Browsing by Subject "LC-MS/MS"

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  • Avela, Henri F.; Siren, Heli (2020)
    The present article examines recently published literature on lipids, mainly focusing on research involving glycero-, glycerophospho- and sphingo-lipids. The primary aim is identification of distinct profiles in biologic lipidomic systems by ultra-high-performance liquid chromatography (UHPLC) coupled with mass spectrometry (MS, tandem MS) with multivariate data analysis. This review specifically targets lipid biomarkers and disease pathway mechanisms in humans and artificial targets. Different specimen matrices such as primary blood derivatives (plasma, serum, erythrocytes, and blood platelets), faecal matter, urine, as well as biologic tissues (liver, lung and kidney) are highlighted.
  • Junttila, Ilkka S.; Vuorio, Alpo; Budowle, Bruce; Laukkala, Tanja; Sajantila, Antti (2018)
    Diabetes mellitus (DM) could cause pilot incapacitation and result in aviation fatalities. The mechanisms could be directly as a consequence of acute hypoglycemia/subacute diabetic ketoacidosis (DKA) or indirectly as an acute cardiovascular event by contributing to the development of atherosclerosis in coronary or carotid and cerebral arteries. In this study, DM-related fatal flight accidents in the US National Transport Bureau's database between years 2011-2016 were analyzed with special emphasis on postmortem (PM) glucose levels and correlation of toxicological reports with anamnestic information on DM. Additionally, autopsy results on coronary arteries were reviewed. In 43 out of 1491 (similar to 3%) fatal accidents pilots had DM. Postmortem glucose or glycated hemoglobin percentage (Hb1Ac) was measured in 12 of the 43 cases; while antidiabetic medication was found in 14 of the cases (only two of the cases had both glucose measurements and medication). With the increasing prevalence of DM, a possibility of pilot incapacitation due to DM or complications of DM should be actively studied, even if no anamnestic information of DM was available. While PM hypoglycemia is difficult to assess, we propose a systematic investigation based on measurement of glucose, Hb1Ac%, and ketone bodies, and documentation of atherosclerotic lesions in major arteries to identify or rule out DM as a cause of pilot incapacitation.
  • Becker, Anna; Backman, Janne T.; Itkonen, Outi (2020)
    Introduction: Life-long monitoring of immunosuppressive drugs (ISDs) in blood is essential after organ transplantation. However, the ISD concentrations vary depending on the assay employed. ISDs are strongly bound to cytoplasmic proteins in erythrocytes in circulation. Therefore, the relatively rapid sedimentation of blood cells in whole blood samples may affect the results when using liquid handling robots. Methods: We used 1115 blood samples from outpatients and ward patients with kidney (n = 373), liver (n = 101), heart (n = 29) and bone marrow (n = 155) transplant. Whole blood samples were pretreated by protein precipitation. Alternatively, the samples were hemolyzed by freezing prior to precipitation. ISDs were analyzed by a 2-plexing liquid chromatography tandem mass spectrometry (LC-MS/MS) assay and commercial chemiluminescent microparticle immunoassays (CMIA). Results: The difference between the two sample preparation practices was negligible (<2%). Overall, the measured ISD concentrations in patient samples were lower by LC-MS/MS than by CMIA. The difference was the largest (20.2%) and the smallest (9.1%) in samples from liver and from heart transplant patients, respectively. Conclusions: CMIA overestimates blood ISD concentrations as compared to LC-MS/MS. The extent of the difference was found to be organ transplant dependent. The ISDs can be quantitated either from intact or hemolyzed blood samples.
