Solvent-free MALDI-MS for the analysis of a membrane protein via the mini ball mill approach: case study of bacteriorhodopsin

A mini ball mill (MBM) solvent-free matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) method allows for the analysis of bacteriorhodopsin (BR), an integral membrane protein that previously presented special analytical problems. For well-defined signals in the molecular ion region of the analytes, a desalting procedure of the MBM sample directly on the MALDI target plate was used to reduce adduction by sodium and other cations that are normally attendant with hydrophobic peptides and proteins as a result of the sample preparation procedure. Mass analysis of the intact hydrophobic protein and the few hydrophobic and hydrophilic tryptic peptides available in the digest is demonstrated with this robust new approach. MS and MS/MS spectra of BR tryptic peptides and intact protein were generally superior to the traditional solvent-based method using the desalted "dry" MALDI preparation procedure. The solvent-free method expands the range of peptides that can be effectively analyzed by MALDI-MS to those that are hydrophobic and solubility-limited.     

Producing highly charged ions without solvent using laserspray ionization: a total solvent-free analysis approach at atmospheric pressure

First examples of highly charged ions in mass spectrometry (MS) produced from the solid state without using solvent during either sample preparation or mass measurement are reported. Matrix material, matrix/analyte homogenization time and frequency, atmospheric pressure (AP) to vacuum inlet temperature, and mass analyzer ion trap conditions are factors that influence the abundance of the highly charged ions created by laserspray ionization (LSI). LSI, like matrix-assisted laser desorption/ionization (MALDI), uses laser ablation of a matrix/analyte mixture from a surface to produce ions. Preparing the matrix/analyte sample without the use of solvent provides the ability to perform total solvent-free analysis (TSA) consisting of solvent-free ionization and solvent-free gas-phase separation using ion mobility spectrometry (IMS) MS. Peptides and small proteins such as non-β-amyloid components of Alzheimer's disease and bovine insulin are examples in which LSI and TSA were combined to produce multiply charged ions, similar to electrospray ionization, but without the use of solvent. Advantages using solvent-free LSI and IMS-MS include simplicity, rapid data acquisition, reduction of sample complexity, and the potential for an enhanced effective dynamic range. This is achieved by more inclusive ionization and improved separation of mixture components as a result of multiple charging.     

MALDI MS sample preparation by using paraffin wax film: systematic study and application for peptide analysis

Recently developed sample preparation techniques employing hydrophobic sample support have improved the detection sensitivity and mass spectral quality of matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). These methods concentrate the samples on target by minimizing the sample area via the solvent repellent effect of the target surface. In the current study, we employed the use of paraffin wax film (Parafilm M) for improved MALDI MS analysis of low-abundance peptide mixtures, including neuronal tissue releasate and protein tryptic digests. This thin film was found to strongly repel polar solvents including water, methanol, and acetonitrile, which enabled the application of a wide range of sample preparation protocols that involved the use of various organic solvents. A "nanoliter-volume deposition" technique employing a capillary column has been used to produce tiny ( approximately 400 microm) matrix spots of 2,5-dihydroxybenzoic acid on the film. By systematically optimizing the sample volume, solvent composition, and film treatment, the Parafilm M substrate in combination with the nanoliter-volume matrix deposition method allowed dilute sample to be concentrated on the film for MALDI MS analysis. Peptide mixtures with nanomolar concentrations have been detected by MALDI time-of-flight and MALDI Fourier transform ion cyclotron resonance mass spectrometers. Overall, the use of Parafilm M enabled improved sensitivity and spectral quality for the analysis of complex peptide mixtures.     

Sample preparation for MALDI mass spectrometry using an elastomeric device reversibly sealed on the MALDI target

A new method for improving low-concentration sample recovery and reducing sample preparation steps in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is presented. In the conventional approach, samples are typically desalted and/or concentrated with various techniques and deposited on the MALDI target as small droplets. In this work, we describe a new approach in which an elastomeric device is reversibly sealed on the MALDI target to form a multi-well plate with the MALDI target as the base of the plate. The new format allows a larger volume (5-200 microL) of samples to be deposited on each spot and a series of sample handling processes, including desalting and concentrating, to be performed directly on the MALDI target. Several advantages have been observed: (i) multiple sample transferring steps are avoided; (ii) recovery of low-concentration peptides during sample preparation is improved using a novel desalting method that utilizes the hydrophobic surface of the elastomeric device; and (iii) sequence coverage of the peptide mass fingerprinting map is improved using a novel method in which proteins are immobilized on the hydrophobic surface of the elastomeric device for in-well trypsin digestion, followed by desalting and concentrating the digestion products in the same well.     

