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The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a conserved domain and a target for neutralizing antibodies. We defined the carbohydrate content of the recombinant RBD produced in different mammalian cells. We found a higher degree of complex-type N-linked glycans, with less sialylation and more fucosylation, when the RBD was produced in human embryonic kidney cells compared to the same protein produced in Chinese hamster ovary cells. The carbohydrates on the RBD proteins were enzymatically modulated, and the effect on antibody reactivity was evaluated with serum samples from SARS-CoV-2 positive patients. Removal of all carbohydrates diminished antibody reactivity, while removal of only sialic acids or terminal fucoses improved the reactivity. The RBD produced in Lec3.2.8.1-cells, which generate carbohydrate structures devoid of sialic acids and with reduced fucose content, exhibited enhanced antibody reactivity, verifying the importance of these specific monosaccharides. The results can be of importance for the design of future vaccine candidates, indicating that it is possible to enhance the immunogenicity of recombinant viral proteins.

Background There is a lack of early and precise biomarkers for personalized respiratory medicine. Breath contains an aerosol of droplet particles, which are formed from the epithelial lining fluid when the small airways close and re-open during inhalation succeeding a full expiration. These particles can be collected by impaction using the PExA method (Particles in Exhaled Air), and are derived from an area of high clinical interest previously difficult to access, making them a potential source of biomarkers reflecting pathological processes in the small airways. Research question Our aim was to investigate if PExA method is useful for discovery of biomarkers that reflect pathology of small airways. Methods and analysis Ten healthy controls and 20 subjects with asthma, of whom 10 with small airway involvement as indicated by a high lung clearance index (LCI ≥ 2.9 z-score), were examined in a cross-sectional design, using the PExA instrument. The samples were analysed with the SOMAscan proteomics platform (SomaLogic Inc.). Results Two hundred-seven proteins were detected in up to 80% of the samples. Nine proteins showed differential abundance in subjects with asthma and high LCI as compared to healthy controls. Two of these were less abundant (ALDOA4, C4), and seven more abundant (FIGF, SERPINA1, CD93, CCL18, F10, IgM, IL1RAP). sRAGE levels were lower in ex-smokers (n = 14) than in never smokers (n = 16). Gene Ontology (GO) annotation database analyses revealed that the PEx proteome is enriched in extracellular proteins associated with extracellular exosome-vesicles and innate immunity. Conclusion The applied analytical method was reproducible and allowed identification of pathologically interesting proteins in PEx samples from asthmatic subjects with high LCI. The results suggest that PEx based proteomics is a novel and promising approach to study respiratory diseases with small airway involvement.

The receptor binding domain (RBD) of the SARS-CoV-2 spike protein is a conserved domain and a target for neutralizing antibodies. We defined the carbohydrate content of recombinant RBD produced in different mammalian cells. We found a higher degree of complex type N-linked glycans, with less sialylation and more fucosylation, when the RBD was produced in Human embryonic kidney cells compared to the same protein produced in Chinese hamster ovary cells. The carbohydrates on the RBD proteins were enzymatically modulated and the effect on antibody reactivity was evaluated with serum samples from SARS-CoV-2 positive patients. Removal of all carbohydrates diminished antibody reactivity while removal of only sialic acids or terminal fucoses improved the reactivity. The RBD produced in Lec3.2.8.1-cells, which generate carbohydrate structures devoid of sialic acids and with reduced fucose content, exhibited enhanced antibody reactivity verifying the importance of these specific monosaccharides. The results can be of importance for the design of future vaccine candidates, indicating that it might be possible to enhance the immunogenicity of recombinant viral proteins.

Like most other cell surface proteins, αHere, we have established the first comprehensive site-specific glycan map of αWe expect that the glycoproteomics data of the current study will serve as a resource for the exploration of structural mechanisms by which glycans control αGlycosylation of α

Heterologous protein production is widely used in industrial biotechnology. However, using non-native production hosts can lead to enzymes with altered post-translational modifications, such as glycosylation. We have investigated how production in a non-native host affects the physicochemical properties and enzymatic activity of a feruloyl esterase from Myceliophthora thermophila, MtFae1a. The enzyme was produced in two microorganisms that introduce glycosylation (M. thermophila and Pichia pastoris) and in Escherichia coli (non-glycosylated). Mass spectrometric analysis confirmed the presence of glycosylation and revealed differences in the lengths of glycan chains between the enzymes produced in M. thermophila and P. pastoris. The melting temperature and the optimal temperature for activity of the non-glycosylated enzyme were considerably lower than those of the glycosylated enzymes. The three MtFae1a versions also exhibited differences in specific activity and specificity. The catalytic efficiency of the glycosylated enzymes were more than 10 times higher than that of the non-glycosylated one. In biotechnology, immobilization is often used to allow reusing enzyme and was investigated on mesoporous silica particles. We found the binding kinetics and immobilization yield differed between the enzyme versions. The largest differences were observed when comparing enzymes with and without glycosylation, but significant variations were also observed between the two differently glycosylated enzymes. We conclude that the biotechnological value of an enzyme can be optimized for a specific application by carefully selecting the production host. Electronic supplementary material The online version of this article (10.1186/s13568-019-0852-z) contains supplementary material, which is available to authorized users.