  • Becker, Anna; Schalin-Jäntti, Camilla; Itkonen, Outi (2021)
    Context: Patients with serotonin-secreting neuroendocrine neoplasms (NENs) have increased serum 5-hydroxyindoleacetic acid (5HIAA) concentrations. Serum 5HIAA thus serves as a biomarker in NEN. Objective: To evaluate an improved tandem mass spectrometric serum 5HIAA assay for diagnosis and follow-up of NEN in a clinical cohort. Design: A retrospective study during 2016-2018 at the Diagnostic Center and Department of Endocrinology at Helsinki University Hospital, Finland. Methods: Detailed patient data was obtained from 116 patients. Serum 5HIAA was analyzed by 2 different liquid chromatography with tandem mass spectrometry (LC-MS/MS) assays with samples prepared either by protein precipitation or solid phase extraction. Twenty-four-hour urine 5HIAA samples (n = 33) were analyzed by amperometric LC, and the results were compared. Specificity and sensitivity were calculated by receiver operating characteristic (ROC) analysis. Results: We achieved 5 to 10 000 nmol/L linearity and Conclusion: Serum 5HIAA by LC-MS/MS after protein precipitation performs equally well for the diagnosis of NEN as urinary 5HIAA LC assay. The outcome and sensitivity for serum and 24-h urine assays are convergent. Due to much more reliable and convenient sampling, we recommend serum instead of 24-h urine 5HIAA for diagnosis and follow-up of NEN patients.
  • Lindström, Mikael; Tohmola, Niina; Renkonen, Risto; Hämäläinen, Esa; Schalin-Jäntti, Camilla; Itkonen, Outi (2018)
    Background: Serotonin (5-hydroxytyramine) is a mediator of gastrointestinal smooth muscle contraction, and is secreted by neuroendocrine neoplasms (NENs). We developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for serum serotonin to be used in NEN diagnostics and follow-up. Methods: We used serum samples from healthy volunteers (n = 31) and patients suspected or monitored for NEN (n = 98). Serotonin-D-4 internal standard was added to samples before solid phase extraction (SPE) and quantification by LC-MS/MS. The effects of sample handling and preparation on serotonin stability were studied. Finally, we established a provisional reference range for serum serotonin and compared our assay with serum 5hydroxyindoleacetic acid (5-HIAA) for detection of NENs. Results: Our assay is sensitive and has a wide linear range (10-10,000 nmo1/1). Serum serotonin is stable for 7 days at room temperature and for 3 months at -20 degrees C. Sampling temperature is not critical. Normal range for serum serotonin was 270-1490 nmo1/1. We found that serum serotonin and 5-HIAA performed equally well as diagnostic tests for NENs. Conclusions: Our LC-MS/MS assay for serum serotonin is well suited for clinical research and patient diagnostics. Our results confirm that it can complement 5-HIAA in diagnosis of NENs.
  • Sivaranjani, Murugesan; Leskinen, Katarzyna; Aravindraja, Chairmandurai; Saavalainen, Päivi; Pandian, Shunmugiah Karutha; Skurnik, Mikael; Ravi, Arumugam Veera (2019)
    Background: Alpha-mangostin (alpha-MG) is a natural xanthone reported to exhibit rapid bactericidal activity against Gram-positive bacteria, and may therefore have potential clinical application in healthcare sectors. This study sought to identify the impact of alpha-MG on Staphylococcus epidermidis RP62A through integrated advanced omic technologies. Methods: S. epidermidis was challenged with sub-MIC (0.875 mu g/ml) of alpha-MG at various time points and the differential expression pattern of genes/proteins were analyzed in the absence and presence of alpha-MG using RNA sequencing and LC-MS/MS experiments. Bioinformatic tools were used to categorize the biological processes, molecular functions and KEGG pathways of differentially expressed genes/proteins. qRT-PCR was employed to validate the results obtained from these analyses. Results: Transcriptomic and proteomic profiling of alpha-MG treated cells indicated that genes/proteins affected by alpha-MG treatment were associated with diverse cellular functions. The greatest reduction in expression was observed in transcription of genes conferring cytoplasmic membrane integrity (yidC2, secA and mscL), cell division (ftsY and divlB), teichoic acid biosynthesis (tagG and dltA), fatty-acid biosynthesis (accB, accC, fabD, fabH, fabl, and fabZ), biofilm formation (icaA) and DNA replication and repair machinery (polA, polC, dnaE, and uvrA). Those with increased expression were involved in oxidative (katA and sodA) and cellular stress response (clpB, clpC, groEL, and asp23). The qRT-PCR analysis substantiated the results obtained from transcriptomic and proteomic profiling studies. Conclusion: Combining transcriptomic and proteomic methods provided comprehensive information about the antibacterial mode of action of alpha-MG. The obtained results suggest that alpha-MG targets S. epidermidis through multifarious mechanisms, and especially prompts that loss of cytoplasmic membrane integrity leads to rapid onset of bactericidal activity.
