Global kinase screening. Applications of frontal affinity chromatography coupled to mass spectrometry in drug discovery

Utilizing frontal affinity chromatography with mass spectrometry detection (FAC-MS), we have identified novel applications in the discovery of small-molecule hits to protein targets that are difficult if not impossible to accomplish using traditional assays. We demonstrate for the first time an ability to distinguish between competitive ligands for the ATP and substrate sites of protein kinase C independently in the same experiment and show that ATP competitive ligands using a functionally inactive receptor tyrosine kinase can be identified. This ability of FAC-MS to simultaneously monitor binding at the ATP and substrate binding sites, as well as measure ligand binding to both active and inactive kinases, suggests that FAC-MS can be used as a "global kinase binding assay".     

Hydrophilic interaction chromatography-electrospray mass spectrometry analysis of polar compounds for natural product drug discovery

For the drug discovery efforts currently taking place within the pharmaceutical industry, natural product extracts have been found to provide a valuable source of molecular diversity which is complementary to that provided by traditional synthetic organic methods or combinatorial chemistry. However, there exists a need for analytical tools that can facilitate the separation and characterization of components from these sources in a rapid manner. Specifically, the evaluation of highly polar compounds (i.e., compounds that cannot be retained on traditional reversed-phase stationary phases) has been challenging, and a hydrophilic interaction chromatography-electrospray ionization mass spectrometry (HILIC-ESI-MS) method was developed to meet this need. In this investigation, amide-, Polyhydroxyethyl Aspartamide-, and cyclodextrin-based packings provided superior performance for the analysis of a set of polar natural product compounds. The properties of the mobile-phase buffers also greatly impacted the separations, and relative to other volatile buffering agents, ammonium acetate at a concentration of approximately 6.5 mM was determined to facilitate optimal HILIC retention, reproducibility, and durability. An optimized HILIC-ESI-MS system was successfully applied for the analysis of complex natural product mixtures. The techniques described in this report should also prove useful for the analysis of polar compounds from synthetic sources of molecular diversity such as combinatorial chemistry.     

Detection, characterization, and screening of heme-binding molecules by mass spectrometry for malaria drug discovery

Drug screening for antimalarials uses heme biocrystallization inhibition methods as an alternative to parasite cultures, but they involve complex processes and cannot detect artemisinin-like molecules. The described method detects heme-binding compounds by mass spectrometry, using dissociation of the drug-heme adducts to evaluate putative antiplasmodial activity. Applied to a chemical library, it showed a good hit-to-lead ratio and is an efficient early stage screening for complex mixtures like natural extracts.     

Mass spectrometric techniques for label-free high-throughput screening in drug discovery

High-throughput screening (HTS) is an important tool for finding active compounds to initiate medicinal chemistry programs in pharmaceutical discovery research. Traditional HTS methods rely on fluorescent or radiolabeled reagents and/or coupling assays to permit quantitation of enzymatic target inhibition or activation. Mass spectrometry-based high-throughput screening (MS-HTS) is an alternative that is not susceptible to the limitations imposed by labeling and coupling enzymes. MS-HTS offers a selective and sensitive analytical method for unlabeled substrates and products. Furthermore, method development times are reduced without the need to incorporate labels or coupling assays. MS-HTS also permits screening of targets that are difficult or impossible to screen by other techniques. For example, enzymes that are challenging to purify can lead to the nonspecific detection of structurally similar components of the impure enzyme or matrix of membraneous enzymes. The high selectivity of tandem mass spectrometry (MS/MS) enables these screens to proceed with low levels of background noise to sensitively discover interesting hits even with relatively weak activity. In this article, we describe three techniques that we have adapted for large-scale (approximately 175,000 sample) compound library screening, including four-way parallel multiplexed electrospray liquid chromatography tandem mass spectrometry (MUX-LC/MS/MS), four-way parallel staggered gradient liquid chromatography tandem mass spectrometry (LC/MS/MS), and eight-way staggered flow injection MS/MS following 384-well plate solid-phase extraction (SPE). These methods are capable of analyzing a 384-well plate in 37 min, with typical analysis times of less than 2 h. The quality of the MS-HTS approach is demonstrated herein with screening data from two large-scale screens.     

