Natrium-kaliumpumpen spelar en väsentlig roll i en rad fysiologiska funktioner då den upprätthåller den elektrokemiska gradienten över cellmembranet. Ytterligare så är störningar i dess funktion associerade med flera neurologiska sjukdomar. Proteinet är en heterodimer av α– och β–subenheter, ibland även associerat med en tredje γ (FXYD) subenhet, vilket gör det problematiskt att studera dess högre ordningens organisation i cellmembranet med hjälp av konventionella, relativt storskaliga inmärkiningsprober såsom antikroppar. Inkorporering av icke-kanoniska aminosyror är ett nyutvecklat och växande område som erbjuder en lösning. Genom CuAAC– och SPIEDAC–klickkonjugationsreaktioner kan organiska färgämnen (fluoroforer) snabbt och specifikt fästas i sidokedjor med motsvarande reaktiva grupper på jonpumpen, vilket skapar en liten och icke-invasiv inmärkningsprob för fluorescensmikroskopi. För att specifikt studera alla tre subenheter samtidigt krävs inmärkning med tre olika fluoroforer). Syftet med detta projekt var att lyckas med trefärgsinmärkning genom inkorporering av icke-kanoniska aminosyror, och därigenom underlätta studerandet av hur natrium-kaliumpumpens subenheter ordnar sig i cellmembranet. Transient transfekterade HEK293T-celler med membraninmärkta jonpumpar studerades med hjälp av fluorescensmikroskopi, vilket kompletterades med gelfluorescensavbildning och immunoblotting. Samtidigt gjordes proteinuttryck och tvåfärgsinmärkning av alla nonsenskodonmuterade subenheter i kombination med varandra och var synlig i proteingel, där endast α och β tidigare hade samuttryckts. α/γ parinmärkning visade sig framgångsrik när de samtransfekterades med β av vildtyp. En autofluorescenseffekt i en av färgkanalerna påverkade resultaten för mikroskopin. Trefärgsinmärkning observerades inte i gelen, och uttrycket av subenheterna (varav α var ersatt för detta experiment) var i stort sett obefintligt. Otydlighet består därmed huruvida trefärgsinmärkning eller trippelsamuttryck är möjligt med de bioortogonala translationssystemen som användes i detta projekt på jonpumpen. / Na+/K+-ATPase is an essential ion pump protein in a host of physiological functions as it maintains the electrochemical gradient across cell membranes. Additionally, its dysfunction is implicated in several neurological diseases. The protein is a heterodimer of α and β subunits, occasionally associated with a third γ (FXYD) subunit, which makes studying its higher order organization in the cell membrane difficult using conventional, relatively large scale labeling probes such as antibodies. Non-canonical amino acid incorporation is an emerging field which offers a solution. Via CuAAC and SPIEDAC click conjugation reactions, organic fluorophores can be specifically attached to the side chains of residues of the ion pump with corresponding reactive moieties, creating a small and noninvasive probe for fluorescence microscopy imaging. In order to specifically image all three subunits concurrently, three color labeling is required. The objective of this project was to achieve three color labeling via non-canonical amino acid incorporation to aid in the study of the cell membrane localization of the subunits of Na+/K+-ATPase. Fluorescence microscopy of transiently transfected and live cell labeled HEK293T cells was complemented by in gel fluorescence imaging and immunoblotting. Coexpression and two color labeling of all nonsense codon subunit mutants in combination was shown in gel, of which only α and β had previously been coexpressed. α/γ dual labeling proved successful when cotransfected with wild type β. An autofluorescent effect in one of the color channels compromised the microscopy results. Three color labeling was not observed in gel, and expression of the subunits (including a substitute for α) was middling to absent. It remains unclear whether three color labeling or triple coexpression is a possibility with the bioorthogonal translation systems used in this project.

Sample preparation as an essential step in mass spectrometry-based analysis, plays a critical role in proteomics studies. Magnetic nanoparticles (MNPs) have been widely used in protein and peptide sample preparation due to their magnetic properties, biocompatibility, easy synthesis and surface functionalization. MNPs loaded with analyte or analyte modification reagent can be easily separated from the reaction medium by an externally applied magnetic field. The small size of MNPs provides high analyte loading and extraction capacity. Additionally, MNP can be decorated with different functional groups to achieve selective modification or extraction of analyte. In this study we have utilized silica coated iron oxide magnetic nanoparticles (Fe3O4@SiO2 MNPs) for protein and peptide sample preparation. Fluorescence-based methods were utilized for quantitative and qualitative characterization of N-hydrosucccinimidyl (NHS) ester groups on the surface of Fe3O4@SiO2 MNPs. Fluorophore Dansylcadaverine was conjugated to NHS ester functional groups. Fluorometric measurement of cleaved dansylcadaveine was employed to determine the number of NHS ester groups per MNPs that was found to be 2.6 × 102 and 3.4 × 103for 20 nm and 100 nm Fe3O4@SiO2 MNPrespectively. The efficiency of labeling native bovine serum albumin (BSA) by NHS ester coated Fe3O4@SiO2 MNPs was also explored in terms of maximizing the number of MNPs conjugated per BSA molecule or maximizing the number of BSA molecules conjugated per each MNP. Lysine residues of apolipoprotein B-100 (apoB-100) on the surface of intact human low density lipoprotein (LDL) were labeled by NHS ester modified Fe3O4@SiO2 MNPs in aqueous solvents at room temperature. The MNP labeledapoB-100 was treated by SDS to remove lipids and then digested using trypsin. Tryptic peptides were eluted from MNPs by cleaving disulfide linkage between labeled peptides and MNPs. LC-MS/MS analysis found 28 peptides containing labeled lysine residues. These lysine residues should be on the solvent exposed surface of LDL since the large size of MNPs prevents contact of the labeling reagent to those lysines embedded inside the structure of LDL. TCEP- immobilized Fe3O4@SiO2MNPs were fabricated and utilized for reduction of disulfide bonds in bovine pancreas insulin and two different cyclic peptides. Disulfide bonds were efficiently cleaved at room temperature in both organic and aqueous solvents confirmed by LC-MS/MS analysis of reduced/alkylated protein and peptides. Disulfide reduction and alkylation reactions was performed in one step and the reducing agent was simply separated from peptide and protein solution by magnetic separation.