  • Syvähuoko, Jenna (Helsingin yliopisto, 2015)
    The literature review focused on the chemical properties of Fusarium mycotoxins and their masked forms, analytical methods for their determination and the toxicological and legislative aspects. In the experimental study, a multi-method was developed and validated for the simultaneous quantification of several Fusarium toxins and their masked forms in barley, oats and wheat using liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique. The simple “dilute-and-shoot” sample preparation procedure was applied, where the extraction was performed with a mixture of acetonitrile, water and acetic acid (79:20:1, v/v/v). Moreover, the aim was to obtain new data on the occurrence of the masked mycotoxins in barley, oats and wheat by analysing 95 cereal grain samples. The type A trichothecenes T-2 and HT-2 toxins (T-2 and HT-2) and the type B trichothecenes deoxynivalenol (DON) and nivalenol (NIV) as well as zearalenone (ZEN), together with 11 masked forms of them, were included based on their importance for the food safety in northern Europe. The analytes were separated on a reversed-phase column and detected in selected reaction monitoring (SRM) mode. Better peak shapes for the early eluting compounds and shorter analysis time were obtained with acetonitrile than methanol as the organic phase, thus it was chosen for the method. The method was validated according to the criteria set in the legislation. The limits of quantification varied from 0.3 to 15.9 ?g/kg. The recoveries were 92?115%, thus being within the tolerable ranges established in the legislation. The inter-day precisions (4?27%) were under the maximum permissible values. Therefore, the method proved to fit for the purpose. In this study, occurrence data on the masked mycotoxins in Finland were obtained for the first time. The presence of ZEN-16-glucoside (ZEN-16-G) and NIV-3-glucoside (NIV-3-G) were reported for the first time worldwide in some of the cereals. The most frequently found toxins were DON, NIV and HT-2. All of the masked mycotoxins included in the method were determined, the most common being DON-3-glucoside (DON-3-G), HT-2-glucoside (HT-2-G) and NIV-3-G.
  • Varis, Vera (Helsingin yliopisto, 2020)
    Protein kinases are signaling molecules that regulate vital cellular and biological processes by phosphorylating cellular proteins. Kinases are linked to variety of diseases such as cancer, immune deficiencies and degenerative diseases. This thesis work aimed to identify direct substrates for protein kinases in the CMGC family, which consists of the cyclin-depended kinases (CDK), mitogen activated protein kinases (MAPK), glycogen synthase kinase-3 (GSK3) and CDC-like kinases (CLK). CMGC kinases have been identified as cancer hubs in interactome studies, but large-scale identification of direct substrates has been difficult due to the lack of efficient methods. Here, we present a heavy-labeled 18O-ATP-based kinase assay combined with LC-MS/MS analysis for direct substrate identification. In the assay, HEK and HeLa cell lysates are treated with a pan-kinase inhibitor FSBA which irreversibly blocks endogenous kinases. After the removal of FSBA, cell lysates are incubated with the kinase of interest and a heavy-labeled ATP, which contains 18O isotope at the γ-phosphate position. Resulting phosphopeptides are enriched with Ti4+- IMAC before the LC-MS/MS analysis, which distinguishes the desired phosphorylation events based on a mass shift caused by the heavy 18O. With this pipeline of methods, we managed to quantify and identify direct substrates for 26 members of CMGC kinase family. A total of 1345 substrates and 3841 interacting kinase-substrate pairs were identified in cytosolic cell lysates, from which 165 were annotated in the PhosphoSitePlus® database. To identify substrates for kinases with nuclear localization, ten kinases were tested with nuclear HEK cell lysate. We identified 194 kinase-substrate pairs, 141 of which were unique to the nuclear fraction and 27 annotated in the PhosphoSitePlus® database. Finally, kinases with outstandingly high amounts of novel substrates were subjected to gene ontology analysis. We were able to link the gene ontology classifications of novel substrates to the biological processes regulated by the kinase of interest. These results indicate that heavy-labeled 18O-ATP-based kinase assay linked LC-MS/MS is a useful tool for large-scale direct kinase substrate identification.