Mixed-mode anion-cation exchange/hydrophilic interaction liquid chromatography-electrospray mass spectrometry as an alternative to reversed phase for small molecule drug discovery

Within pharmaceutical drug discovery, significant needs currently exist for the analysis and purification of structurally diverse samples prior to or immediately following high-throughput screening. These processes are required to facilitate rapid and accurate biological profiling, structural determination, and resupply of new drug candidates. Reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS) for both analytical and preparative applications has become the small molecule separation/detection tool of choice for meeting many of these needs. However, the separation selectivity provided by RP-HPLC has been limited to the hydrophobicity-based resolution of relatively nonpolar sample components, and for high-throughput drug discovery applications, no sufficient alternative procedures have been identified. In this investigation, a mixed-mode anion-cation exchange/hydrophilic interaction chromatography (ACE-HILIC) method has been developed to provide both direct compatibility with ESI-MS and evaporative light-scattering detection (ELSD) and separation selectivity highly orthogonal to RP-HPLC. The technique employed silica-based small-pore weak ion exchange resins eluted with a combined aqueous and pH gradient. A diverse set of dipeptide probes was employed for the elucidation of the relative contributions of three retention mechanisms. ACE-HILIC-ESI-MS-ELSD should prove useful for the analysis and purification of compounds from both biological (e.g., natural products) and synthetic (e.g., combinatorial chemistry) sources of molecular diversity.     

The use of magnetic techniques in the development of a hydrogenation process

Finely divided Raney Nickel is widely used as a hydrogenation catalyst in the chemical industry and in the refining or hardening of oils and fats. Catalyst removal from process streams is usually carried out by filtration or by centrifugation. Catalyst recycle is difficult owing to the abrasive and pyrophoric nature of the catalyst, general solids handling problems and the propensity of the catalyst to form blockages in linework and in processing equipment. This paper describes the development of a novel design of reactor using magnetic techniques to overcome these problems. An electromagnetic coil is used at the end of a batch to induce sufficient magnetism within the Nickel particles suspended in the process liquor to cause flocculation or agglomeration of the catalyst particles into clusters. These agglomerates settle much faster than the original finely divided material and within minutes most of the catalyst settles out on the base of the vessel. The slight haze of catalyst which remains is removed using an external HGMS unit which is backflushed into the reactor by the following charge. Settled catalyst is easily resuspended by an agitator. Alternative ways of applying the magnetic flocculation technique are described and ways of improving the HGMS unit are discussed.     

Investigation of enzyme kinetics using quench-flow techniques with MALDI-TOF mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is combined off-line with rapid chemical quench-flow methods to investigate the pre-steady-state kinetics of a protein-tyrosine phosphatase (PTPase). PTPase kinetics are generally interrogated spectrophotometrically by the employment of an artificial, chromophoric substrate. However, that methodology places a constraint on the experiment, hampering studies of natural, biochemically relevant substrates that do not incorporate a chromophore. The mass spectrometric assay reported herein is based on the formation of a covalent phosphoenzyme intermediate during substrate turnover. This species is generated in the reaction regardless of the substrate studied and has a molecular weight 80 Da greater than that of the native enzyme. By following the appearance of this intermediate in a time-resolved manner, we can successfully measure pre-steady-state kinetics, regardless of the incorporation of a chromophore. The strengths of the mass-spectrometric assay are its uniform response to all substrates, simple and direct detection of covalent enzyme-substrate intermediates, and facile identification of enzyme heterogeneities that may affect enzymatic activity.     

Application of quantitative chemometric analysis techniques to direct sampling mass spectrometry

This paper explores the use of direct sampling mass spectrometry coupled with multivariate chemometric analysis techniques for the analysis of sample mixtures containing analytes with similar mass spectra. Water samples containing varying mixtures of toluene, ethyl benzene, and cumene were analyzed by purge-and-trap/direct sampling mass spectrometry. Multivariate calibration models were built using partial least-squares regression (PLS), trilinear partial least-squares regression (tri-PLS), and parallel factor analysis (PARAFAC), with the latter two methods taking advantage of the differences in the temporal profiles of the analytes. The prediction errors for each model were compared to those obtained with simple univariate regression. Multivariate quantitative methods were found to be superior to univariate regression when a unique ion for quantitation could not be found. For prediction samples that contained unmodeled, interfering compounds, PARAFAC outperformed the other analysis methods. The uniqueness of the PARAFAC model allows for estimation of the mass spectra of the interfering compounds, which can be subsequently identified via visual inspection or a library search.     

