High-throughput one-bead-one-compound approach to peptide-encoded combinatorial libraries: MALDI-MS analysis of single TentaGel beads

The identification of pharmacologically promising compounds (lead compounds) from combinatorial libraries is frequently limited by the throughput of the analytical technique employed. Fourier transform mass spectrometry (FTMS) offers high sensitivity, mass accuracy (m/Deltam > 500 000), and sequencing capabilities. A rapid and efficient method for high-throughput analysis of single beads from peptide-encoded combinatorial libraries with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is presented. Encoding peptides on single beads are identified and structurally characterized by MALDI time-of-flight (TOF) and ultrahigh-resolution MALDI Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry. A strategy of on-probe sample preparation is developed to minimize handling of the beads.     

Multisample preparation methods for the solvent-free MALDI-MS analysis of synthetic polymers

A limitation of any current approach using solvent-free MALDI mass spectrometry is that only one sample at a time can be prepared and transferred to the MALDI-plate. For this reason, multiple-sample preparation approaches for solvent-free MALDI MS analysis of synthetic polymers were developed that are simple and practical. One approach multiplexed sample preparation by simultaneously preparing multiple samples. With this approach, as many as 384 samples could be prepared by addition of analyte, matrix, salt, and 1-mm metal beads to each well of a 384-well disposable bacti plate, capping the plate with the lid and homogenizing all samples simultaneously using a common laboratory vortex device. Besides the time savings achieved by a single vortex step for multiple samples, an additional advantage of this method relative to previously reported solvent-free preparation methods is that the mixing volume per sample is reduced, which allows a reduction in the amount of analyte required. This method, however, still requires the transfer of each homogenized sample to the MALDI plate for subsequent analysis. Here we report a novel approach that combines multiple simultaneous solvent-free sample preparation with automatic sample transfer to the MALDI target plate. This approach reduces the possibility of cross-contamination, the amount of sample and matrix consumed for an analysis, and the time required for preparation of multiple samples. These methods were shown to provide high-quality mass spectra for various synthetic polymer standards with M(n) values to 10 kDa. The methods are efficient in that small sample amounts are required, the sample/salt/matrix ratio is not critical, and the time necessary to achieve sufficient homogenization of multiple samples is less than 5 min.     

Evaluation of 384-well formatted sample preparation technologies for regulated bioanalysis

The capabilities and limitations of 384-well formatted sample preparation technologies applied to regulated bioanalysis were evaluated by developing two assays for the simultaneous quantitation of lopinavir and ritonavir, the active ingredients of Kaletra. One method used liquid-liquid extraction (LLE), and the other used solid-phase extraction (SPE). The steps and apparatuses employed by the two methods covered most of those used for bioanalysis. Briefly, the previously validated 96-well formatted assays were adapted to the 384-format with minor modifications. Because the wells of a 384-well plate are clustered together, cross-contamination between adjacent wells was evaluated critically, along with sensitivity, assay throughput, and ruggedness. Samples (35 microL) containing plasma samples (15 microL), internal standard (10 microL), and sodium carbonate (0.5 M, 10 microL to basify the sample) were placed in a 384-well microtiter plate that may contain saquinavir or amprenavir as contamination markers. For LLE preparation, the samples were placed in a deep 384-well plate (300-microL well volume) and extracted with 150 microL of ethyl acetate. Approximately 50 microL of the extracts were removed from each well after phase separation for analysis. For SPE preparation, the fortified samples were transferred to a 384-formatted SPE plate (C18, 5 mg packing). The extracts were eluted from the plate with basified 2-propanol. The LLE or SPE extracts were dried and reconstituted for column-switching high-performance liquid chromatography with tandem mass spectrometric detection (HPLC/MS/MS). The lower limit of quantitation and the assay range were the same as the 96-well formatted assay. If combined with appropriate automation, sample preparation in the 384-well format would be up to five times more efficient than the 96-well format.     