Neurexins are presynaptic adhesion molecules that shape the molecular composition of synapses. Diversification of neurexins in numerous isoforms is believed to confer synapse‐specific properties by engaging with distinct ligands. For example, a subset of neurexin molecules carry a heparan sulfate (HS) glycosaminoglycan that controls ligand binding, but how this post‐translational modification is controlled is not known. Here, we observe that CA10, a ligand to neurexin in the secretory pathway, regulates neurexin‐HS formation. CA10 is exclusively found on non‐HS neurexin and CA10 expressed in neurons is sufficient to suppress HS addition and attenuate ligand binding and synapse formation induced by ligands known to recruit HS. This effect is mediated by a direct interaction in the secretory pathway that blocks the primary step of HS biosynthesis: xylosylation of the serine residue. NMR reveals that CA10 engages residues on either side of the serine that can be HS‐modified, suggesting that CA10 sterically blocks xylosyltransferase access in Golgi. These results suggest a mechanism for the regulation of HS on neurexins and exemplify a new mechanism to regulate site‐specific glycosylations., CA10 binds the presynaptic adhesion molecules neurexin in the secretory pathway and inhibits their modification with heparan sulfate, thereby modifying the ligand‐binding properties of neurexin.

The structural study of glycans and glycoconjugates is essential to assign their roles in homeostasis, health, and disease. Once dominated by nuclear magnetic resonance spectroscopy, mass spectrometric methods have become the preferred toolbox for the determination of glycan structures at high sensitivity. The patterns of such structures in different cellular states now allow us to interpret the sugar codes in health and disease, based on structure-function relationships. Dr. Catherine E. Costello was the 2017 recipient of the American Society for Mass Spectrometry’s Distinguished Contribution Award. In this Perspective article, we describe her seminal work in a historical and geographical context and review the impact of her research accomplishments in the field. 8

To identify pathological changes in the composition of the lining fluid of small airways, it is important to know what factors that may influence it, also in healthy individuals. The aim of the present study was to 1) identify determinants of Albumin, Surfactant Protein A (SP-A) and the profile of phospholipids in PEx in healthy subjects, and 2) to explore if the composition is altered in asthma and smokers in a small number of subjects. Methods: 166 non-smoking healthy individuals, 17 current smokers and 16 subject reporting asthma-symptoms within the last year, from the European Community Respiratory Health Survey III, was examined with the PExA® method, i.e. sampling of droplets of lining fluid from small airways in breath. PEx samples were analyzed with ELISA (Albumin and SP-A) and the di-palmitoyl-phosphatidylcholine (DPPC), palmitoyl-oleoyl-glycero-phosphocholine (POPC), PC14:0/16:0 and PC16:0/18:2 with LC-MS-MS. Concentrations of all biomarkers in PEx are expressed as weight percent (wt%). statistical analysis: Spearmann correlations and quantile regression were used. Results: Increasing age was associated with an increase in albumin (rs =0.23, p=0.001). The levels of DPPC and POPC in PEx were decreasing with age (rs=-0.35, p=0.006 and rs =-0.28, p=0.008, respectively, and not in women. In asthma, albumin was significantly lower in PEx than in healthy subjects. In females with asthma, PC14:0/16:0 was also lower and PC16:0/18:2 higher, but not in men. The levels of SP-A were not different from that in healthy subjects. Smokers had higher levels of DPPC, POPC as well as SP-A, but lower levels of albumin in PEx. Conclusion: Age and gender, but also smoking and asthma, seem to significantly influence surfactant composition.

Introduction At present, there are few methods available for monitoring respiratory diseases affecting distal airways. Bronchoscopy is the golden standard for sampling the lower airways. The recently developed method for collecting non-volatile material from exhaled air – PExA (Particles in Exhaled air) is a promising new tool, but no direct comparison between the two methods has yet been performed. The aim of the present study was to compare sampling using PExA with bronchial wash (BW) representing the larger more proximal airways and broncho-alveolar lavage (BAL) representing the distal airways. Methods 15 healthy non-smoking subjects (7 female/8 male), age 28 ± 4 years, with normal lung function were included in the study. PExA-sampling (2 × 250 ng particles) and bronchoscopy with BW (2 × 20 ml) and BAL (3 × 60 ml sterile saline) was performed. Albumin and Surfactant Protein A (SP-A) were analyzed with ELISA, and analyses of correlation were performed. Results A significant association was found between BAL-fluid albumin and PExA-albumin (rs:0.65 p = 0.01). There was also an association between SP-A in PExA and BAL, when corrected for albumin concentration (rs:0.61, p = 0.015). When correlating concentrations of albumin and SP-A in bronchial wash and PExA respectively, no associations were found. Conclusions This is the first direct comparison between the bronchoscopy-based BW/BAL-fluids and material collected using the PExA methodology. Both albumin and albumin-corrected SP-A concentrations were significantly associated between BAL and PExA, however, no such association was found in either marker between BW and PExA. These results indicate that the PExA method samples the distal airways. PExA is thus considered a new promising non-invasive assessment for monitoring of the distal airways.

