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Fingerprinting metal-containing biomolecules after reductive displacement of iron by gallium and subsequent column-switched LC-ICPMS analysis applied on siderophores
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
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2004 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 76, no 9, 2618-2622 p.Article in journal (Refereed) Published
Abstract [en]

Column-switching liquid chromatography followed by low-resolution ICPMS was evaluated as a tool for speciation analysis of metal-containing biomolecules. The strategy was applied on siderophores, strong iron chelators of low molecular weight (M(w) < 1500). Prior to the LC-ICPMS analysis, reductive displacement of iron by gallium was performed using ascorbate as the reducing agent to increase the sensitivity. Different experimental conditions during the exchange reaction were tested using ferrichrysin and ferrichrome for evaluation. A reaction time of 30 min and a pH of 3.9 gave an exchange yield of 27 and 83% for ferrichrysin and ferrichrome, respectively. A gradient elution profile was also developed to separate gallium-chelated siderophores on a PGC column. Detection limits for standard solutions of ferrichrysin and ferrichrome in the low-nanomolar range were obtained by monitoring the gallium-69 isotope. The combined use of LC-ICPMS and LC-ESI-MS/MS was also evaluated as a tool to identify unknown metal complexes, here siderophores, in field soil solution samples.

Place, publisher, year, edition, pages
2004. Vol. 76, no 9, 2618-2622 p.
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-94665DOI: 10.1021/ac0355000PubMedID: 15117206OAI: oai:DiVA.org:uu-94665DiVA: diva2:168606
Available from: 2006-08-30 Created: 2006-08-30 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Liquid Chromatography Coupled to Mass Spectrometry: Implementation of Chemometric Optimization and Selected Applications
Open this publication in new window or tab >>Liquid Chromatography Coupled to Mass Spectrometry: Implementation of Chemometric Optimization and Selected Applications
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Liquid chromatography (LC) coupled to mass spectrometry (MS) offers highly selective and sensitive analysis of a wide variety of compounds. However, the use of hyphenated experimental set-ups implies that many parameters may have an effect on the studied response. Therefore, in order to determine optimized experimental conditions it is of vital importance to incorporate systematic procedures during method development. In this thesis, a generic stepwise optimization strategy is proposed that aims at high chromatographic quality, as well as high mass spectrometric response. The procedure comprises (i) screening experiments to identify the most important parameters, (ii) LC studies to ensure sufficient chromatographic separation, (iii) extended infusion experiments in order to maximize precursor signal(s), and in the case of tandem MS (iv) extended infusion experiments to determine optimal conditions for collision induced dissociation and when applicable also ion trap settings. Experimental design and response surface methodology is used throughout the procedure.

Further, the general applicability of LC-MS is demonstrated in this thesis. Specifically, a novel quantitative column-switched LC-MS method for ferrichrome, ferrichrysin and ferricrocin determination is presented. Using the method it was shown how the siderophore content varies with depth in podzolic soil profiles in the north and south of Sweden. The parallel approach using LC coupled to both inductively coupled plasma (ICP) mass spectrometry, and electrospray ionization (ESI) tandem MS is also evaluated as a tool to identify unknown siderophores in a sample. Additionally, different trypsin digestion schemes used for LC-ESI-MS peptide mapping were compared. By multivariate data analysis, it was clearly shown that the procedures tested induce differences that are detectable using LC-ESI-MS. Finally, the glutathione S-transferase catalyzed bioactivation of the prodrug azathioprine was verified using LC-MS.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2006. 64 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 201
Keyword
Analytical chemistry, liquid chromatography, mass spectrometry, tandem mass spectrometry, electrospray ionization, inductively coupled plasma, chemometrics, optimization, multivariate data analysis, design of experiments, response surface model, siderophore, azathioprine, peptide, Analytisk kemi
Identifiers
urn:nbn:se:uu:diva-7071 (URN)91-554-6612-5 (ISBN)
Public defence
2006-09-28, Room B:21, BMC, Uppsala, 10:15 (English)
Opponent
Supervisors
Available from: 2006-08-30 Created: 2006-08-30 Last updated: 2009-03-24Bibliographically approved
2. Coupled Liquid Separation and Spectrometric Detection of Organic Compounds Containing Hetero-atoms
Open this publication in new window or tab >>Coupled Liquid Separation and Spectrometric Detection of Organic Compounds Containing Hetero-atoms
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis exemplifies the strength in the combination of inductively coupled plasma (ICP) spectrometry and electrospray ionization tandem mass spectrometry ESI-MS/MS as detection techniques for liquid chromatography (LC) in the search for and investigation of compounds that bind or can bind hetero-atoms. Furthermore, some aspects involved in the coupling of LC and ICP spectrometry and quantification without identical standards have been studied.

The importance of using a separation step in combination with ICP spectrometry was shown for urine and blood plasma samples from patients treated with boron neutron capture therapy. In addition to the carrier molecule used in the therapy, one major and a few possible minor metabolites were found in the urine samples. One fragment mass of the major metabolite was obtained with LC-ESI-MS/MS. Liquid chromatography coupled to ICP-MS was also shown to be a valuable tool for fingerprinting metal-binding compounds in complex matrices, such as siderophores (iron-complexing compounds) in soil. The presence of at least two siderophores in a field soil solution sample could be revealed by LC-ICP-MS. Their identities could thereafter be determined by LC-ESI-MS/MS.

The non-UV-absorbing o-carboranylalanine could be quantified in relation to its degradation products by LC-ICP-AES, which provided information about the mechanism behind the degradation. Moreover, LC-ICP spectrometry was shown to provide an accurate quantification of biomolecules (bias < 10 %) when evaluated from external calibration graphs based on inorganic elemental standards.

Finally, the causes of the large decrease in boron signal seen when adding acetonitrile to the LC mobile phase in LC-ICP-MS was investigated in some detail. Space charge effects might explain a large part of the depression from carbon species on the boron signal.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2004. 47 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 933
Keyword
Analytical chemistry, Analytisk kemi
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-3977 (URN)91-554-5863-7 (ISBN)
Public defence
2004-02-27, B42, BMC, Husargatan 3, Uppsala, 10:15
Opponent
Supervisors
Available from: 2004-01-30 Created: 2004-01-30 Last updated: 2013-09-24Bibliographically approved

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