A combined ion source for fast switching between electrospray and matrix-assisted laser desorption/ionization in Fourier transform ion cyclotron resonance mass spectrometry

A new ion source has been developed for Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) that enables quick changes between matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) modes. When operating as an ESI source, the sample solution is sprayed through an angled nebulizer. The generated ions pass through a glass capillary followed by a skimmer and three sequential hexapole ion guides. Ions can be accumulated in the third hexapole (storage hexapole) before they are injected into the ICR trap. The second hexapole is mounted on a movable platform which also carries the MALDI sample plate. During the switch from ESI to MALDI, this platform moves the second hexapole out of the hexapole series and locates a MALDI sample plate with 384 sample positions into the area directly in front of the storage hexapole. The storage hexapole is in a medium pressure chamber (MPC) which has windows both for the incoming laser beam and for the observation optics, as well as a gas tube for pulsing collision gas into the chamber. During the MALDI operation the focused laser beam enters the MPC, passes between the hexapole rods and irradiates a MALDI sample on the target plate. The sample molecules are desorbed/ionized into the storage hexapole and simultaneously cooled by collisions with the pulsed gas. Ions desorbed from multiple laser shots can be accumulated in this hexapole before they are transferred to the ICR trap. With the combined ion source a computer-controlled switch between MALDI and ESI modes is possible in less than a minute, depending on the position of the MALDI target on the 384-spot plate. Immediate acquisition of mass spectra is possible after mode switching without the need for tuning or re-calibration.     

A clinical trial on a plate? The potential of 384-well format solid phase extraction for high-throughput bioanalysis using liquid chromatography/tandem mass spectrometry

The application of 384-well format solid phase extraction (SPE) for bioanalysis using liquid chromatography/tandem mass spectrometry (LC/MS/MS) is reported and a 384-well SPE method for the 5-HT agonist sumatriptan in human plasma described. Plasma samples were extracted on a prototype low-density polyethylene 384-well SPE block using a packed bed of 5 mg Oasistrade mark HLB. Liquid handling was automated by a combination of a robotic sampler processor and a 96/384 multi-channel dispensing station. Samples and SPE reagents were drawn through the SPE block by centrifugation. The extracts were analysed by LC/MS/MS with thermally and pneumatically assisted electrospray ionisation and selected reaction monitoring. The method is used to illustrate and discuss the feasibility and viability of sample preparation techniques in high-density microtitre plate format for routine bioanalysis.     

High-performance liquid chromatography with on-line coupled UV, mass spectrometric and biochemical detection for identification of acetylcholinesterase inhibitors from natural products

A high-performance liquid chromatography (HPLC) method with on-line coupled ultraviolet (UV), mass spectrometry (MS) and biochemical detection for acetylcholinesterase (AChE) inhibitory activity has been developed. By combining the separation power of HPLC, the high selectivity of biochemical detection, and the ability to provide molecular mass and structural information of MS, AChE inhibitors can be rapidly identified. The biochemical detection was based on a colorimetric method using Ellman's reagent. The detection limit of galanthamine, an AChE inhibitor, in the HPLC-biochemical detection is 0.3 nmol. The three detector lines used, i.e., UV, MS and Vis for the biochemical detection were recorded simultaneously and the delay times of the peaks obtained were found to be consistent. This on-line post-column detection technique can be used for the identification of AChE inhibitors in plant extracts and other complex mixtures such as combinatorial libraries.     

Evaluation of an on-target sample preparation system for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in conjunction with normal-flow peptide high-performance liquid chromatography for peptide mass fingerprint analyses

