Self-aliquoting microarray plates for accurate quantitative matrix-assisted laser desorption/ionization mass spectrometry.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a fast analysis tool employed for the detection of a broad range of analytes. However, MALDI-MS has a reputation of not being suitable for quantitative analysis. Inhomogeneous analyte/matrix co-crystallization, spot-to-spot inhomogeneity, as well as a typically low number of replicates are the main contributing factors. Here, we present a novel MALDI sample target for quantitative MALDI-MS applications, which addresses the limitations mentioned above. The platform is based on the recently developed microarray for mass spectrometry (MAMS) technology and contains parallel lanes of hydrophilic reservoirs. Samples are not pipetted manually but deposited by dragging one or several sample droplets with a metal sliding device along these lanes. Sample is rapidly and automatically aliquoted into the sample spots due to the interplay of hydrophilic/hydrophobic interactions. With a few microliters of sample, it is possible to aliquot up to 40 replicates within seconds, each aliquot containing just 10 nL. The analyte droplet dries immediately and homogeneously, and consumption of the whole spot during MALDI-MS analysis is typically accomplished within few seconds. We evaluated these sample targets with respect to their suitability for use with different samples and matrices. Furthermore, we tested their application for generating calibration curves of standard peptides with α-cyano-4-hdydroxycinnamic acid as a matrix. For angiotensin II and [Glu(1)]-fibrinopeptide B we achieved coefficients of determination (r(2)) greater than 0.99 without the use of internal standards.

Binding constant determination of high-affinity protein-ligand complexes by electrospray ionization mass spectrometry and ligand competition.

We describe an approach for the determination of binding constants for protein-ligand complexes with electrospray ionization mass spectrometry, based on the observation of unbound ligands competing for binding to a protein target. For the first time, dissociation constants lower than picomolar could be determined with good accuracy by electrospray ionization mass spectrometry. The presented methodology relies only on the determination of signal intensity ratios for free ligands in the low mass region. Therefore, all the advantages of measuring low masses with mass spectrometry, such as high resolution are preserved. By using a reference ligand with known binding affinity, the affinity of a second ligand can be determined. Since no noncovalently bound species are observed, assumptions about response factors are not necessary. The method is validated with ligands binding to avidin and applied to ligands binding to p38 mitogen-activated protein kinase.

Determination of protein-ligand binding constants of a cooperatively regulated tetrameric enzyme using electrospray mass spectrometry.

This study highlights the benefits of nano electrospray ionization mass spectrometry (nanoESI-MS) as a fast and label-free method not only for determination of dissociation constants (KD) of a cooperatively regulated enzyme but also to better understand the mechanism of enzymatic cooperativity of multimeric proteins. We present an approach to investigate the allosteric mechanism in the binding of inhibitors to the homotetrameric enzyme fructose 1,6-bisphosphatase (FBPase), a potential therapeutic target for glucose control in type 2 diabetes. A series of inhibitors binding at an allosteric site of FBPase were investigated to determine their KDs by nanoESI-MS. The KDs determined by ESI-MS correlate very well with IC50 values in solution. The Hill coefficients derived from nanoESI-MS suggest positive cooperativity. From single-point measurements we could obtain information on relative potency, stoichiometry, conformational changes, and mechanism of cooperativity. A new X-ray crystal structure of FBPase tetramer binding ligand 3 in a 4:4 stoichiometry is also reported. NanoESI-MS-based results match the current understanding of the investigated system and are in agreement with the X-ray structural data, but provide additional mechanistic insight on the ligand binding, due to the better dynamic resolution. This method offers a powerful approach for studying other proteins with allosteric binding sites, as well.

Critical evaluation of mass spectrometric measurement of dissociation constants: accuracy and cross-validation against surface plasmon resonance and circular dichroism for the calmodulin-melittin system.

We present a comprehensive study for determining the binding affinity of a protein-ligand complex, using mass spectrometric methods. Mass spectrometry has been used to study noncovalent interactions for a number of years. However, the use of soft ionization mass spectrometry for quantitative analysis of noncovalently bound complexes is not widely accepted. This paper reports a comparison of MS methods against established methods such as surface plasmon resonance (SPR) and circular dichroism (CD) whose suitability for the quantitative assessment of noncovalent interactions is well known. ESI titration and MALDI-SUPREX were used as representative mass spectrometric methods for this work. We chose to study the calmodulin-melittin complex that presents three challenges: (i) it exhibits a high affinity (low nanomolar KD); (ii) complexes are formed only in the presence of a coactivator, calcium ions in this case; and (iii) the protein and the complex show a different ionization efficiency. Dissociation constants were obtained from each method for the selected system and compared thoroughly to elucidate pros and cons of the selected methodologies in terms of their ability for the determination of binding constants of protein-ligand complexes. ESI titration, SPR, CD and MALDI-SUPREX yielded KD values in the low nanomolar range that are in general agreement with an older value reported in the literature. We also critically evaluated the limitations in particular of the MS methods and the associated data evaluation procedures. We present an improved evaluation of SUPREX data, as well as a detailed error analysis for all methods used.

Immobilization of biomolecules on polyurethane membrane surfaces.

Lipophilic polymer membranes incorporating binding sites are widely used in various potentiometric, amperometric, and optical sensors. Here, we report on the biofunctional modification of the surface of a Ca(2+)-selective membrane. A photoactivatable biotin derivative was synthesized and covalently immobilized on a soft polyurethane membrane. The modified polymer was characterized by X-ray photoelectron spectroscopy (XPS) as well as by potentiometric measurements. The selective binding of streptavidin by the photo-cross-linked biotin derivative was demonstrated. The surface coverage obtained with different experimental protocols was analyzed by autoradiography using [(35)S]-streptavidin. The new approach may significantly extend the scope of applicability of potentiometric sensors.