison of analytical techniques of cysteine-linked ADCs.

Antibody-drug conjugates (ADCs) are an emerging type of biotherapeutics that utilize multiple tissue-specific antibodies combined with a range of linker designs to enable the transportation and selective release of cytotoxic drugs in close proximity to tumours. Consisting of antibodies conjugated to small drug molecules through a variety of linkers, ADCs are chemically complex analytes. Here we present a unique experimental comparison of four techniques for ADC analysis: hydrophobic interaction chromatography (HIC-UV/Vis), reversed phase liquid chromatography mass spectrometry (RPLC-MS), using either a QToF or an Orbitrap analyser, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Four different ADCs consisting of Trastuzumab, monomethyl auristatin E (MMAE) and a peptidic linker moiety differing in their respective stoichiometric ratios in regard to drug-to-antibody ratio (DAR) were used for the comparison. We found that the determined DAR from all techniques was comparable, while the accuracy of the molecular weights for the conjugated light and heavy chain differed more extensively. This indicates that the choice of a mass analyser is more crucial for determining the accurate weights of the light and heavy chains than to evaluate the DAR of a given batch. However, ambiguous DAR assignment in HIC-UV/Vis or bias for either the light or heavy chain fragments in the mass spectrometry-based techniques can influence the obtained average DAR value and the use of complementary techniques is advisable. Out of the four techniques evaluated, HIC-UV/Vis and MALDI required less time to obtain an average DAR value and would therefore be good for initial screenings in the early stages of the discovery phase of new ADCs.

Time-resolved proteomics of adenovirus infected cells.

Viral infections cause large problems in the world and deeper understanding of the disease mechanisms is needed. Here we present an analytical strategy to investigate the host cell protein changes during human adenovirus type 2 (HAdV-C2 or Ad2) infection of lung fibroblasts by stable isotope labelling of amino acids in cell culture (SILAC) and nanoLC-MS/MS. This work focuses on early phase of infection (6 and 12 h post-infection (hpi)) but the data is combined with previously published late phase (24 and 36 hpi) proteomics data to produce a time series covering the complete infection. As many as 2169 proteins were quantitatively monitored from 6 to 36 hpi, while some proteins were time-specific. After applying different filter criteria, 2027 and 2150 proteins were quantified at 6 and 12 hpi and among them, 431 and 544 were significantly altered at the two time points. Pathway analysis showed that the De novo purine and pyrimidine biosynthesis, Glycolysis and Cytoskeletal regulation by Rho GTPase pathways were activated early during infection while inactivation of the Integrin signalling pathway started between 6 and 12 hpi. Moreover, upstream regulator analysis predicted MYC to be activated with time of infection and protein and RNA data for genes controlled by this transcription factor showed good correlation, which validated the use of protein data for this prediction. Among the identified phosphorylation sites, a group related to glycolysis and cytoskeletal reorganization were up-regulated during infection. The results show specific aspects on how the host cell proteins, the final products in the genetic information flow, are influenced by Ad2 infection, which would be overlooked if only knowledge derived from mRNA data is considered.

Acute sleep loss results in tissue-specific alterations in genome-wide DNA methylation state and metabolic fuel utilization in humans.

Curtailed sleep promotes weight gain and loss of lean mass in humans, although the underlying molecular mechanisms are poorly understood. We investigated the genomic and physiological impact of acute sleep loss in peripheral tissues by obtaining adipose tissue and skeletal muscle after one night of sleep loss and after one full night of sleep. We find that acute sleep loss alters genome-wide DNA methylation in adipose tissue, and unbiased transcriptome-, protein-, and metabolite-level analyses also reveal highly tissue-specific changes that are partially reflected by altered metabolite levels in blood. We observe transcriptomic signatures of inflammation in both tissues following acute sleep loss, but changes involving the circadian clock are evident only in skeletal muscle, and we uncover molecular signatures suggestive of muscle breakdown that contrast with an anabolic adipose tissue signature. Our findings provide insight into how disruption of sleep and circadian rhythms may promote weight gain and sarcopenia.

Parallel Proteomic Workflow for Mass Spectrometric Analysis of Tissue Samples Preserved by Different Methods.