Large-scale mass spectrometry (MS)-based proteomic analyses require high-throughput sample preparation techniques due to the increasing numbers of samples that make up a typical proteomics experiment. Moreover, extensive sample pre-treatment steps are necessary prior to MS acquisition for even the most rapid and robust MS-based proteomics methodology, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS followed by peptide mass fingerprinting (PMF) analysis. These include sample purification and fractionation, removal of digestion buffers or solvents, and spotting of sample with matrix onto the MALDI target. These multiple steps of time-consuming sample handling can result in high overall analysis costs and the likelihood of sample contamination and loss. In order to overcome some of these limitations in sample processing, we have investigated the use of a novel, simple, inexpensive 96-well elastomeric array that affixes to a MALDI target to create an on-target 96-well plate that accommodates a high solution volume (ca. 200 microL), thereby enabling the on-target processing of samples for MALDI-TOFMS. We explored several factors that influence MALDI sample preparation: type of matrix, solution volume, solution organic composition, solution drying rates and matrix/analyte co-crystallization methods. We also investigated the use of the 96-well elastomeric device for coupling MALDI-TOFMS analysis directly to high flow rate (1 mL/min) reversed-phase (rp)-HPLC. By developing an optimized, robust sample preparation protocol, we were able to obtain mass spectra with a high signal-to-noise ratio from peptide standards present at the 50-fmol level in large starting volumes of solution. PMF analyses were possible from 1-pmol and 500-fmol protein-digest standards. Coupling the device to high-flow HPLC (750 microL/min) yielded a robust and semi-automated means to obtain enhanced MALDI-TOFMS data at 500 ng of protein digest. These methodologies developed for this simple, on-target, elastomeric device show promise for streamlining the sample preparation process from HPLC to MALDI-MS.     

Increasing sensitivity and decreasing spot size using an inexpensive, removable hydrophobic coating for matrix-assisted laser desorption/ionisation plates

Spot size reduction and increased detection sensitivity in matrix-assisted laser desorption/ionisation (MALDI) of small molecules are accomplished by using an inexpensive and removable hydrophobic coating for MALDI targets, based on 3M Scotch Gard surface treatment. Several variations in sample preparation were explored, such as surface coating technique, identity of the matrix, solvent composition, and the type of metal support plate used. These were investigated on both uncoated and coated surfaces and their impact on spot size, crystal coverage, and sensitivity is presented here. Additionally, crystallisation behaviour obtained on coated plates is compared with that on uncoated plates using scanning electron microscope analysis. To demonstrate the potential of the new coating technique, erythromycin A and valinomycin are studied to determine the increase in detection sensitivity of coated plates in comparison to uncoated plates, and to reveal the suitability of the plates for application in combined high-performance liquid chromatography/MALDI (HPLC/MALDI), where widely varying solvent compositions and droplet volumes are observed. It is shown that enhancements in detection sensitivities correlate very well with the achieved spot size reduction. The versatility of the coated plates is also exhibited by the ease of removing the surface layer, after which the plates can be rigorously cleaned without worry about damaging the hydrophobic surface, followed by a quick reapplication of new hydrophobic coating material. This makes the non-polar coating superior to more expensive commercial hydrophobic-coated targets, which are much more delicate to clean. Furthermore, cleaning and reapplication eliminate potential carry-over effects and the easy application procedure also makes the fabrication of inexpensive, disposable MALDI targets readily possible.     

Self-aspirating atmospheric pressure chemical ionization source for direct sampling of analytes on surfaces and in liquid solutions

A self-aspirating heated nebulizer probe is described and demonstrated for use in the direct analysis of analytes on surfaces and in liquid samples by atmospheric pressure chemical ionization (APCI) mass spectrometry. Functionality and performance of the probe as a self-aspirating APCI source is demonstrated using reserpine and progesterone as test compounds. The utility of the probe to sample analytes directly from surfaces was demonstrated first by scanning development lanes of a reversed-phase thin-layer chromatography plate in which a three-component dye mixture, viz., Fat Red 7B, Solvent Green 3, and Solvent Blue 35, was spotted and the components were separated. Development lanes were scanned by the sampling probe operated under computer control (x, y plane) while full-scan mass spectra were recorded using a quadrupole ion trap mass spectrometer. In addition, the ability to sample the surface of pharmaceutical tablets (viz., Extra Strength Tylenol and Evista tablets) and to detect the active ingredients (acetaminophen and raloxifene, respectively) selectively was demonstrated using tandem mass spectrometry (MS/MS). Finally, the capability to sample analyte solutions from the wells of a 384-well microtiter plate and to perform quantitative analyses using MS/MS detection was illustrated with cotinine standards spiked with cotinine-d3 as an internal standard.     