  • Tervahauta, Tuomas (Helsingfors universitet, 2015)
    Prodrugs are pharmacologically inactive molecules which undergo metabolic bioactivation in vivo to form pharmaceutically active agents. Prodrugs have been designed to improve so called drug-like properties of active parent compounds (APC) i.e. to increase solubility or absorption and to reduce first-pass metabolism etc. In this master's thesis the goal was to establish non-cell-based in vitro methods to study prodrug bioactivation. Four commercially available prodrugs (bambuterol, olmesartan medoxomil (OM), candesartan cilexetil (CC) and famciclovir) were used as test compounds. The prodrugs were incubated in liver and intestinal S9 fractions and blood plasma to study in vitro bioactivation of these prodrugs. Other metabolism of the prodrug and APC (nonproductive metabolism) was studied by comparing incubation with and without cofactors of metabolizing enzymes. Species differences was studied using human, rat and dog matrices. Prodrug concentrations were quantified from the incubation samples using liquid chromatography- tandem mass spectrometry (LC-MSMS) methods developed for this study. Additionally the effect of promoiety on passive permeability was studied with parallel artificial membrane permeability assay (PAMPA). All of the studied prodrugs produced at least low concentrations of APC in one or more incubations. Terbutaline (APC of bambuterol) formation was observed in human plasma and was concentration dependent which is consisted with the literature. Olmesartan and candesartan were formed in S9 fraction in high rate, but not in buffer: indicating enzyme mediated hydrolysis. However, based on literature CC hydrolysis was not expected to occur in intestinal S9 fractions. Penciclovir (APC of famciclovir) was formed only in presence of human or rat liver S9 fraction which was in line with the pre-existing literature. With the method used the nonproductive metabolism could not be estimated. In PAMPA bambuterol, famciclovir and OM had higher permeability than corresponding APCs whereas CC was only more permeable than candesartan in pH 7.4. The in vitro incubation used in this study can be used for screening prodrugs. However both low and high activation rates were observed thus the clinically relevant in vivo APC formation can be achieved with both high and low bioactivation in vitro. Studying the rate of prodrug formation alone estimations about clinically relevant bioactivation rates cannot be concluded. No clear signs of nonproductive could be seen with the prodrugs studied with current method. For the estimation of nonproductive metabolism, metabolite screening studies would need to be developed and conducted parallel to studies prescribed in this master's thesis.
  • Söderholm, Sandra; Hintsanen, Petteri; Ohman, Tiina; Aittokallio, Tero; Nyman, Tuula A. (2014)
  • Ranta, Merja; Ketola, Raimo (2016)
    A liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the analysis of seven common sartans (candesartan, eprosartan, losartan, olmesartan, telmisartan, valsartan, and losartan carboxylic acid (EXP3174), the active metabolite of losartan) in postmortem blood samples with liquid-liquid extraction. Detection was accomplished by triple-quadrupole MS in the selected reaction monitoring mode. The validation procedure showed low limits of quantitation of 5 ng/mL for all compounds with good accuracy and precision. The matrix effect was evaluated from the variation of peak areas and concentrations. The method shows some matrix effect but the effect was efficiently compensated by using an internal standard method. The method was applied to the analysis of sartans in postmortem blood samples, and produced 534 positive findings during 2014-2015. Nine deaths were encountered where the concentration of a sartan was very high, indicating intoxication together with other causes of death or multi-drug intoxication. The quantitative results indicate that sartans do not show significant postmortem redistribution; thus concentrations higher than the therapeutic range can be considered as being toxic or fatal.