Characterization of an insoluble poly(9,9-diphenyl-2,7-fluorene) by solvent-free sample preparation for MALDI-TOF mass spectrometry

The application of solvent-free sample preparation for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) allowed the characterization of an insoluble fraction of poly(9,9-diphenylfluorene) that was previously hindered by the lack of suitable characterization methods. The MALDI mass spectrometric analysis gives valuable mechanistic information about the heterogeneous polymerization process of the insoluble high molecular weight fraction of the polymer. The fragmentation appearing even under moderate desorption and ionization conditions of this rigid backbone analyte is identified as a multiple loss of the bulky phenyl side groups and can be avoided by applying the new MALDI matrix 7,7,8,8-tetracyanoquinodimethane. A specialized fragmentation study by postsource decay MALDI-TOF MS reveals a molecular weight dependent change in fragmentation mechanism from an exclusive cleavage of side groups from long polymer chains to an additional cleavage of the polymer backbone of short polymer chains.     

In situ liquid-liquid extraction as a sample preparation method for matrix-assisted laser desorption/ionization MS analysis of polypeptide mixtures

A novel liquid-liquid extraction (LLE) procedure was investigated for preparation of peptide and protein samples for matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). LLE using ethyl acetate as the water-immiscible organic solvent enabled segregation of hydrophobic and hydrophilic polypeptides in mixtures, thereby reducing the complexity of mass spectra obtained by MALDI MS. The LLE technique was optimized for rapid and sensitive in situ (on-target) sample preparation for MALDI MS analysis of proteins and peptides at low-picomole and subpicomole levels. Addition of MALDI matrix to the organic solvent enhanced the efficiency of the LLE-MALDI MS method for analysis of hydrophobic peptides and proteins. LLE-MALDI MS enabled the detection of the hydrophobic membrane protein bacteriorhodopsin as a component in a simple protein mixture. Peptide mixtures containing phosphorylated, glycosylated, or acylated peptides were successfully separated and analyzed by the in situ LLE-MALDI MS technique and demonstrate the potential of this method for enhanced separation and structural analysis of posttranslationally modified peptides in proteomics research.     

Combining liquid chromatography with MALDI mass spectrometry using a heated droplet interface

A novel interfacing technology is described to combine solution-based separation techniques such as liquid chromatography (LC) with matrix-assisted laser desorption ionization (MALDI) mass spectrometry. The interface includes a transfer tube having an inlet and an outlet, the inlet being adapted to accept the LC effluents and the outlet being adapted to form continuously replaced, hanging droplets of the liquid stream, and a MALDI sample plate mounted below the outlet of the transfer tube for collecting the droplets. The liquid stream in the transfer tube is heated to a temperature sufficient to cause partial evaporation of the carrier solvent from the hanging droplets. The droplets are dislodged to the MALDI plate, which is heated to above the boiling point of the carrier solvent to cause further evaporation of the carrier solvent from the collected droplets. It is found that analytes can be fractionated and deposited to a sample spot of 0.8 mm in diameter when a liquid flow rate of up to 50 microL/min and a fractionation interval of 1 min/spot are used. Flow rate of up to 200 microL/min can be used with a deposition sample spot of 2.4 mm in diameter on a commercial MALDI target. This heated droplet interface does not introduce sample loss, and the detection sensitivity of LC/MALDI is similar to that of standard MALDI, i.e., low femtomoles for peptide analysis with a microliter sample deposition. It is compatible with microbore and narrow-bore column separation, thus allowing the injection of a larger amount of sample for separation and analysis, compared to a capillary column LC/MALDI system. The detection dynamic range is shown to be in the order of 10(6) for peptide mixture analysis, which is 4 orders of magnitude greater than standard MALDI. The application of this interface for combining LC with MALDI MS/MS is demonstrated in the proteome analysis of water-soluable protein components of E. coli K12 extracts.     