On‐plate deposition of oxidized proteins to facilitate protein footprinting studies by radical probe mass spectrometry

The on‐plate deposition of oxidized proteins is described to advance footprinting applications by radical probe mass spectrometry (RP‐MS). An electrospray ionization (ESI) needle assembly mounted vertically over a 384‐target matrix‐assisted laser desorption/ionization (MALDI) plate enabled the limited oxidation of proteins as they were released in the charged droplets ahead of their deposition on the plate. This method combined with on‐plate proteolytic digestion protocols expedites the analysis of proteins oxidized by RP‐MS, and avoids the need to collect and reconstitute samples prior to analysis by MALDI mass spectrometry. Oxidation of peptides from solutions in water as well as an ammonium bicarbonate solution was investigated to test the optimal conditions required for on‐plate oxidation of proteins. These comprised of peptides with a wide range of reactive amino acids including Phe, Tyr, Pro, His, Leu, Met and Lys that were previously shown to oxidize in both electrospray discharge and synchrotron radiolysis based footprinting experiments. The on‐plate deposition of lysozyme oxidized at electrospray needle voltages of 6 and 9 kV were carried out to demonstrate conditions suitable for footprinting experiments as well as those that induce the onset of protein damage. Copyright © 2012 John Wiley & Sons, Ltd.     

A Cleavable Scaffold Strategy for the Synthesis of One-Bead One-Compound Cyclic Peptoid Libraries That Can Be Sequenced By Tandem Mass Spectrometry

Many macrocyclic depsipeptides or related compounds have interesting medicinal properties and often display more favorable pharmacokinetic properties than linear analogues. Therefore, there is considerable interest in the development of large combinatorial libraries of macrocyclic peptidomimetic compounds. However, such molecules cannot be easily sequenced by tandem mass spectrometry, making it difficult to identify hits isolated from library screens using one bead one compound libraries. Here we report a strategy to solve this problem by placing a methionine in both the linker connecting the cyclic molecule to the bead as well as within the cycle itself. Treatment with CNBr both linearizes the molecule at a specific position and releases the molecule from the bead, making its characterization by tandem MALDI mass spectrometry straightforward.     

A simple MALDI target plate with channel design to improve detection sensitivity and reproducibility for quantitative analysis of biomolecules

Overcoming the detrimental effects of sweet spots during crystallization is an important step to improve the quantitative abilities of matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry. In this study, we introduce MALDI targets, which exhibit a channel design to reduce sweet spot phenomena and improve reproducibility. The size of the channels was 3.0 mm in length, 0.35 mm in depth, and 0.40 mm in width, adjusted to the width of the implemented laser beam. For sample deposition, the matrix/sample mixture was homogenously deposited into the channels using capillary action. To demonstrate the proof‐of‐principle, the novel plates were used for the quantification of acetyl‐L‐carnitine in human blood plasma using a combined standard addition and isotope dilution method. The results showed that the reproducibility of acetyl‐L‐carnitine detection was highly improved over a conventional MALDI‐MS assay, with RSD values of less than 5.9% in comparison with 15.6% using the regular MALDI method. The limits of quantification using the new plates were lowered approximately two‐fold in comparison with a standard rastering approach on a smooth stainless‐steel plate. Matrix effects were also assessed and shown to be negligible. The new assay was subsequently applied to the quantification of acetyl‐L‐carnitine in human plasma samples.     