We present a new, quantification-driven proteomic approach to identifying biomarkers. In contrast to the identification-driven approach, limited in scope to peptides that are identified by database searching in the first step, all MS data are considered to select biomarker candidates. The endopeptidome of cerebrospinal fluid from 40 Alzheimer’s disease (AD) patients, 40 subjects with mild cognitive impairment, and 40 controls with subjective cognitive decline was analyzed using multiplex isobaric labeling. Spectral clustering was used to match MS/MS spectra. The top biomarker candidate cluster (215% higher in AD compared to controls, area under ROC curve = 0.96) was identified as a fragment of pleiotrophin located near the protein’s C-terminus. Analysis of another cohort (n = 60 over four clinical groups) verified that the biomarker was increased in AD patients while no change in controls, Parkinson’s disease or progressive supranuclear palsy was observed. The identification of the novel biomarker pleiotrophin 151–166 demonstrates that our quantification-driven proteomic approach is a promising method for biomarker discovery, which may be universally applicable in clinical proteomics.

An exaggerated aging process of the lung is suggested to play role in development of respiratory disease. Detailed information on the macro molecular make up of the lining fluid and how that changes over time and with disease is still scarce. When analyzing the protein composition of exhaled particles which have been described to originate from the lining fluid of small airways, many proteins were found to be associated with age and were further explored. Method: 10 healthy controls and 20 subjects with asthma (age 20-68 years) were included. Samples were collected using the PExA® method and analyzed using an analytical platform capable of measuring the relative quantity of 207 different proteins (SomaLogic Inc.). Results: The relative quantity of 106 out of 207 detected proteins was associated with age (R ±0.36-±0.8, q Conclusion: Protein composition of lining fluid from small airways seem to change with age. More proteins were associated with age than with asthma, but some were also independently associated with both. The non-invasive nature of the PExA® method provide the means for new insight into the pathobiology of the aging lung.

Introduction: At present, there are few methods available for monitoring respiratory diseases affecting distal airways. Bronchoscopy is the golden standard for sampling the lower airways. The recently developed method for collecting non-volatile material from exhaled air (Particles in Exhaled air – PExA) is a promising new tool, but no direct comparison between the two methods have yet been performed. The aim of the present study was to validate sampling using PExA with bronchoscopy with bronchoalveolar lavage (BAL) representing distal airways and bronchial wash (BW) representing proximal airways. Methods: 15 healthy subjects (7 female/8 male), age 28 ± 4 with normal lung function were included in the study. PExA-sampling (2x250ng particles) and bronchoscopy with BAL (3x60ml sterile saline) and BW (2x20ml) were performed. Albumin and surfactant protein A (SPA) were analyzed and correlation analyses were performed. Results: Albumin was the most abundant protein in the respiratory tract lining fluid. There was a significant correlation between albumin concentrations in BAL-fluid and PExA (p=0.01; r2=0.65) and between SPA levels in PExA and BAL, when corrected for albumin concentrations (p=0.015; r2=0.62). There was no correlation between concentrations of albumin and SPA in bronchial wash and PExA respectively, indicating that the PExA sample originates from the distal airways. Conclusions: This is the first direct comparison between levels of airway markers in BAL-fluid and in particles collected with PExA. Strong correlations were found between both albumin and SPA in those matrixes. PExA is considered a new promising non-invasive assessment for monitoring of the lower airways.

Although highly desired, there are currently no non-invasive methods to collect samples from distal airways for robust biomarker analysis. Breath contains particles which are formed in small airways when the lining fluid bursts during inhalation. These particles can be collected by impaction technology (PExA AB). We have identified a method that allows us to examine the protein content of these particles and we have addressed the following questions: - How many different proteins can be detected and accurately quantified? - Are the detected proteins relevant in respiratory disease? - Is it possible to identify protein signatures that can be used to sub-phenotype asthma? Method: 10 healthy controls and 20 subjects with asthma, of whom 10 with normal small airway function as defined by lung-clearance index (LCI), were included. Particles in exhaled air samples were analyzed using a proteomic platform assessing the relative quantity of 1310 different proteins (SomaLogic, Inc). Results: Up to 207 different proteins could be detected in a robust way. The relative quantity of 36 proteins, including several S100 proteins and proteins involved in coagulation and antioxidant response, were different in asthma as compared to controls (p Conclusion: Our findings demonstrate how protein biomarkers originating from the distal airway can be assessed in a non-invasive way. The obtained protein profile unique to a subgroup within asthma suggest that the method captures pathobiologically relevant proteins and hold potential to boost development of Precision Medicines in respiratory disease.

Exhaled particles constitute a micro-sample of respiratory tract lining fluid. Inhalations from low lung volumes generate particles in small airways by the airway re-opening mechanism. Forced exhalations are assumed to generate particles in central airways by mechanisms associated with high air velocities. To increase knowledge on how and where particles are formed, different breathing manoeuvres were compared in 11 healthy volunteers. Particles in the 0.41-4.55μm diameter range were characterised and sampled. The surfactant lipid dipalmitoylphosphatidylcholine (DPPC) was quantified by mass spectrometry. The mass of exhaled particles increased by 150% (95% CI 10-470) for the forced exhalation and by 470% (95% CI 150-1190) for the airway re-opening manoeuvre, compared to slow exhalations. DPPC weight percent concentration (wt%) in particles was 2.8wt% (95%CI 1.4-4.2) and 9.4wt% (95%CI 8.0-10.8) for the forced and the airway re-opening manoeuvres, respectively. In conclusion, forced exhalation and airway re-opening manoeuvres generate particles from different airway regions having different DPPC concentration.