  • Mannelli, Petriina (Helsingin yliopisto, 2019)
    Psykotrooppiset lääkeaineet vaikuttavat potilaan psyykeeseen. Tällaisiä lääkeaineita ovat esimerkiksi antipsykoottiset ja antidepressiiviset lääkeaineet. Ne ovat tehokkaita tautien hoidossa, mutta vakavien haittavaikutusten välttämäiseksi lääkeaineenpitoisuuden tasoa tulee seurata. Terapeuttisessa lääkkeen monitoroinnissa on tärkeää toteuttaa seurantasuunnitelmaa, etenkin klotsapiiniä sekä norklotsapiini käyttävien potilaiden kohdalla. Tämän maisterin tutkielman kirjallisessa osassa tarkastastellaan psykotrooppisten lääkeaineiden luokituksia ja molekyylirakennetta. Lisäksi siinä esitellään neste- ja kaasukromatografisia erotusmenetelmiä eri detektoreilla, sekä nestekromatografinen laitteisto. Kromatografiset menetelmät ovat tärkeässä roolissa nykyaikana määritettäessä psykotrooppisia lääkeaineita. Massaspektrometrin käyttö detektorina on yleistynyt huomattavasti, mutta kliinisessä työssä suositaan myös UV-Vis- detektoreja tai diodirividetektoreja. Usein näytteen esikäsittelyssä käytetään aikaa vievää sekä kallista neste-neste uuttoa. Joskus käytetään myös kapillaarissa tapahtuvaa kiinteäfaasiuuttoa tai saostusta. Tutkielman kokeellisessa osassa validoitiin analyysimenetelmä, LC-MS/MS, joka oli lineaarinen, selektiivinen, tarkka ja yksityiskohtainen, sekä toistettava. Lyhyt retentioaika, tehokas ja helppo näytteenesikäsittely saostamalla ja Tecan nesteensiirtorobotilla tehtävä näytteensiirto rutiinianalysointia varten oli suuri etu verrattuna käytössä olleeseen HPLC-UV/Vis- menetelmään. Nykyisellä kuoppalevyllä voidaan tehdä 96- näytteen sarja kun aiemmin pystyttiin analysoimaan vain 50 näytteen sarja. Menetelmä paransi myös muiden analysoitavien näytteiden häiritsevyyttä tarkemmalla detektorilla. Lisäksi tarkoituksena oli vähentää orgaanisten liuottimien kulutusta muuttamalla neste-nesteuutto proteiinien saostukseksi.
  • TEDDY Study Grp; Stanfill, Bryan A.; Nakayasu, Ernesto S.; Bramer, Lisa M.; Knip, Mikael (2018)
    Liquid chromatography-mass spectrometry (LC-MS)-based proteomics studies of large sample cohorts can easily require from months to years to complete. Acquiring consistent, high-quality data in such large-scale studies is challenging because of normal variations in instrumentation performance over time, as well as artifacts introduced by the samples themselves, such as those because of collection, storage and processing. Existing quality control methods for proteomics data primarily focus on post-hoc analysis to remove low-quality data that would degrade downstream statistics; they are not designed to evaluate the data in near real-time, which would allow for interventions as soon as deviations in data quality are detected. In addition to flagging analyses that demonstrate outlier behavior, evaluating how the data structure changes over time can aide in understanding typical instrument performance or identify issues such as a degradation in data quality because of the need for instrument cleaning and/or re-calibration. To address this gap for proteomics, we developed Quality Control Analysis in Real-Time (QC-ART), a tool for evaluating data as they are acquired to dynamically flag potential issues with instrument performance or sample quality. QC-ART has similar accuracy as standard post-hoc analysis methods with the additional benefit of real-time analysis. We demonstrate the utility and performance of QC-ART in identifying deviations in data quality because of both instrument and sample issues in near real-time for LC-MS-based plasma proteomics analyses of a sample subset of The Environmental Determinants of Diabetes in the Young cohort. We also present a case where QC-ART facilitated the identification of oxidative modifications, which are often underappreciated in proteomic experiments.