Effect of local matrix crystal variations in matrix-assisted ionization techniques for mass spectrometry

Intense intact molecular ion signals have been obtained from phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidyiinositol using matrix-enhanced secondary ion mass spectrometry (ME-SIMS). It was found that the high-mass (m/z >500) regions of the ME-SIMS spectra closely resembled those obtained using matrix-assisted laser desorption/ionization (MALDI). Using high spatial resolution SIMS, a detailed investigation of dried-droplet samples was performed. Based on the detected Na+ and 2,5-DHB matrix signal intensities, different crystal types were distinguished, in addition to different sizes of crystals. Spatially mapping the pseudomolecular and fragment ions of the phospholipids revealed that the nature of the pseudomolecular ions formed, as well as the ratio of intact molecular to fragment ion, was dependent on the type and surface composition of the crystal. The observed chemical bias effects due to crystal heterogeneity and the resulting variation in desorption/ionization efficiency will complicate the interpretation of data obtained from matrix-assisted mass spectrometric (imaging) techniques and is an important factor in the "hot spot" phenomenon frequently encountered in MALDI experiments. In this respect, imaging SIMS was found to be a versatile tool to investigate the effects of the local physicochemical conditions on the detected molecular species.     

Evaluations of the stability of sheathless electrospray ionization mass spectrometry emitters using electrochemical techniques

The processes that cause the failure of sheathless electrospray ionization (ESI) emitters, based on different kinds of gold coatings on fused-silica capillaries, are described and explained. The methods chosen for this study include electrochemical methods, ICPMS analysis of the electrolytes used, SEM studies, and electrospray experiments. Generally, the failure occurs by loss of the conductive coating. It is shown that emitters with sputter-coated gold lose their coatings because of mechanical stress caused by the gas evolution accompanying water oxidation or reduction. Emitters with gold coatings on top of adhesion layers of chromium and nickel alloy withstand this mechanical stress and have excellent durability when operating as cathodes. When operating as anodes, the adhesion layer is electrochemically dissolved through the gold film, and the gold film then flakes off. It is shown that the conductive coating behaves as a cathode even in the positive electrospray mode when the magnitude of a superimposed reductive electrophoretic current exceeds that of the oxidative electrospray current. Fairy-dust coatings developed in our laboratory (see Barnidge, D. R.; etal.Anal. Chem. 1999, 71, 4115-4118,) bygluing gold dust onto the emitter, are unaffected by the mechanical stress due to gas evolution. When oxidized, the fairy-dust coatings show an increased surface roughness and decreased conductivities due to the formation of gold oxide. The resistance of this oxide layer is however negligible in comparison with that of the gas phase in ESI. Furthermore, since no flaking and only negligible electrochemical etching of gold was found, practically unlimited emitter lifetimes may be achieved with fairy-dust coatings.     

Comprehensive gas chromatography-time-of-flight mass spectrometry using soft and selective photoionization techniques

The hyphenation of gas chromatography and mass spectrometry (GC/MS) revolutionized organic analysis. In GC/MS coupling, usually electron impact ionization is applied, and molecules are identified by their fragment pattern. Although mass spectrometry in principle is a separation method, it is used predominantly as a spectrometric technique. However, if soft (i.e., fragmentation-free) ionization techniques are applied, the inherent separation character of MS is emphasized, which has similarities to a GC boiling point separation. By combining polar column GC separation and fast soft ionization time-of-flight mass spectrometry technology, a comprehensive separation of complex petrochemical samples can be obtained (GC x MS approach). Compounds of comparable physical-chemical properties are characteristically grouped together in a two-dimensional retention time-m/z representation. This resembles the separation characteristics of comprehensive two-dimensional gas chromatography (GC x GC) and, thus, represents a novel multidimensional separation approach. In this work, a gas chromatograph equipped with a polar separation column was coupled to a home-built laser ionization time-of-flight mass spectrometer. Laser-based, single-photon ionization was used for universal soft ionization and resonance-enhanced multiphoton ionization for selective ionization of aromatic compounds. A novel capillary-jet inlet system was used for the coupling. Multidimensional comprehensive analysis of complex petrochemical hydrocarbon samples using gas chromatography coupled to mass spectrometry with soft and selective photo ionization sources is first demonstrated.     