Background: many full-time welders experience some sort of respiratory disorder e.g., asthma, bronchitis and metal fume fever. Thus, welding aerosols are thought to cause airway inflammation. There is a need for markers of welding aerosols in exposure assessments, and as most welding aerosols contain manganese and iron, these metals may possibly be used as an indicator. We have previously developed a novel non-invasive technique to collect endogenous particles in exhaled air (PEx). This study is designed to (i) develop a method for analysis of manganese and iron in PEx and (ii) investigate whether the manganese and/or iron content of PEx changes after exposure to welding aerosols. Methods: nine individuals were experimentally exposed to welding fumes. PEx was collected at three time points for each individual; before, after and 24 hours after exposure. Analyses of PEx samples were performed using Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Results: four out of nine individuals showed an increase in manganese and iron levels after exposure to welding aerosols. The mean manganese and iron concentration increased from

Background Nocturnal gastroesophageal reflux (nGER) is associated with respiratory symptoms and sleep-disordered breathing (SDB), but the pathogenesis is unclear. We aimed to investigate the association between nGER and respiratory symptoms, exacerbations of respiratory symptoms, SDB and airway inflammation. Methods Participants in the European Community Respiratory Health Survey III in Iceland with nGER symptoms (n = 48) and age and gender matched controls (n = 42) were studied by questionnaires, exhaled breath condensate (EBC), particles in exhaled air (PEx) measurements, and a home polygraphic study. An exacerbation of respiratory symptoms was defined as an episode of markedly worse respiratory symptoms in the previous 12 months. Results Asthma and bronchitis symptoms were more common among nGER subjects than controls (54 % vs 29 %, p = 0.01; and 60 % vs 26 %, p

Background: Nocturnal gastroesophageal reflux (nGER) is associated with respiratory symptoms and sleep-disordered breathing (SDB), but the pathogenesis is unclear. We aimed to investigate the association between nGER and respiratory symptoms, exacerbations of respiratory symptoms, SDB and airway inflammation. Methods: Participants in the European Community Respiratory Health Survey III in Iceland with nGER symptoms (n = 48) and age and gender matched controls (n = 42) were studied by questionnaires, exhaled breath condensate (EBC), particles in exhaled air (PEx) measurements, and a home polygraphic study. An exacerbation of respiratory symptoms was defined as an episode of markedly worse respiratory symptoms in the previous 12 months. Results: Asthma and bronchitis symptoms were more common among nGER subjects than controls (54 % vs 29 %, p = 0.01; and 60 % vs 26 %, p < 0.01, respectively), as were exacerbations of respiratory symptoms (19 % vs 5 %, p = 0.04). Objectively measured snoring was more common among subjects with nGER than controls (snores per hour of sleep, median (IQR): 177 (79-281) vs 67 (32-182), p = 0.004). Pepsin (2.5 ng/ml (0.8-5.8) vs 0.8 ng/ml (0.8-3.6), p = 0.03), substance P (741 pg/ml (626-821) vs 623 pg/ml (562-676), p < 0.001) and 8-isoprostane (3.0 pg/ml (2.7-3.9) vs 2.6 pg/ml (2.2-2.9), p = 0.002) in EBC were higher among nGER subjects than controls. Albumin and surfactant protein A in PEx were lower among nGER subjects. These findings were independent of BMI. Conclusion: In a general population sample, nGER is associated with symptoms of asthma and bronchitis, as well as exacerbations of respiratory symptoms. Also, nGER is associated with increased respiratory effort during sleep. Biomarker measurements in EBC, PEx and serum indicate that micro-aspiration and neurogenic inflammation are plausible mechanisms. Erratum in: Respiratory Research 17 article number: 130 DOI: 10.1186/s12931-016-0448-y

Surfactant protein A (SP-A) is the major lung surfactant protein important for surfactant function and host-defense. Exhaled SP-A was suggested as a marker of airway inflammation in COPD patients 1 . However, no data on expected normal values of exhaled SP-A is available. The aim of the present study was to estimate normal values for exhaled SP-A and albumin in a healthy Swedish population and investigate relationships between the observed protein concentrations and age, gender, body size as well as spirometry data. Healthy individuals were selected from the participants of ECRHS III, examined at the Gothenburg center (N=154, age:40-66). In addition to the standard evaluation, all participants provided exhaled breath samples collected using the PExA method 2 . Exhaled proteins were measured by ELISA and reported as mass percent of exhaled particles (%, w/w). Log transformed data were evaluated by multiple linear regression analysis. The median exhaled SP-A was 3.22% (Q1=2.59, Q3=4.07). No associations were observed between SP-A and the evaluated variables. The median exhaled albumin was 6.41% (Q1=5.30, Q3=7.91). Albumin was associated to age with 0.8% increase per age year (p SP-A is not associated with age, gender and body size in a healthy population. This is attractive for a candidate marker as no corrections for these variables is required. For albumin, the age of the subject has to be taken into account when evaluating patient data. 1. Larstad M, Plos One 2015, 10(12) 2. Almstrand AC, Anal Chem 2009, 81:662.