  • Eskelin, Katri; Varjosalo, Markku; Ravantti, Janne; Makinen, Kristiina (2019)
    Nicotiana benthamiana is an important model plant for plant-microbe interaction studies. Here, we compared ribosome profiles and riboproteomes of healthy and infected N. benthamiana plants. We affinity purified ribosomes from transgenic leaves expressing a FLAG-tagged ribosomal large subunit protein RPL18B of Arabidopsis thaliana. Purifications were prepared from healthy plants and plants that had been infiltrated with Agrobacterium tumefaciens carrying infectious cDNA of Potato virus A (PVA) or firefly luciferase gene, referred to here as PVA- or Agrobacterium-infected plants, respectively. Plants encode a number of paralogous ribosomal proteins (r-proteins). The N. benthamiana riboproteome revealed approximately 6600 r-protein hits representing 424 distinct r-proteins that were members of 71 of the expected 81 r-protein families. Data are available via ProteomeXchange with identifier PXD011602. The data indicated that N. benthamiana ribosomes are heterogeneous in their r-protein composition. In PVA-infected plants, the number of identified r-protein paralogues was lower than in Agrobacterium-infected or healthy plants. A. tumefaciens proteins did not associate with ribosomes, whereas ribosomes from PVA-infected plants co-purified with viral cylindrical inclusion protein and helper component proteinase, reinforcing their possible role in protein synthesis during virus infection. In addition, viral NIa protease-VPg, RNA polymerase NIb and coat protein were occasionally detected. Infection did not affect the proportions of ribosomal subunits or the monosome to polysome ratio, suggesting that no overall alteration in translational activity took place on infection with these pathogens. The riboproteomic data of healthy and pathogen-infected N. benthamiana will be useful for studies on the specific use of r-protein paralogues to control translation in infected plants.
  • Ketola, Raimo A.; Ojanperä, Ilkka (2019)
    Concentration distributions for 183 drugs and metabolites frequently found in post-mortem (PM) femoral venous blood were statistically characterized based on an extensive database of 122 234 autopsy cases investigated during an 18-year period in a centralized laboratory. The cases represented all causes of death, with fatal drug poisonings accounting for 8%. The proportion of males was 74% with a median age of 58 years compared with 26% females with a median age of 64 years. In 36% of these cases, blood alcohol concentration was higher than or equal to 0.2 parts per thousand, the median being 1.6 parts per thousand. The mean, median, and upper percentile (90th, 95th, 97.5th) drug concentrations were established, as the median PM concentrations give an idea of the "normal" PM concentration level, and the upper percentile concentrations indicate possible overdose levels. A correspondence was found between subsets of the present and the previously published PM drug concentrations from another laboratory that grouped cases according to the cause of death. Our results add to the knowledge for evidence-based interpretation of drug-related deaths.
