Browsing by Subject "Massenspektrometrie"

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External nutrition stimuli induced proteome and phosphoproteome responses of maize root hairs and arabidopsis root microsomal fraction
(2021) Li, Zhi; Schulze, Waltraud
This work studied how the proteome from young maize root hair cells responds to different nutrition deprivation, and gives perspectives to the possible involvement of NRT1.1 and NRT2.1 in regulating root membrane phosphoproteome responses. This work also proposes a phospho-switch model that may explain how the NRT2.1 activity was regulated.
Quantitative Proteomanalyse von Pseudomonaden zur Aufklärung biotechnologisch relevanter Stoffwechselwege
(2013) Simon, Oliver; Huber, Armin
The main focus of this work was a quantitative proteome analysis of a variety of Pseudomonas strains with respect to the biotechnological synthesis of the base chemicals glyoxylic acid, butanol and vanillin. In addition, effects of the terpene citronellol on the proteome of P. aeruginosa were investigated. A second key aspect of this work involved the establishment of proteomics methods for the analysis of complex samples, especially for the analysis of membrane proteins. Using carbonate extraction followed by label-free MS-based quantification allowed the identification and quantification of a significant number of hydrophobic proteins which were not covered by the 2D-DIGE approach. In addition, the GeLCMSMS workflow was found to be a simple and efficient method for the analysis of total bacterial lysates. Using this method, about 30% of all proteins encoded by the P. putida KT2440 genome could be identified and quantified. In conclusion, this work demonstrated that different proteomics methods can substantially contribute to biotechnological strain development and the understanding of cellular networks.
The formation of an apoplastic diffusion barrier in Arabidopsis seeds is regulated by peptide hormone signaling
(2022) Royek, Stefanie; Schaller, Andreas
Diffusion barrier formation is a critical factor in plant development. The most well described diffusion barriers in Arabidopsis are the Casparian strip and the cuticle. They function in the formation of organ boundaries, prevent water and molecule loss, and protect the plant against environmental stresses. The Casparian strip surrounds the root vascular tissue, whereas the cuticle covers aerial plant organs and is formed de novo during seed development. Embryonic cuticle formation is regulated by a peptide hormone signaling pathway, involving the leucine rich repeat receptor like kinases GASSHO1 (GSO1), GASSHO2 (GSO2) (Tsuwamoto et al. 2008) and the subtilisin-like serine protease ABNORMAL LEAF SHAPE 1 (ALE1). Whereas the latter pathway components have been identified in 2001 and 2008, the peptide hormone mediating the signaling has remained elusive. One aim of this work was to identify the missing pathway element. It was hypothesized that the peptide hormone is released from a larger precursor by ALE1 protease activity to trigger cuticle formation via interaction with the GSO receptors. To uncover the unknown element, the signaling pathway for Casparian strip formation, prooved to be a useful lead. Remarkably, Casparian strip and embryonic cuticle formation employ the same receptor (GSO1), and for Casparian strip formation the GSO1 ligands are known to be members of the CASPARIAN STRIP INTEGRITY FACTOR (CIF) protein family (Doblas et al. 2017, Nakayama et al. 2017). Based on its similarity to the mature CIF peptides and on its phenotypic appearance, it was speculated that a seed expressed protein, called TWISTED SEED1 (TWS1), could serve as the sought ALE1 substrate. As it can be challenging to link proteases to their physiological substrates, this work describes methods how to identify protease specific cleavage sites. One of them was applied to test if TWS1 serves as ALE1 substrate. GFP-tagged TWS1 was transiently coexpressed with ALE1 in Nicotiana benthamiana via agroinfiltration. An ALE1-specific TWS1 cleavage product was detected in the protein extract of coinfiltrated leaves. It was identified by pull down via GFP immunoprecipitation, subsequent separation by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MS) analysis. Another method, described in this work, is the identification of protease cleavage sites by in-gel reductive dimethylation: cleavage product-containing gel bands are treated with formaldehyde and cyanoborohydride, prior to in-gel tryptic digest, to achieve a dimethylation of N-terminal free amino groups. The chemically modified N-termini can rapidly be identified and assigned to previous cleavage by