We exhale endogenous particles during breathing that may carry information on the status of the respiratory tract. The aim of the study was to investigate particle formation during forced exhalations and cough, by evaluating particle size distribution and surfactant content. A reference maneuver that emits a minimum number of particles, was compared with a forced exhalation, a cough maneuver and an airway opening maneuver. These maneuvers were studied in a group of 11 healthy subjects using the PExA method. Particle size distribution and number concentrations in the range of 0.41-4.55 µm in diameter were measured using an optical particle counter before sampling by inertial impaction. Concentration of the main surfactant lipid dipalmitoylphosphatidylcholine (DPPC) was determined by mass spectrometry. The exhaled particle mass for the forced and cough maneuvers were 2.5 (95% CI 1.1-5.7, p=0.03) and 7.4 (95% CI 3.3-16.7, p The results are consistent with particle formation in central airways during forced exhalation and in the upper airways during cough. There was no increase in exhaled DPPC by forced exhalations or cough, despite the increase in exhaled particles, these particles have a very low concentrations of surfactant compared to particles from small airways.

Background : In asthma, clinical measures like FEV1 are a poor surrogate for detection of sub-groups and disease progression, and more efficient screening methods are needed. The PExA method (particles in exhaled air) non-invasively counts and collects exhaled particles, which reflect the composition of the fluid lining the peripheral airways and contains possible information about lung status. Collected particle mass is in the ng range and currently only single protein analysis is carried out routinely. The pilot study aimed to investigate Multiplex technology for simultaneous analysis of acute-phase proteins in exhaled particles. Method : Particles were collected during 30 minutes from two groups: Population 1: 12 healthy volunteers. Population 2: 6 subjects with asthma and 6 age-matched controls. Albumin, fibronectin, fibrinogen, SAP, A2M, CRP, haptoglobin and AGP were analysed using two Multiplex-kits (MILLIPLEX® SKINMAG-50K, MILLIPLEX® HCVD3-67K). Results: Albumin, fibrinogen, fibronectin, haptoglobin and AGP could be quantified in the majority of samples (>60%) and A2M in 7 samples, all regardless of subject9s health status. CRP and SAP were detected in one asthmatic and one control, respectively. Median particle mass collected in Population 1 was 340 ng (143-739). In Population 2, sampled mass in asthmatics (median 295 ng, 116-430) was significantly decreased compared to controls (median 535 ng, 234-1060) (P Conclusion : Multiplex technology can improve analysis possibilities by facilitating simultaneous quantification of multiple proteins in exhaled particles. Asthma may lead to a decrease in exhaled particles and less sample material.

Non-invasive and quantitative sampling of particles in exhaled air may provide valuable information on the status of the peripheral airways. Studies of particles have confirmed the presence of pulmonary surfactant, including both important phospholipids and surfactant proteins. The aim of this study was to develop a reliable method for the quantification of the major phosphatidylcholines (PCs) in exhaled particles. Methods: Exhaled particles were counted and collected on membranes using the PExA method. The particles were extracted with methanol:chloroform prior to flow injection into an electrospray ionization tandem mass spectrometer. A quantitative method was developed for dipalmitoylphosphatidylcholine (DPPC) and palmitoyl-oleoylphosphatidylcholine (POPC) using selected reaction monitoring. The method was validated by investigating within- and between-batch precision, accuracy, carry-over, stock stability, short-term and long-term stability, freeze-thaw stability and processed sample integrity. The validated method was used to investigate the inter-individual variation in exhaled particles from 6 healthy volunteers. Results: The method had a linear response for DPPC and POPC with a limit of quantification of 0.005 µM. The DPPC and POPC concentration, expressed as weight % in exhaled particles, were 13.9 (median, range 8.8-16%) and 4.1 (median, range 2.4-4.9%), respectively, of the total particle sample. Conclusion: We have developed a reliable method for the quantification of surfactant PCs in exhaled particles using a simple sample extraction and mass spectrometry. The method can readily be applied in clinical studies to investigate the role of PCs in different lung related diseases.

Phosphorylation is one of the key mechanisms that regulate centrosome biogenesis, spindle assembly, and cell cycle progression. However, little is known about centrosome-specific phosphorylation sites and their functional relevance. Here, we identified phosphoproteins of intact Drosophila melanogaster centrosomes and found previously unknown phosphorylation sites in known and unexpected centrosomal components. We functionally characterized phosphoproteins and integrated them into regulatory signaling networks with the 3 important mitotic kinases, cdc2, polo, and aur, as well as the kinase CkIIβ. Using a combinatorial RNA interference (RNAi) strategy, we demonstrated novel functions for P granule, nuclear envelope (NE), and nuclear proteins in centrosome duplication, maturation, and separation. Peptide microarrays confirmed phosphorylation of identified residues by centrosome-associated kinases. For a subset of phosphoproteins, we identified previously unknown centrosome and/or spindle localization via expression of tagged fusion proteins in Drosophila SL2 cells. Among those was otefin (Ote), an NE protein that we found to localize to centrosomes. Furthermore, we provide evidence that it is phosphorylated in vitro at threonine 63 (T63) through Aurora-A kinase. We propose that phosphorylation of this site plays a dual role in controlling mitotic exit when phosphorylated while dephosphorylation promotes G(2)/M transition in Drosophila SL2 cells.

