The Application Notebook
In this study, we aimed to use a charge modulated HILIC stationary phase (iHILIC®-Fusion) to separate different siderophores of the Pseudomonas taiwanensis VLB120 bacteria.
Pseudomonas bacteria are a genus of rod-shaped, ubiquitous, and gram-negative bacteria, which are resistant against commonly used antibiotics and disinfectants. Pseudomonads are able to survive in extreme conditions (1) and are increasingly used as whole-cell biocatalysts. The range of their biotechnological applications is not limited by the toxicity of used chemicals because of their manifold survival strategies (2).
Pseudomonads require iron(III) for metabolism and growth. Under aerobic conditions, the iron(III) is almost insoluble and the pseudomonas bacteria synthesize yellow-green fluorescent molecules called pyoverdines. These molecules can react with iron(III) and form stable hexadentate iron(III)-complexes in the extracellular space and transport the metal (1). The pyoverdines consist of three structural parts: (i) chromophoric unit derived from 2,3-diamino-6,7-dihydroxyqunoline; (ii) strain specific peptide moiety consisting of 6–14 proteinogenic and nonproteinogenic amino acids; (iii) acyl side chain derived from malic acid, succinic acid, their mono amides, α-ketoglutaric acid, or glutamic acid.
Pyoverdines are highly polar molecules that conventional reversed-phase high performance liquid chromatography (HPLC) is problematic due to insufficient retention. Furthermore, we intended to separate and identify intact metal complexes in our study. Hydrophilic interaction liquid chromatography (HILIC) is well suited for the separation of polar compounds and enables the separation of polar non-covalent metal species (3,4). In this study, we aimed to use a charge modulated HILIC stationary phase (iHILIC®-Fusion) to separate different siderophores of the Pseudomonas taiwanensis VLB120 bacteria.
Experimental
LC–MS System: Agilent 1100er LC system and Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™ mass spectrometer, equipped with a HESI II source, operated in positive ionization mode (ESI+).
Column: 150 × 2.1 mm, 3.5 µm 100 Å, iHILIC®-Fusion (P/N 110.152.0310, HILICON AB, Sweden)
Eluent: A) acetonitrile; B) ammonium bicarbonate (5 mM, pH 7) and acetonitrile (95:5, v/v);
Gradient Elution: 0–0.75 min, 80% A; 0.75–20 min, from 80% A to 40% A; 20–25 min, 40% A; 25–27 min, from 40% A to 80% A; 27–40 min, 80% A
Flow Rate: 0.3 mL/min
Column Temperature: 40 °C
Injection Volume: 5 µL
P. taiwanensis VLB120 Cell Supernatants: Cell supernatants of the P. taiwanensis VLB120 bacteria were provided by Dr. Till Tiso at iAMB (Institute of Applied Microbiology) and ABBt (Aachen Biology and Biotechnology, RWTH Aachen University, Aachen, Germany) and purified by solid-phase extraction as described in reference 5. The samples for injection contained five pyoverdines that differ in their acyl side chain and in the peptide moiety (6), shown in Figure 1.
Results and Conclusion
The different siderophores of the P. taiwanensis VLB120 bacteria and their non-covalent iron and aluminium complexes can be simultaneously separated and determined by an iHILIC®-Fusion column hyphenated with electrospray ionization mass spectrometry (ESI-MS) detection (Figures 2 and 3). Pyoverdines with various acyl side chains exhibit different levels of hydrophilic partitioning and electrostatic interaction with the stationary phase. The extracted ion chromatograms in Figure 3 also demonstrate that the iHILIC®âFusion column is even capable of separating the iron(III) and aluminium(III) complexes of each siderophore. Because of the smaller radius, aluminium has a stronger polarizing effect on the siderophores. Therefore, aluminium(III) complex has higher retention on the HILIC stationary phase. This work demonstrates that intact non-covalent metal complexes can be separated and identified by a HILIC–MS method using an iHILIC®-Fusion column.
References
HILICON AB
Tvistevägen 48, SE-90736, Umeå, Sweden
Tel.: +46 (90) 193469
E-mail: info@hilicon.com
Website: www.hilicon.com
2024 EAS Awardees Showcase Innovative Research in Analytical Science
November 20th 2024Scientists from the Massachusetts Institute of Technology, the University of Washington, and other leading institutions took the stage at the Eastern Analytical Symposium to accept awards and share insights into their research.
Inside the Laboratory: The Richardson Group at the University of South Carolina
November 20th 2024In this edition of “Inside the Laboratory,” Susan Richardson of the University of South Carolina discusses her laboratory’s work with using electron ionization and chemical ionization with gas chromatography–mass spectrometry (GC–MS) to detect DBPs in complex environmental matrices, and how her work advances environmental analysis.
AI and GenAI Applications to Help Optimize Purification and Yield of Antibodies From Plasma
October 31st 2024Deriving antibodies from plasma products involves several steps, typically starting from the collection of plasma and ending with the purification of the desired antibodies. These are: plasma collection; plasma pooling; fractionation; antibody purification; concentration and formulation; quality control; and packaging and storage. This process results in a purified antibody product that can be used for therapeutic purposes, diagnostic tests, or research. Each step is critical to ensure the safety, efficacy, and quality of the final product. Applications of AI/GenAI in many of these steps can significantly help in the optimization of purification and yield of the desired antibodies. Some specific use-cases are: selecting and optimizing plasma units for optimized plasma pooling; GenAI solution for enterprise search on internal knowledge portal; analysing and optimizing production batch profitability, inventory, yields; monitoring production batch key performance indicators for outlier identification; monitoring production equipment to predict maintenance events; and reducing quality control laboratory testing turnaround time.
Infographic: Be confidently audit ready, at any time and reduce failures in pharma QC testing
November 20th 2024Discover how you can simplify the audit preparation process with data integrity dashboards that provide transparency to key actions, and seamlessly track long-term trends and patterns, helping to prevent system suitability failures before they occur with waters_connect Data Intelligence software.
Critical Role of Oligonucleotides in Drug Development Highlighted at EAS Session
November 19th 2024A Monday session at the Eastern Analytical Symposium, sponsored by the Chinese American Chromatography Association, explored key challenges and solutions for achieving more sensitive oligonucleotide analysis.