the protease of interest. With the method described above, it was found that TWS1 is c-terminally cleaved by ALE1. The two amino acids directly flanking the cleavage site were found to be important for ALE1 cleavage site selection, as their substitution caused a loss of ALE1- dependent cleavage. Our cooperation partners demonstrated an interaction of mature TWS1 with the GSO receptors. The binding affinity of mature TWS1 was reduced by a 3 amino acid C-terminal extension, demonstrating the biological relevance of ALE1-mediated TWS1 processing. Like the CIFs, TWS1 contains a DY tyrosine sulfation motive at its N-terminal processing site. The role of tyrosine sulfation in precursor processing is largely unexplored and was addressed in this work by comparing in-vitro cleavage of different sulfated versus nonsulfated TWS1 precursors. SBT1.8 was found to cleave TWS1 at the N-terminal processing site, and cleavage site selection was influenced by the sulfation state of TWS1 P2´ tyrosine. A homology based 3D model of SBT1.8 was created, which suggested that SBT1.8 interacts with the negatively charged sulfate via a positively charged arginine residue (R302). The role of R302 in substrate binding and recognition was confirmed by in-vitro cleavage assays with mutated SBT1.8 versions, in which R302 was replaced. N-terminal TWS1 cleavage was no longer observed when R302 was substituted. Likewise, no N-terminal cleavage was observed for two other seed expressed Arabidopsis subtilases (SBT1.1 and SBT5.4) that feature an arginine at the corresponding position, indicating that the sole presence of R302 is not sufficient for N terminal cleavage site recognition.
Trace analysis of acrylamide by high-performance thin-layer chromatography coupled to mass spectrometry
(2011) Alpmann, Alexander; Schwack, Wolfgang
Planar-chromatography (High-Performance Thin-Layer Chromatography, HPTLC) is a rapid and cost-effective offline separation method. Through advances in the automatization of each step the system reproducibility, from application and development to detection, has been improved. This makes planar-chromatography a highly reliable technique. HPTLC shows a couple of features that make it unique. There is great flexibility concerning application, development and detection that distinguishes HPTLC from other techniques. Especially the parallel development of up to 36 tracks per plate, the possibility of pre-chromatographic derivatization on the stationary phase, application volumes from nL up to mL, two-dimensional development, automated single or multiple development, and the multiple detection with different methods (UV, fluorescence, bioluminescence, etc.) have to be emphasized. A further advantage over column- (LC) and gas-chromatography (GC) is the single use of the stationary phase. This leads to a high tolerance towards sample matrix and allows for reducing sample preparation. Because of these aspects, planar-chromatography is an interesting tool for each analyst. However, in the last years hyphenation with mass spectrometry (MS) did not make great advancements in comparison to HPLC and GC: thus, planar-chromatography became less attractive. Therefore an existing universal hyphenation (ChromeXtract by Dr. Luftmann), that was based upon a plunger for elution, was improved (publication 1). The original version of the plunger did not allow any elution from glass backed plates, since they broke easily under the pressure applied during clamping. It was difficult to adjust the pressure depending on the experience of the operator. Furthermore, solvent leakage was possible because of insufficient sealing of the cup-point. For a reproducible contact pressure that was independent from the experience of the operator, a commercial torque wrench was used for clamping of the plates. This guaranteed reproducible contact pressure. The installation of a small plastic buffer into the plunger ensured a slight kind of attenuation. This decreased the frequency of leakage from over 50 % to below 5 %. An important criterion of applicability of this hyphenation is the repeatability of the extractions and thus the measurements. Thus, zones of xanthylethylcarbamat (XEC) and dansylpropanamid (DPA) were extracted after chromatographic development. Their specific masses were detected in positive ESI-mode. The relative standard deviation of the signal in single-ion-monitoring (SIM) mode was 18.6 % for XEC and 8.7 % for DPA. Linearity was given in the range of 10 to 200 ng/zone with a very good correlation coefficient (r > 0.9919). The limit of quantification at an S/N-ratio of 10 was calculated by means of the blank signal and amounted 52 and 160 pg/zone for