Summary In this study we test the hypothesis that endogenous particles in exhaled air (PEx), non-invasively sampled from lower airways, are well suited for the analysis of respiratory tract lining fluid (RTLF) proteins, i.e., surfactant protein A (SP-A) and albumin. Ten healthy volunteers were included in the study and participated in two sampling sessions. Blood, exhaled breath condensate (EBC) and PEx were collected at each session. 100 L of breath were collected for each exhaled sample. Serum and exhaled samples were analyzed for SP-A using an in-house ELISA. Albumin was analyzed in exhaled samples using a commercial ELISA kit. SP-A detection rates were 100%, 21%, and 89% for PEx, EBC and serum, respectively. Albumin was detected in PEx, but not in EBC. SP-A measurements in PEx showed good repeatability with an intra-individual coefficient of variation of 13%. Both SP-A and albumin showed significant correlation to mass of PEx ( r s = 0.93, p r s = 0.86, p = 0.003, respectively). Sampling and analysis of PEx is a valid non-invasive method to monitor RTLF proteins sampled from the lower respiratory tract, as demonstrated here by example of SP-A and albumin analysis.

BACKGROUND We recently developed a novel, noninvasive method for sampling nonvolatile material from the distal airways. The method is based on the collection of endogenous particles in exhaled air (PEx). The aim of this study was to characterize the protein composition of PEx and to verify that the origin of PEx is respiratory tract lining fluid (RTLF). METHOD Healthy individuals exhaled into the sampling device, which collected PEx onto a silicon plate inside a 3-stage impactor. After their extraction from the plates, PEx proteins were separated by SDS-PAGE and then analyzed by LC-MS. Proteins were identified by searching the International Protein Index human database with the Mascot search engine. RESULTS Analysis of the pooled samples identified 124 proteins. A comparison of the identified PEx proteins with published bronchoalveolar lavage (BAL) proteomic data showed a high degree of overlap, with 103 (83%) of the PEx proteins having previously been detected in BAL. The relative abundances of the proteins were estimated according to the Mascot exponentially modified protein abundance index protocol and were in agreement with the expected protein composition of RTLF. No amylase was detected, indicating the absence of saliva protein contamination with our sampling technique. CONCLUSIONS Our data strongly support that PEx originate from RTLF and reflect the composition of undiluted RTLF.

Rationale Asthma is often characterised by inflammation, damage and dysfunction of the small airways, but no standardised biomarkers are available. Objectives Using a novel approach-particles in exhaled air (PExA)-we sought to (a) sample and analyse abundant protein biomarkers: surfactant protein A (SPA) and albumin in adult asthmatic and healthy patients and (b) relate protein concentrations with physiological markers using phenotyping. Methods 83 adult asthmatics and 21 healthy volunteers were recruited from a discovery cohort in Leicester, UK, and 32 adult asthmatics as replication cohort from Sweden. Markers of airways closure/small airways dysfunction were evaluated using forced vital capacity, impulse oscillometry and multiple breath washout. SPA/albumin from PEx (PExA sample) were analysed using ELISA and corrected for acquired particle mass. Topological data analysis (TDA) was applied to small airway physiology and PExA protein data to identify phenotypes. Results PExA manoeuvres were feasible, including severe asthmatic subjects. TDA identified a clinically important phenotype of asthmatic patients with multiple physiological markers of peripheral airway dysfunction, and significantly lower levels of both SPA and albumin. Conclusion We report that the PExA method is feasible across the spectrum of asthma severity and could be used to identify small airway disease phenotypes.

The technique of sampling exhaled air is attractive because it is noninvasive and so allows repeated sampling with ease and no risk for the patient. Knowledge of the biomarkers' origin is important to correctly understand and interpret the data. Endogenous particles, formed in the airways, are exhaled and reflect chemical composition of the respiratory tract lining fluid. However, the formation mechanisms and formation sites of these particles are unknown. We hypothesize that airway opening following airway closure causes production of airborne particles that are exhaled. The objective of this study was to examine production of exhaled particles following varying degrees of airway closure. Ten healthy volunteers performed three different breathing maneuvers in which the initial lung volume preceding an inspiration to total lung capacity was varied between functional residual capacity (FRC) and residual volume (RV). Exhaled particle number concentrations in the size interval 0.30–2.0 μm were recorded. Number concentrations of exhaled particles showed a 2- to 18-fold increase after exhalations to RV compared with exhalations where no airway closure was shown [8,500 (810–28,000) vs. 1,300 (330–13,000) particles/expired liter, P = 0.012]. The difference was most noticeable for the smaller size range of particles (

Trichomonas vaginalis causes the most common non-viral sexually transmitted infection linked to increased risk of premature birth, cervical cancer and HIV. This study defines molecular domains of the parasite surface glycoconjugate lipophosphoglycan (LPG) with distinct functions in the host immunoinflammatory response. The ceramide phospho-inositol glycan core (CPI-GC) released by mild acid had Mr of approximately 8,700 Da determined by MALDI-TOF MS. Rha, GlcN, Gal and Xyl and small amounts of GalN and Glc were found in CPI-GC. N-acetyllactosamine repeats were identified by endo-beta-galactosidase treatment followed by MALDI-MS and MS/MS and capLC/ESI-MS/MS analyses. Mild acid hydrolysis led to products rich in internal deoxyhexose residues. The CPI-GC induced chemokine production, NF-kappaB and extracellular signal-regulated kinase (ERK)1/2 activation in human cervicovaginal epithelial cells, but neither the released saccharide components nor the lipid-devoid LPG showed these activities. These results suggest a dominant role for CPI-GC in the pathogenic epithelial response to trichomoniasis.