  • Ahmed, Mohamed Adel (Helsingin yliopisto, 2015)
    Lactobacillus rhamnosus GG (GG) is one of the most studied probiotics worldwide and it has proved to confer health benefits to human. However, the exact mechanism of its probiotic action is still not fully understood. Some proteins secreted by probiotics, such as p75 (Msp1) and p40 (Msp2) in GG, are reported to play an important role in gut epithelial homeostasis. The aim of this work was to develop a static biofilm plate model for analyzing exoproteomes (all secreted proteins) produced by L. rhamnosus GG biofilms. First, different culture volumes (7 mL and 10 mL), incubation times and protein precipitation methods were tested to optimize conditions in order to maximize the protein yield. Next, the impact of different sugars on the exoproteome composition of the GG cells during planktonic and biofilm mode of growth was explored. Finally, the planktonic and biofilm exoproteins were subjected to antigen profiling and protein identification. The 6-well Polystyrene Microtiter plate was used as the static biofilm model for inducing the biofilm formation of GG. Biofilms were formed for 24 h and 48 h and the protein secretion from each time point was assessed by precipitating the supernatant proteins using 10% trichloroacetic acid (TCA)/acetone or 2-D Clean Up kit (GE Healthcare). The purified proteins were subjected to one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1-DE) and the proteins were visualized using Coomassie blue staining. 1-DE immunoblotting using antibodies raised against whole GG cells was used to analyze antigens produced by the GG biofilms under the optimized biofilm formation conditions. The antigens produced by the GG biofilm cells were compared to those produced by the GG cells during planktonic growth (from over- night cultures) and the cross-reacting proteins were visualized using an IR800-conjugated secondary antibody. The immunoblots were scanned using an Odyssey Infrared Imaging System (Licor). In addition, the impact of two different carbohydrate sources on the antigen profiles was also explored. Finally, in-liquid tryptic digestion coupled with Liquid Chromatography- tandem Mass Spectrometry analysis (LC-MS/MS) was used to identify and compare exoproteomes produced by the biofilm cells cultured in the presence of the tested carbohydrates. The results showed that the commercial 2-D Clean Up kit is better than 10% TCA protein precipitation/acetone washing, because it produced clear protein patterns with less background. However, the TCA/acetone–protocol resulted in detection of higher number of proteins in 1-DE gels. Comparative immunoblot analyses of the planktonic and biofilm exoproteins at 24 h and 48 h time points revealed a clear difference in antigen profiles between the two modes of growth. In addition, the utilized carbon source was found to have a great impact on the antigen abundances and/or export. Using LC-MS/MS, 36 exoproteins were identified from the GG biofilm cultures (identification score ≥ 40, p < 0.05). Most of the identified proteins were associated with cell wall/membrane biogenesis, peptide and sugar transporters and transcription.
  • Ven, Katharina (Helsingin yliopisto, 2019)
    Lipid droplets (LDs) are ubiquitous intracellular storage organelles, consisting of a core of energy rich neutral lipids surrounded by a phospholipid monolayer. Research in the past decade has expanded the view on LDs from simple, passive cytosolic inclusions to dynamic organelles which play an important role in many cellular processes. Furthermore, there is mounting evidence for links between LD biology and human pathologies, such as metabolic disorders, non-alcoholic fatty liver disease and cardiovascular diseases. Thus, understanding the basic biology of LD formation is crucial. LD biogenesis is thought to occur in the microdomains of endoplasmic reticulum (ER), due to the accumulation of neutral lipids between the two leaflets of the ER bilayer before budding into the cytosol. Many proteins are involved in this early formation, but no single indispensable protein has been discovered. After assembly, these early LDs grow through lipid deposition from the ER, and with lipid synthesis on the droplet monolayer. During LD growth, LDs are thought to retain connection to the ER. A protein important for LD biogenesis is seipin. This oligomeric ER protein has been found to localize at contact sites between the ER and LDs. Mutations in seipin give rise to three distinct diseases in humans; BSCL2, seipinopathy and Celia’s encephalopathy. The role of seipin in the formation of LDs and the pathogenesis of these diseases is still unknown. Work from numerous model systems has shown seipin to be important for LD biogenesis and adipocyte differentiation. LD formation is a complex process which is still poorly understood, and seipin likely collaborates with other proteins during LD assembly. In this thesis, APEX2-mediated proteome mapping combined with LC-MS/MS, is set up to identify proteins involved in LD biogenesis. In this technology, an engineered ascorbate peroxidase, APEX2, is genetically inserted to the intracellular region of interest where it rapidly biotinylates nearby endogenous proteins upon exposure to biotin-phenol and hydrogen peroxide. Biotinylated proteins can then be enriched by using streptavidin beads and identified with a mass spectrometry. The aim using this technology is to unravel new interaction partners of seipin and proteins important for LD formation, which is a crucial step for understanding LD formation and diseases related to it.