Ultra-performance liquid chromatography coupled to time-of-flight mass spectrometry for robust, high-throughput quantitative analysis of an automated metabolic stability assay, with simultaneous determination of metabolic data

The application of ultra-performance liquid chromatography coupled to time-of-flight mass spectrometry (UPLC/TOFMS) for high-throughput analysis of a 96-well plate based metabolic stability assay has been investigated. Full-scan data were acquired, with run times of 2.5-3.5 min, from which narrow window extracted ion chromatograms were generated, producing quantitative data for the test compound equivalent to that obtained by high-performance liquid chromatography with tandem mass spectrometric detection on a triple quadrupole instrument (HPLC/MS/MS). Sensitivity and mass accuracy were maintained over the analysis of >300 samples. Additionally, the UPLC/TOFMS datasets obtained gave access to metabolic route information, at no cost in terms of sensitivity for the test compound.     

Mass Spectrometry Detection of Nitrobenzoic Acids and Their Salts on the Surface of Construction Materials

The application of mass spectrometry to the detection of m-nitrobenzoic and 3,5-dinitrobenzoic acids and their salts on the surface of construction materials used in rocketry is described. Analytes are washed with acetonitrile from the studied surface and then analyzed by HPLC?MS with electrospray ionization or the matrix assisted laser desorption/ionization (MALDI). For electrospray ionization, the limit of detection is 6 μg/L and for MALDI ionization, 2 μg/L. The MALDI technique also ensures the direct investigation of samples without washing out; in this case, mass spectra can be visualized by constructing 2D diagrams of the distribution of nitrobenzoic acids over the surface.     

Disposable polymeric high-density nanovial arrays for matrix assisted laser desorption/ionization-time of flight-mass spectrometry: I. Microstructure development and manufacturing.

In order to meet the expected enormous demand for mass spectrometry (MS) throughput as a result of the current efforts to completely map the human proteome, this paper presents a new concept for low-cost high-throughput protein identification by matrix assisted laser desorption/ionization-time of flight-(MALDI-TOF)-MS peptide mapping using disposable polymeric high-density nanovial MALDI target plates. By means of microfabrication technology precision engineered nanovial arrays are fabricated in polymer substrates such as polymethylmethacrylate (PMMA) and polycarbonate (PC). The target plate fabrication processes investigated were precision micromilling, cold embossing and injection moulding (work in progress). Nanovial dimensions were 300, 400 or 500 microm. Typical array densities were 165 nanovials/cm2, which corresponds to 3,300 vials on a full Applied Biosystems MALDI target plate. Obtained MALDI data displayed equal mass resolution, accuracy, signal intensity for peptide standards as compared to high-density silicon nanovial arrays previously reported by our group [7], as well as conventional stainless steel or gold targets.     

A fast,reproducible and low‐cost method for sequence deconvolution of ‘on‐bead’ peptides via ‘on‐target’ maldi‐TOF/TOF mass spectrometry

A novel approach to high‐throughput sequence deconvolution of on‐bead small peptides (MW < 2000 Da) using on‐target MALDI‐TOF/TOF instrumentation is presented. Short peptides of pentamer and octamer length, covalently attached to TentaGel polystyrene beads through a photolabile linker, were placed onto the MALDI target, apportioned with suitable matrix (2,5‐dihydroxybenzoic acid) and then hit with the instrument laser (Nd : YAG, 355 nm). This induced easy and highly reproducible photochemical cleavage, desorption (MS mode) and fragmentation (MS/MS mode). Peptide fragments were identified with a mass accuracy of 0.1 Da of the expected values. This technique significantly accelerates the sequence determination of positive peptide hits obtained from random combinatorial libraries when screening against biological targets, paving the way for a rapid and efficient method to identify molecular imaging ligands specific to pathological targets in cancer and other diseases. Copyright © 2009 John Wiley & Sons, Ltd.     