Exploring the importance of the relative solubility of matrix and analyte in MALDI sample preparation using HPLC

New insight into the role of solubility in the sample preparation process for MALDI MS is reported. Reversed-phase gradient HPLC conditions were developed that enable the analysis of a broad range of analyte polarities with a single method. This HPLC method was used to establish a relative polarity scale for a series of 15 MALDI matrix materials, a set of example peptides, and a series of model polymer materials with a broad range of polarity. Examples of each polymer type within the range of 6000-10,000 were analyzed with six matrixes that cover a broad range of polarity using MALDI TOFMS. With regard to polymer signal-to-noise ratio, the matrix and polymer combinations that had a close match of HPLC retention time produced the best MALDI spectra. Conversely, the matrix and polymer combinations that have a large difference in HPLC retention time produced poor MALDI spectra. The results suggest that there is a relationship between polarity (solubility) and effective MALDI sample preparation. The relative HPLC retention time of an unknown polymer can serve as a starting point for predicting the matrix (or range of matrixes) that would be most effective.     

Localization and analyses of small drug molecules in rat brain tissue sections

Traditional detection of drugs in tissue requires tissue homogenization, which precludes the mapping and localization of drugs. The use of autoradiography could compensate for such shortcoming. However, it requires expensive custom-synthesized radioactive drugs. Recent improvement in sample preparation for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and MALDI-MS/MS provides an alternative approach for in situ drug detection. In this work, rat brains were collected after intracranial injection of chlorisondamine or intraperitoneal injection of cocaine and snap frozen. MALDI matrixes were applied directly to 14-mum brain cryosections and spectra acquired. The identity of the drugs was further confirmed by MS/MS. Careful matrix selection and tissue preparation allows for the successful detection of drugs and the mapping of their relative abundance across various regions of the brain. This new method is simple, safe, accurate, fast, cost-effective, and low in sample consumption and shows potential for imaging, pharmacokinetics, and toxicology applications.     

MALDI MS peptide mapping performance by in-gel digestion on a probe with prestructured sample supports

Matrix-assisted laser desorption/ionization (tandem) mass spectrometry (MALDI MS) is widely used in protein chemistry and proteomics research for the identification and characterization of proteins isolated by polyacrylamide gel electrophoresis. In an effort to minimize sample handling and increase sample throughput, we have developed a novel in-gel digestion protocol where sample preparation is performed directly on a MALDI probe with prestructured sample support. The protocol consists of few sample-handling steps and has minimal consumption of reagents, making the protocol sensitive, timesaving, and cost-efficient. Performance of the on-probe sample preparation protocol was demonstrated by analysis of a set of rat liver proteins obtained from a fluorescently stained (Cy3 and SyproRuby) two-dimensional polyacrylamide gel. The success rate of protein identification by on-probe tryptic digestion and MALDI peptide mass mapping was 89%. The on-probe in-gel digestion procedure provided superior sensitivity and peptide mass mapping performance as compared to our standard in-gel digestion protocol. The on-probe digestion technique resulted in significantly improved amino acid sequence coverage of proteins, mainly due to efficient recovery and detection of large (>1.5 kDa) hydrophobic peptides. These observations indicate that numerous tryptic peptides are lost when using the standard in-gel digestion methods and sample preparation techniques for MALDI MS. This study also demonstrates that the on-probe digestion protocol combined with MALDI tandem mass spectrometry provides a robust platform for proteomics research, including protein identification and determination of posttranslational modifications.     

Two-layer sample preparation method for MALDI mass spectrometric analysis of protein and peptide samples containing sodium dodecyl sulfate

Sodium dodecyl sulfate (SDS) is widely used in protein sample workup. However, many mass spectrometric methods cannot tolerate the presence of this strong surfactant in a protein sample. We present a practical and robust technique based on a two-layer matrix/sample deposition method for the analysis of protein and peptide samples containing SDS by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The two-layer method involves the deposition of a mixture of sample and matrix on top of a thin layer of matrix crystals. It was found that for SDS-containing samples, the intensity of the MALDI signals can be affected by the conditions of sample preparation: on-probe washing, choice of matrix, deposition method, solvent system, and protein-to-SDS ratio. However, we found that, under appropriate conditions, the two-layer method gave reliable MALDI signals for samples with levels of SDS up to approximately 1%. The applications of this method are demonstrated for MALDI analysis of hydrophobic membrane proteins as well as bacterial extracts. We envision that this two-layer method capable of handling impure samples including those containing SDS will play an important role in protein molecular weight analysis as well as in proteome identification by MALDI-MS and MS/MS.     