BACKGROUND Exhaled breath contains nonvolatile substances that are part of aerosol particles of submicrometer size. These particles are formed and exhaled as a result of normal breathing and contain material from distal airways of the respiratory system. Exhaled breath can be used to monitor biomarkers of both endogenous and exogenous origin and constitutes an attractive specimen for medical investigations. CONTENT This review summarizes the present status regarding potential biomarkers of nonvolatile compounds in exhaled breath. The field of exhaled breath condensate is briefly reviewed, together with more recent work on more selective collection procedures for exhaled particles. The relation of these particles to the surfactant in the terminal parts of the respiratory system is described. The literature on potential endogenous low molecular weight compounds as well as protein biomarkers is reviewed. The possibility to measure exposure to therapeutic and abused drugs is demonstrated. Finally, the potential future role and importance of mass spectrometry is discussed. SUMMARY Nonvolatile compounds exit the lung as aerosol particles that can be sampled easily and selectively. The clinical applications of potential biomarkers in exhaled breath comprise diagnosis of disease, monitoring of disease progress, monitoring of drug therapy, and toxicological investigations.

Exhaled breath contains suspended particles of respiratory tract lining fluid from the small airways. The particles are formed when closed airways open during inhalation. We have developed a method called Particles in Exhaled air (PExA

Determining which proteins are unique among one or several protein populations is an often-encountered task in proteomics. To this purpose, we present a new method based on trypsin-catalyzed incorporation of the stabile isotope (18)O in the C-termini of tryptic peptides, followed by LC-MALDI MS analysis. The analytical strategy was designed such that proteins unique to a given population out of several can be assigned in a single experiment by the isotopic signal intensity distributions of their tryptic peptides in the recorded mass spectra. The method is demonstrated for protein-protein interaction analysis, in which the differential isotope labeling was used to distinguish endogenous human brain proteins interacting with a recombinant bait protein from nonbiospecific background binders.

We report the development of a robust interface for off-line coupling of nano liquid chromatography (LC) to matrix-assisted laser desorption/ionisation-mass spectrometry (MALDI-MS) and its application to the analysis of proteolytic digests of proteins, both isolated and in mixtures. The interface makes use of prestructured MALDI sample supports to concentrate the effluent to a small sample plate area and localize the MALDI sample to a predefined array, thereby enriching the analyte molecules and facilitating automated MALDI-MS analysis. Parameters that influence the preparation of MALDI samples from the LC effluent were evaluated with regard to detection sensitivity, spectra quality, and reproducibility of the method. A procedure for data processing is described. The presented nano LC MALDI-MS system allowed the detection of several peptides from a tryptic digest of bovine serum albumin, at analyzed amounts corresponding to one femtomole of the digested protein. For the identification of native proteins isolated from mouse brain by two-dimensional gel electrophoresis, nano LC MALDI-MS increased the number of detected peptides, thereby allowing identification of proteins that could not be identified by direct MALDI-MS analysis. The ability to identify proteins in complex mixtures was evaluated for the analysis of Escherichia coli 50S ribosomal subunit. Out of the 33 expected proteins, 30 were identified by MALDI tandem time of flight fragment ion fingerprinting.

To gain insights into complex biological processes, such as transcription and replication, the analysis of protein-DNA interactions and the determination of their sequence requirements are of central importance. In this study, we probed protein microarray technology and ultraviolet crosslinking combined with mass spectrometry (MS) for their practicability to study protein-DNA interactions. We chose as a model system the well-characterized interaction of bacterial replication initiator DnaA with its cognate binding site, the DnaA box. Interactions of DnaA domain 4 with a high-affinity DnaA box (R4) and with a low-affinity DnaA box (R3) were compared. A mutant DnaA domain 4, A440V, was included in the study. DnaA domain 4, wt, spotted onto FAST slides, revealed a strong signal only with a Cy5-labeled, double-stranded, 21-mer oligonucleotide containing DnaA box R4. No signals were obtained when applying the mutant protein. Ultraviolet crosslinking combined with nanoLC/MALDI-TOF MS located the site of interaction to a peptide spanning amino acids 433- 442 of Escherichia coli DnaA. This fragment contains six residues that were identified as being involved in DNA binding by recently published crystal structure and nuclear magnetic resonance (NMR) analysis. In the future, the technologies applied in this study will become important tools for studying protein-DNA interactions.

Sustained off resonance irradiation (SORI) collision-induced dissociation (CID) is a commonly used method of collisionally activating ions for fragmentation in Fourier transform mass spectrometric experiments. To achieve the degree of fragmentation desired, both the irradiation frequency and amplitude must be optimized. This is a time-consuming procedure, particularly when the m/z values of the precursor ions vary over a broad range. We present an approach that simplifies this optimization by precalculating the irradiation frequency of the ions to be fragmented as a constant percentage of the reduced cyclotron frequency. Using this approach, the optimal amplitude was found to be significantly less dependent on the m/z value of the precursor ion, and therefore required little or no adjustment. This method considerably simplified optimization of SORI-CID for analysis of carbohydrates, glycoconjugates, and peptides over the mass range m/z 300–3500, requiring optimization of only a single experimental parameter, the irradiation amplitude, and only for the first MS/MS stage.

