Kaempferol rhamnoside catabolism in rosette leaves of senescing Arabidopsis and postharvest stored radish.

Flavonol rhamnosides including kaempferitrin (i.e., kaempferol 3-O-α-rhamnoside-7-O-α-rhamnoside) occur throughout the plant kingdom. Mechanisms governing flavonol rhamnoside biosynthesis are established, whereas degradative processes occurring in plants are relatively unknown. Here, we investigated the catabolic events affecting kaempferitrin status in the rosette leaves of Arabidopsis thaliana L. Heynh. (Arabidopsis) and Raphanus sativus L. (radish), respectively, in response to developmental senescence and postharvest handling. On a per plant basis, losses of several kaempferol rhamnosides including kaempferitrin were apparent in senescing leaves of Arabidopsis during development and postharvest radish stored at 5 °C. Conversely, small pools of kaempferol 7-O-α-rhamnoside (K7R), kaempferol 3-O-α-rhamnoside (K3R), and kaempferol built up in senescing leaves of both species. Evidence is provided for ⍺-rhamnosidase activities targeting the 7-O-α-rhamnoside of kaempferitrin and K7R in rosette leaves of both species. An HPLC analysis of in vitro assays of clarified leaf extracts prepared from developing Arabidopsis and postharvest radish determined that these metabolic shifts were coincident with respective 237% and 645% increases in kaempferitrin 7-O-⍺-rhamnosidase activity. Lower activity rates were apparent when these ⍺-rhamnosidase assays were performed with K7R. A radish ⍺-rhamnosidase containing peak eluting from a DEAE-Sepharose Fast Flow column hydrolyzed various 7-O-rhamnosylated flavonols, as well as kaempferol 3-O-ß-glucoside. Together it is apparent that the catabolism of 7-O-α-rhamnosylated kaempferol metabolites in senescing plant leaves is associated with a flavonol 7-O-α-rhamnoside-utilizing α-rhamnosidase.

In through the out door: Biochemical mechanisms affecting flavonoid glycoside catabolism in plants.

Plants are the sole source of flavonoids, a chemical category that includes flavonols. For the most part, flavonols occur as glycosides with numerous postulated biological roles in plants, including photoprotection, modulation of hormone translocation, and sequestration of reactive oxygen species. Flavonol glycosides are often considered as dead-end metabolites because related flavonoids (i.e., anthocyanins) occur in terminal tissues such as flowers and fruit, but recent evidence points to their turnover in planta, including developing photosynthetic tissues. Although microbial degradation pathways for flavonol glycosides of plant origin are well described, plant catabolic pathways are little studied by comparison. This review will address our current understanding of biochemical processes leading to the loss of flavonol glycosides in plants, with a specific emphasis on the evidence for flavonol-specific ß-glucosidases. Complete elucidation of these catabolic pathways is dependent on the identification of regiospecific modifying steps, including enzymes associated with the hydrolysis of rhamnosylated flavonols, as well as flavonol peroxidation and their encoding genes. Herein, we highlight challenges for the identification of hypothetical plant α-rhamnosidases and peroxidases involved in flavonol glycoside degradation, and the potential biological role of this catabolism in mitigating oxidative stress in developing and postharvest plant tissues.

Membrane-Based SDS Depletion Ahead of Peptide and Protein Analysis by Mass Spectrometry.

SDS interferes with both bottom-up and top-down MS analysis, requiring removal prior to detection. Filter-aided sample preparation (FASP) is favored for bottom-up proteomics (BUP) while acetone precipitation is popular for top-down proteomics (TDP). We recently demonstrated acetone precipitation in a membrane filter cartridge. Alternatively, our automated electrophoretic device, termed transmembrane electrophoresis (TME), depletes SDS for both TDP and BUP studies. Here TME is compared to these two alternative methods of SDS depletion in both BUP and TDP workflows. To do so, a modified FASP method is described applicable to the SDS purification and recovery of intact proteins, suitable for LC/MS. All three methods reliably deplete >99.8% SDS. TME provide higher sample yields (average 90%) than FASP (55%) or acetone precipitation (57%), translating into higher total protein identifications (973 vs 877 FASP or 890 acetone) and higher spectral matches (2.5 times) per protein. In a top down workflow, each SDS-depletion method yields high-quality MS spectra for intact proteins. These results show each of these membrane-based strategies is capable of depleting SDS with high sample recovery and high spectra quality for both BUP and TDP studies.

Accelerated SDS depletion from proteins by transmembrane electrophoresis: Impacts of Joule heating.

SDS plays a key role in proteomics workflows, including protein extraction, solubilization and mass-based separations (e.g. SDS-PAGE, GELFrEE). However, SDS interferes with mass spectrometry and so it must be removed prior to analysis. We recently introduced an electrophoretic platform, termed transmembrane electrophoresis (TME), enabling extensive depletion of SDS from proteins in solution with exceptional protein yields. However, our prior TME runs required 1 h to complete, being limited by Joule heating which causes protein aggregation at higher operating currents. Here, we demonstrate effective strategies to maintain lower TME sample temperatures, permitting accelerated SDS depletion. Among these strategies, the use of a magnetic stir bar to continuously agitate a model protein system (BSA) allows SDS to be depleted below 100 ppm (>98% removal) within 10 min of TME operations, while maintaining exceptional protein recovery (>95%). Moreover, these modifications allow TME to operate without any user intervention, improving throughput and robustness of the approach. Through fits of our time-course SDS depletion curves to an exponential model, we calculate SDS depletion half-lives as low as 1.2 min. This promising electrophoretic platform should provide proteomics researchers with an effective purification strategy to enable MS characterization of SDS-containing proteins.