Ammonium dodecyl sulfate as an alternative to sodium dodecyl sulfate for protein sample preparation with improved performance in MALDI mass spectrometry

Sodium dodecyl sulfate (SDS) is a strong surfactant that is widely used in protein sample preparation. While protein and peptide samples containing up to approximately 1% SDS can be analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) using a two-layer matrix/sample deposition method, the presence of SDS in a protein sample generally degrades mass resolution and mass measurement accuracy. This degradation in performance is found to be related to the formation of sodium-protein adducts in the MALDI process. If the instrument resolving power is insufficient to separate these adduct peaks from the protonated molecular ion peak, peak broadening is observed in the protein molecular ion region, and as a result, the peak centroid shifts to a higher mass. In this work, we present a method using ammonium dodecyl sulfate as a viable alternative to SDS for protein sample preparation with much improved MALDI MS performance. Three non-sodium-based dodecyl sulfate surfactants, ammonium dodecyl sulfate (ADS), hydrogen dodecyl sulfate, and tris(hydroxymethyl)aminomethane dodecyl sulfate were investigated. Of the three surfactants tested, it is found that ADS gives the best performance in MALDI. For proteins with moderate molecular masses (i.e., up to approximately 25 kDa), the presence of ADS in a protein sample does not result in significant degradation in mass resolution and accuracy, and the protonated molecular ion peak is the dominant peak in the MALDI spectrum. The ammonium adduct ions dominate the MALDI spectra when the protein mass exceeds approximately 25 kDa; however, ADS still gives better results than SDS. The behavior of ADS in gel electrophoresis was also investigated. It is shown that cell extracts dissolved in ADS can be separated by normal SDS-polyacrylamide gel electrophoresis by simply mixing them with the SDS sample buffer. The application of ADS as the surfactant for protein solubilization with improved performance in MALDI analysis is demonstrated in the study of a detergent insoluble fraction from a Raji/CD9 B-cell lymphocyte lysate.     

Higher throughput bioanalysis by automation of a protein precipitation assay using a 96-well format with detection by LC-MS/MS

Generic methodology for the automated preparation and analysis of drug levels in plasma samples within a drug discovery environment was achieved through the redesign of a protein precipitation assay to a microtiter (96-well) plate format and the application of robotic liquid handling for performance of all transfer and pipetting steps. Validation studies revealed that the application of robotics to sample preparation, in general, maintained the analytical accuracy and precision compared with preparing samples manually. The use of rapid gradient LC-MS/MS for analysis coupled with flow diversion of the solvent front allowed the introduction of protein-precipitated samples into the mass spectrometer without the necessity for source cleaning. The problem inherent in automatically pipetting plasma, caused by fibrinogen clots, was overcome by storing samples at -80 degrees C and thus precluding clot formation. The resulting methodology allowed sample preparation for a 96-well plate designed to accommodate 54 unknowns, duplicate 12-point calibration curves, and 6 sets of quality controls at three levels in approximately 2 h. This approach allowed an increase in throughput of sample preparation and analysis to >400 samples per day per LC-MS/MS instrument with minimal manual intervention. Overall, substantial time savings were realized, demonstrating that automation is an increasingly essential tool in a drug discovery bioanalytical environment.     

Electrostatic-spray ionization mass spectrometry

An electrostatic-spray ionization (ESTASI) method has been used for mass spectrometry (MS) analysis of samples deposited in or on an insulating substrate. The ionization is induced by a capacitive coupling between an electrode and the sample. In practice, a metallic electrode is placed close to but not in direct contact with the sample. Upon application of a high voltage pulse to the electrode, an electrostatic charging of the sample occurs leading to a bipolar spray pulse. When the voltage is positive, the bipolar spray pulse consists first of cations and then of anions. This method has been applied to a wide range of geometries to emit ions from samples in a silica capillary, in a disposable pipet tip, in a polymer microchannel, or from samples deposited as droplets on a polymer plate. Fractions from capillary electrophoresis were collected on a polymer plate for ESTASI MS analysis.     