Simplifying CE-MS operation. 2. Interfacing low-flow separation techniques to mass spectrometry using a porous tip

A robust, reproducible, and single-step interface design between low flow rate separation techniques, such as sheathless capillary electrophoresis (CE) and nanoliquid chromatography (nLC), and mass spectrometry (MS) using electrospray ionization (ESI), is introduced. In this design, the electrical connection to the capillary outlet was achieved through a porous tip at the capillary outlet. The porous section was created by removing 1-1.5 in. of the polyimide coating of the capillary and etching this section by 49% solution of HF until it is porous. The electrical connection to the capillary outlet is achieved simply by inserting the capillary outlet containing the porous tip into the existing ESI needle (metal sheath) and filling the needle with the background electrolyte. Redox reactions of water at the ESI needle and transport of these small ions through the porous tip into the capillary provides the electrical connection for the ESI and for the CE outlet electrode. The etching process reduces the wall thickness of the etched section, including the tip of the capillary, to 5-10 microm, which for a 20-30 microm i.d. capillary results in stable electrospray at approximately 1.5 kV. The design is suitable for interfacing a wide range of capillary sizes with a wide range of flow rates to MS via ESI, but it is especially useful for interfacing narrow (<30 microm i.d.) capillaries and low flow rates (<100 nL/min). The advantages of the porous tip design include the following: (1) its fabrication is reproducible, can be automated, and does not require any mechanical tools. (2) The etching process reduces the tip outer diameter and makes the capillary porous in one step. (3) The interface can be used for both nLC-MS and CE-MS. (4) If blocked or damaged, a small section of the tip can be etched off without any loss of performance. (5) The interface design leaves the capillary inner wall intact and, therefore, does not add any dead volume to the CE-MS or nLC-MS interface. (6) Bubble formation due to redox reactions of water at the high-voltage electrode is outside of the separation capillary and does not affect separation or MS performances. The performance of this interface is demonstrated by the analyses of amino acids, peptide, and protein mixtures.     

Integration of mass spectrometry in analytical biotechnology.

Mass spectrometry (MS) has become an indispensable tool for peptide and protein structure analysis because of three unique capabilities that enable it to be used to solve structural problems not easily handled by conventional techniques. First, MS is able to provide accurate molecular weight information on low-picomole amounts of peptides and proteins independent of covalent modifications that may be present. Second, this information is obtainable for peptides present in complex mixtures such as those that result from a proteolytic digest of a protein. Third, by using tandem MS, partial to complete sequence information may be obtained for peptides containing up to 25 amino acid residues, even if the peptides are present in mixtures. Sensitivity and speed of the MS-based approaches now equal (and in some cases exceed) that of Edman-based sequence analysis. In this perspective we discuss how MS, tandem high-performance MS, and on-line liquid chromatography/MS using fast atom bombardment or electrospray ionization have been integrated with more conventional techniques in order to increase the accuracy and speed of peptide and protein structure characterization. The expanding role of matrix-assisted laser desorption MS in protein analysis is also described. The unique niche that MS occupies for locating and structurally characterizing posttranslational modifications of proteins is emphasized. Examples chosen from the authors' laboratory illustrate how MS is used to sequence blocked proteins, define N- and C-terminal sequence heterogeneity, locate and correct errors in DNA- and cDNA-deduced protein sequences, identify sites of deamidation, isoaspartyl formation, phosphorylation, oxidation, disulfide bond formation, and glycosylation, and define the structural class of carbohydrate at specific attachment sites in glycoproteins.     

Effective use of mass spectrometry for glycan and glycopeptide structural analysis

Most proteins are glycosylated. Mass spectrometry methods are used for mapping glycoprotein glycosylation and detailed glycan structural determination. This technology enables precise characterization of recombinant glycoproteins in the pharmaceutical industry and academic biomedicine.     

Sulfur-specific detection of impurities in cimetidine drug substance using liquid chromatography coupled to high resolution inductively coupled plasma mass spectrometry and electrospray mass spectrometry

The use of liquid chromatography coupled to sector field inductively coupled plasma mass spectrometry (SF-ICP-MS) for the specific detection of sulfur-containing compounds is described. In the sulfur-containing drug substance cimetidine, structurally related impurities well below the 0.1% mass fraction level relative to the main drug substance could easily be detected. The structure of most of the impurities was confirmed by electrospray mass spectrometry (ESI-MS), and thus, the complementarity of the two techniques for drug analysis is shown. The limit of detection by SF-ICP-MS for cimetidine in solution was approximately 4-20 ng x g(-1), but it was blank-limited.