XEC and DPA, respectively. Additionally, the influence of the elution solvent on the extraction of the HPTLC-plate and signal intensity was demonstrated with tests using different solvents. The second publication addressed the application of planar-chromatography hyphenated with MS by means of the modified ChromeXtractor on the determination of acrylamide in drinking water. The strict limit within the EU of 0.1 mug/L until then was only controlled through costly methods that were almost exclusively based on GC-MS or LC-MS/MS after applying intensive clean-up procedures. Thus it was aimed to develop a low priced and rapid alternative method for routine analysis based on HPTLC. Therefore a pre-chromatographic in-situ derivatization of acrylamide with a fluorescence marker was used. The product was detected densitometrically after chromatographic separation. During development of the method, the mass of the reaction product was determined for analysis of the derivatization step. With the aid of the modified ChromeXtract the product could be directly extracted from the plate and transferred to MS. The exact mass proved that instead of the originally used fluorescence marker dansylhydrazine the dimethylaminonaphthaline(Dan)-sulfinic acid reacts with acrylamide. Consequently, dansulfinic acid was synthesized and used for derivatization. To take advantage of the high tolerance of planar-chromatography towards various sample matrices, an approach was searched in order to skip sample preparation. However the necessity to use excess of reagent led to high background fluorescence. This allowed only a limit of detection of 20 mug/L. Thus, sample preparation and analyt enrichment was necessary to obtain a method able to control the maximum concentration. In accordance with DIN 38413-6 concerning determination of acrylamide in drinking water, activated carbon was used for analyte enrichment by means of solid phase extraction (SPE). An internal standard (dimethylacrylamide) was added prior sample preparation. The final extract was analysed as described. In spiked samples of drinking water, a 1000-fold lower limit of detection of 0.02 mug/L and a very good mean reproducibility across the whole system was shown, which suffices to control the maximum amount. A comparative study with measurements by LC-MS/MS revealed satisfactory correlation. Thus, for the first time a planar-chromatographic method for the determination of acrylamide at ultra-trace levels were presented. The third publication addresses the application of the developed method on a very complex food matrix like coffee. Several publications reported problems during determination of acrylamide in coffee. Therefore the extremely high tolerance of planar-chromatography towards sample matrix effects was used, allowing for a shortened sample preparation. The idea of a rapid method was followed by the extraction of commercial coffee samples by means of accelerated solvent extraction (ASE). This allowed for higher throughput during sample preparation. To remove a part of the co extracted matrix, the whole ASE-extract was cleaned by SPE with activated carbon and evaporated to a defined volume. This represented a simplification of common multistage extraction methods and clean-up steps, that aim for complete removal of co extracted matrix prior injection into LC- or GC-systems. In accordance with determination of acrylamide in drinking water, the extract was derivatized in-situ with the fluorescence marker Dansulfinic acid and detected densitometrically after chromatographic separation. The concentration of acrylamide was quantified by means of parallel preparation of three standard additions. Systematic errors and the influence of the sample were corrected by the calibration within the matrix. The linearity of the calibration (between r = 0.9825 and 0.9995) were acceptable. Good values were reached for the limit of quantification (48 mug/kg) and repeatability (rsd 3 %). After method development the acrylamide concentration of commercial coffee samples was determined, showing results being consistent with literature findings. Thus the applicability of the newly developed method to complex food samples was demonstrated. In summary, the present work shows the applicability of planar-chromatography hyphenated with mass spectrometry for sensitive determination of acrylamide. It was possible to quantify the analyte at ultra-trace levels using less instrumental effort and time than usual. Quantification in complex sample matrices was feasible in spite of a simplified sample preparation. These applications prove the relevance of planar-chromatography to solve current analytical problems.