Electrospray sample deposition for matrix-assisted laser desorption/ionization (MALDI) and atmospheric pressure MALDI mass spectrometry with attomole detection limits

Electrospray sample deposition was explored for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). In this method, nanoliter volumes of matrix/analyte mixture were electrosprayed from a high voltage biased (1-2 kV) fused-silica capillary onto a grounded MALDI plate mounted 100-500 microm from the capillary outlet. Electrospray deposition with these conditions produced sample spots 200-300 microm in diameter thus matching the laser spot size. Varying spray voltage and distance resulted in different crystal sizes and volatilization rates for alpha-cyano-4-hydroxycinnamic acid matrix. Best results were obtained when the sample was deposited as wet droplets as opposed to deposition as dried solid. Under 'wet-spray' conditions, 2-4 microm diameter crystals were formed and detection limits for several neuropeptides were 0.7-25 amol. Samples could be pre-concentrated on the plate by spraying continuously and allowing sample to evaporate in a small spot. Sample volumes as large as 580 nL were deposited yielding a detection limit of 35 pM for neurotensin 1-11. Electrospray sample deposition yielded similar results when using atmospheric pressure-MALDI coupled with a quadrupole ion trap mass spectrometer, except that the sensitivity was approximately seven-fold worse.     

Microscale sample deposition onto hydrophobic target plates for trace level detection of neuropeptides in brain tissue by MALDI-MS

A sample preparation method that combines a modified target plate with a nanoscale reversed-phase column (nanocolumn) was developed for detection of neuropeptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). A gold-coated MALDI plate was modified with an octadecanethiol (ODT) self-assembled monolayer to create a hydrophobic surface that could concentrate peptide samples into a approximately 200-500-microm diameter spot. The spot sizes generated were comparable to those obtained for a substrate patterned with 200-microm hydrophilic spots on a hydrophobic substrate. The sample spots on the ODT-coated plate were 100-fold smaller than those formed on an unmodified gold plate with a 1-microl sample and generated 10 to 50 times higher mass sensitivity for peptide standards by MALDI-TOF MS. When the sample was deposited on an ODT-modified plate from a nanocolumn, the detection limit for peptides was as low as 20 pM for 5-microl samples corresponding to 80 amol deposited. This technique was used to analyze extracts of microwave-fixed tissue from rat brain striatum. Ninety-eight putative peptides were detected including several that had masses matching neuropeptides expected in this brain region such as substance P, rimorphin, and neurotensin. Twenty-three peptides had masses that matched peaks detected by capillary liquid chromatography with electrospray ionization MS.     

Cleavable hydrophilic linker for one-bead-one-compound sequencing of oligomer libraries by tandem mass spectrometry

We have developed a method for the rapid and unambiguous identification of sequences of hit compounds from one-bead-one-compound combinatorial libraries of peptide and peptoid ligands. The approach uses a cleavable linker that is hydrophilic to help reduce nonspecific binding to biological samples and allows for the attachment of a halogen tag, which greatly facilitates post-screening sequencing by tandem mass spectrometry (MS/MS). The linker is based on a tartaric acid unit, which, upon cleavage from resin, generates a C-terminal aldehyde. This aldehyde can then be derivatized with a bromine-containing amino-oxy compound that serves as an isotope tag for subsequent MS/MS analysis of y-ion fragments. We have applied this linker and method to the syntheses of a number of peptoids that vary in sequence and length and have also demonstrated single-bead sequencing of a peptoid pentamer. The linker is also shown to have very low levels of nonspecific binding to proteins.     

Coupling of nanoflow liquid chromatography to matrix-assisted laser desorption/ionization mass spectrometry: real-time liquid chromatography run mapping on a MALDI plate

The major obstacle in the use of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) instruments in the analysis of complex proteome samples is the lack of a direct coupling of a highly resolving separation technique with the mass spectrometer itself. To overcome this drawback, a spotting device for capillary and nanoflow liquid chromatography (LC) with a special liquid deposition principle for lowest volumes was developed. The instrument is able to perform MALDI spotting in real time in order to deposit the LC run on the MALDI plate, and therefore couples the high resolution power of nano-RP-HPLC separation directly with MALDI-MS. This work describes the development and optimization of a method for spotting with online matrix addition, and illustrates its use in the analysis of a complex proteome sample.