Derivatization of the tricarboxylic acid cycle intermediates and analysis by online solid-phase extraction-liquid chromatography-mass spectrometry with positive-ion electrospray ionization.

The analysis of cellular metabolic processes is of fundamental biological interest. Cellular metabolites, such as the intermediates of the tricarboxylic acid (TCA) cycle, provide essential information about the metabolic state of the cell. Not only is the TCA cycle a key factor in the energy regulation within aerobic cells, it possibly also plays a role in cell signaling. This paper describes a novel derivatization strategy, using the empirically selected N-methyl-2-phenylethanamine as derivatization reagent with a carbodiimide as co-reagent, for the selective derivatization of carboxylic acids, such as the di- and tri-carboxylic acids of the TCA cycle. Optimization of the derivatization protocol is described. This procedure enables analysis of the derivatives using on-line solid-phase extraction and reversed-phase liquid chromatography in combination with sensitive positive-ion electrospray ionization mass spectrometry. The complete procedure, involving the use of core-shell silica column material, allows the rapid analysis of TCA cycle intermediates in sample matrices, here shown for pig heart tissue extracts, with a good linearity over 3-4 orders of magnitude. Detection limits range from 12 to 1000 nM, depending on the analyte.

On-line continuous-flow, multi-protein biochemical assays for the characterization of bioaffinity compounds using electrospray quadrupole time-of-flight mass spectrometry.

The applicability of a homogeneous on-line continuous-flow, multi-protein biochemical assay was demonstrated for the interaction between fluorescein-biotin and streptavidin and for digoxin and anti-digoxigenin using electrospray quadrupole time-of-flight mass spectrometry (Q-TOF MS). In the on-line continuous-flow biochemical MS-based system several receptors (e.g., streptavidin and anti-digoxigenin, respectively) were allowed to react with corresponding reporter ligands (e.g.,fluorescein-biotin and digoxin, respectively). The methodology presented allows the simultaneous measurement of affinity and molecular mass of an active compound. By using automated MS and MS-MS switching functions of the Q-TOF, structure information is obtained allowing the characterization of bioactive compounds. No cross-reactivities were observed between the two model systems fluorescein-biotin/streptavidin and digoxin/anti-digoxigenin.

Continuous-flow, on-line monitoring of biospecific interactions using electrospray mass spectrometry.

A continuous-flow analytical screening system is presented using electrospray mass spectrometry to measure the interaction of biologically active compounds with soluble affinity proteins. The biochemical detection system is based on a solution-phase, homogeneous assay. In a first step, compounds to be screened (e.g., biotinylated compounds, concentration range 10-1,000 nmol/L) are injected into a continuous-flow reaction system and allowed to react with the affinity protein (e.g., streptavidin, concentration range 3-48 nmol/L). Subsequently, a reporter ligand (fluorescein-labeled biotin 96 nmol/L) is added to saturate the remaining free binding sites of the affinity protein and the concentration of unbound reporter ligand is measured using electrospray MS in the selectedion monitoring mode. The presence of active compounds in the sample results in an increase of the concentration of unbound reporter ligands. The feasibility of a homogeneous MS-based biochemical assay is demonstrated using streptavidin/biotin and anti-digoxigenin/digoxin as model systems. Compared to radioactive or fluorescence-based biochemical assays, the present assay format does not require the synthesis and purification of labels. Various analytical conditions were investigated to determine the ability of MS to measure the biochemical interactions. The availability of a single ligand that can be detected at 10-50 nmol/L concentrations by electrospray MS is sufficient to set up the biochemical assay. For the biospecific interactions studies, detection limits of 10-100 nmol/L were obtained.