Formalin-fixed and paraffin-embedded (FFPE) and optimal cutting temperature (OCT)-embedded and frozen tissue specimens in biobanks are highly valuable in clinical studies but proteomic and post-translational modification (PTM) studies using mass spectrometry (MS) have been limited due to structural arrangement of proteins and contaminations from embedding material. This study aims to develop a parallel proteomic workflow for FFPE and OCT/frozen samples that allows for large-scale, quick, reproducible, qualitative, and quantitative high-resolution MS analysis. The optimized protocol gives details on removal of embedding material, protein extraction, and multienzyme digestion using filter-aided sample preparation method. The method was evaluated by investigating the protein expression levels in nonmuscle-invasive and muscle-invasive bladder cancer samples in two cohorts and MS spectra were carefully reviewed for contaminations. More than 2000 and 3000 proteins in FFPE and OCT/frozen samples, respectively, were identified, and samples could be clustered in different tumor stages based on their protein expression. Furthermore, more than 250 and 400 phosphopeptides could be identified from specific patient samples of FFPE and OCT/frozen, respectively, using titanium dioxide enrichment. The paper presents unique data describing the similarities and differences observed in FFPE and OCT/frozen samples and shows the feasibility to detect proteins and site-specific phosphorylation even after long-term storage of clinical samples.

Proteome analysis of bronchoalveolar lavage from calves infected with bovine respiratory syncytial virus-Insights in pathogenesis and perspectives for new treatments.

Human and bovine respiratory syncytial viruses (HRSV/BRSV) are major causes of severe lower respiratory tract infections in children and calves, respectively. Shared epidemiological, clinical, pathological and genetic characteristics of these viruses make comparative research highly relevant. To characterise the host response against BRSV infection, bronchoalveolar lavage supernatant (BAL) from i) non-vaccinated, BRSV-infected ii) vaccinated, BRSV-infected and iii) non-infected calves was analysed by tandem mass spectrometry. Proteins were semi-quantified and protein expression was validated by immunoblotting. Correlations between selected proteins and pathology, clinical signs and virus shedding were investigated. Calves with BRSV-induced disease had increased total protein concentrations and a decreased number of proteins identified in BAL. The protein profile was characterised by neutrophil activation and a reduction in identified antioxidant enzymes. The presence of neutrophils in alveolar septa, the expression level of neutrophil-related or antioxidant proteins and LZTFL1 correlated significantly with disease. Citrullinated histone 3, an indicator of extracellular traps (ETs), was only detected in non-vaccinated, BRSV-infected animals. By bringing disequilibrium in the release and detoxification of reactive oxygen species, generating ETs and causing elastine degradation, exaggerated neutrophil responses might exacerbate RSV-induced disease. Neutrophil-mitigating or antioxidant treatments should be further explored.

Temporal characterization of the non-structural Adenovirus type 2 proteome and phosphoproteome using high-resolving mass spectrometry.

The proteome and phosphoproteome of non-structural proteins of Adenovirus type 2 (Ad2) were time resolved using a developed mass spectrometry approach. These proteins are expressed by the viral genome and important for the infection process, but not part of the virus particle. We unambiguously confirm the existence of 95% of the viral proteins predicted to be encoded by the viral genome. Most non-structural proteins peaked in expression at late time post infection. We identified 27 non-redundant sites of phosphorylation on seven different non-structural proteins. The most heavily phosphorylated protein was the DNA binding protein (DBP) with 15 different sites. The phosphorylation occupancy rate could be calculated and monitored with time post infection for 15 phosphorylated sites on various proteins. In the DBP, phosphorylations with time-dependent relation were observed. The findings show the complexity of the Ad2 non-structural proteins and opens up a discussion for potential new drug targets.

Identification of the adenovirus type 2 C-168 protein.

A hitherto predicted but undetected protein, C-168, in adenovirus type 2 (Ad2) has been identified using mass spectrometry (MS) based proteomics. The gene of this 17.7kDa protein is located on the forward strand in the major late transcription unit between base pairs 9294 and 9797. A tryptic peptide, derived from the C-terminal part of the protein, was identified with high amino acid sequence coverage. A candidate splice site for the corresponding mRNA is also presented. The protein sequence is unusual with repeats of serine, glycine and arginine. A bioinformatics prediction of protein function and localization is presented.