Transiently elevating pressure in a matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) source into the 1-10 mbar range during ionization decreases the metastable fragmentation of gangliosides. This allows detection of the molecular ion species without loss of the highly labile sialic acid residues. In these experiments, gangliosides with up to five sialic acids were ionized by MALDI and detected with the FTMS. In each case, when the high pressure collisional cooling was used, the singly charged molecular ion was the base peak in the spectra, both in the positive and negative ion modes, and minimal metastable fragmentation was observed. This result is promising, as the previously developed TLC separation methods can be coupled to MALDI-FTMS.

Mass spectrometric identification of cysteinsulfinic acid resulting in restoration of activity of chemically modified Glu400 Cys catalytic-base glucoamylase (GA) mutants is described. This oxidation unexpectedly occurred during attempts to carboxyalkylate the Cys400 GA mutant using three different alkylation reagents. However, mass spectrometric peptide mapping did not show the presence of carboxyalkylation of the Cys400 residue but suggested an oxidation to cysteinsulfinic acid based on the observed mass increment. The presence of cysteinsulfinic acid was confirmed by employing matrix-assisted laser desorption/ionization mass spectrometry combined with post-source decay analysis. Furthermore, strong enhancement of metastable fragmentation was observed for peptides containing oxidized Cys in comparison with non-oxidized peptide. Copyright © 1999 John Wiley & Sons, Ltd.

A simple reversed-phase nano-column purification and sample preparation technique is described, which markedly improves the mass spectrometric analysis of complex and contaminated peptide mixtures by matrix-assisted laser desorption/ionization (MALDI). The method is simple, fast and utilizes only low-cost disposables. After loading the sample on the column and a subsequent washing step, the analyte molecules are eluted with 50-100 nl of matrix solution directly on to the MALDI/MS target. The washing step ensures removal of a wide range of contaminants. The small bed volume of the column allows efficient sample concentration and the elution process yields very small sample spots. This simplifies the analysis and minimizes discrimination effects due to sample heterogeneity, because the desorption/ionization laser simultaneously irradiates a large portion of the sample. Taken together, these features of the method significantly improve the sensitivity for MALDI/MS analysis of contaminated peptide samples compared with the commonly used sample preparation procedures. This is demonstrated with in-gel tryptic digests of proteins from human brain that were separated by 2D gel electrophoresis. Furthermore, it is shown that with this method 2,5-dihydroxybenzoic acid (DHB) acts as an efficient matrix for peptide mapping. Both detection sensitivity and sequence coverage are comparable to those obtained with the currently preferred matrix alpha-cyano-4-hydroxycinnamic acid (CHCA). The higher stability of peptide ions generated with DHB compared with CHCA is advantageous when analyzing fragile sample molecules. Therefore, the method described here is also of interest for the use of Fourier transform ion cyclotron resonance (FT-ICR) or ion-trap mass analyzers.

A method for mass spectrometric peptide mapping was developed, based on hydrolysis of a solid protein by acid vapor followed by mass spectrometric analysis of the cleavage products. The method is applicable to lyophilized samples as well as proteins present in gels after separation by SDS−PAGE. The cleavage specificity was established using a number of standard proteins. Three different types of cleavages were observed: specific internal backbone cleavages at Asp, Ser, Thr, and Gly and N- and C-terminal sequence ladders. On the basis of the observed cleavage characteristics, a strategy for protein identification based on the peptide mass maps was developed. The identification strategy utilizes the specific internal backbone cleavages as well as the partial sequence information, obtained from the sequence ladders.

Glucoamylase catalyzes the hydrolysis of glucosidic bonds with inversion of the anomeric configuration. Site-directed mutagenesis and three-dimensional structure determination of the glucoamylase from Aspergillus awamori previously identified Glu179 and Glu400 as the general acid and base catalyst, respectively. The average distance between the two carboxyl groups was measured to be 9.2 A, which is typical for inverting glycosyl hydrolases. In the present study, this distance was increased by replacing the catalytic base Glu400 with cysteine which was then oxidized to cysteinesulfinic acid. Initially, this oxidation occurred during attempts to carboxyalkylate the Cys400 residue with iodoacetic acid, 3-iodopropionic acid, or 4-bromobutyric acid. However, endoproteinase Lys-C digestion of modified glucoamylase followed by high-pressure liquid chromatography in combination with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry on purified peptide fragments demonstrated that all enzyme derivatives contained the cysteinesulfinic acid oxidation product of Cys400. Subsequently, it was demonstrated that treatment of Glu400--Cys glucoamylase with potassium iodide in the presence of bromine resulted in complete conversion to the cysteinesulfinic acid product. As expected, the catalytic base mutant Glu400--Cys glucoamylase had very low activity, i.e., 0.2% compared to wild-type. The oxidation of Cys400 to cysteinesulfinic acid, however, restored activity (kcat) on alpha-1,4-linked substrates to levels up to 160% of the wild-type glucoamylase which corresponded to approximately a 700-fold increase in the kcat of the Glu400--Cys mutant glucoamylase. Whereas Glu400--Cys glucoamylase was much less thermostable and more sensitive to guanidinium chloride than the wild-type enzyme, the oxidation to cysteinesulfinic acid was accompanied by partial recovery of the stability.