Phosphoric acid as a matrix additive for MALDI MS analysis of phosphopeptides and phosphoproteins

Phosphopeptides are often detected with low efficiency by MALDI MS analysis of peptide mixtures. In an effort to improve the phosphopeptide ion response in MALDI MS, we investigated the effects of adding low concentrations of organic and inorganic acids during peptide sample preparation in 2,5-dihydroxybenzoic acid (2,5-DHB) matrix. Phosphoric acid in combination with 2,5-DHB matrix significantly enhanced phosphopeptide ion signals in MALDI mass spectra of crude peptide mixtures derived from the phosphorylated proteins alpha-casein and beta-casein. The beneficial effects of adding up to 1% phosphoric acid to 2,5-DHB were also observed in LC-MALDI-MS analysis of tryptic phosphopeptides of B. subtilis PrkC phosphoprotein. Finally, the mass resolution of MALDI mass spectra of intact proteins was significantly improved by using phosphoric acid in 2,5-DHB matrix.     

Characterization and performance of MALDI on a triple quadrupole mass spectrometer for analysis and quantification of small molecules

The usefulness of MALDI for small-molecule work has been limited by matrix chemical interference in the mass range of interest, tedious sample preparation, and various crystallization and sample deposition issues. We report instrument characterization and small-molecule quantification performance data from a high repetition rate laser MALDI ion source coupled to a triple quadrupole mass spectrometer. The high repetition rate laser improves sensitivity and precision and allows a proportional increase in sample throughput. Tandem mass spectrometry is used to discriminate the signal from the high chemical background caused by the MALDI matrix. Successful quantification requires use of an internal standard and a means of sample cleanup for typical in vitro sample compositions. This instrument combination and analysis technique is relatively insensitive to sample crystal quality and spot homogeneity. Quantitative performance results are characterized for 53 small-molecule pharmaceutical compounds and compared to those obtained by ESI-MS/MS. Further comparison between MALDI and ESI is examined, and the potential for high-throughput MALDI-MS/MS quantification is demonstrated.     

Characterization of synthetic polymers by MALDI-TOF/MS: investigation into new methods of sample target preparation and consequence on mass spectrum finger print

Characterization of synthetic polymers by Matrix assisted laser desorption (MALDI) is limited by the solubility of different oligomers in a suitable solvent, and the fingerprint of the mass spectrum is affected by the properties of solvents employed (eg., pH, secondary solvents, evaporation) during sample target preparation. If solvents are not used during sample target preparation, then solvent properties should not play an important role in determining the quality of the MALDI mass spectrum. We report here two solventless approaches for sample target preparation. It was observed that Poly(ethylene glycol) 6000 (PEG) showed the same molecular mass distribution in different modes of sample target preparation. Fluorinated polymer used in these studies was affected by sample target preparation protocol and by target surface. Pyrolysis of PEG oligomers was observed in all the methods of sample target preparation. The desorbed high mass neutral oligomers fragment to give small oligomers which are then cationized by the desolvation of the cationized matrix clusters. Moreover, the origin of the matrix clusters (i.e., formed in the condensed phase or in the gas phase) determines the relative intensities of PEG oligomers cationized by sodium or potassium.     

A solid sample preparation method that reduces signal suppression effects in the MALDI analysis of peptides

Here we report on the application of a solid-solid (SS) sample preparation protocol for the MALDI analysis of peptides and multicomponent peptide mixtures. Our results with a series of model peptides indicate that a SS MALDI sample preparation protocol is useful for the analysis of peptides in the 1-3 kDa mass range. MALDI mass spectra recorded for peptides in this size range using a SS sample preparation were of a quality comparable to spectra recorded using a conventional dried-droplet (DD) sample preparation. Our results with several model peptide mixtures indicate that one advantage of a SS sample preparation protocol for the MALDI analysis of peptides is that it can significantly reduce signal suppression effects in multicomponent mixtures. MALDI results obtained using a SS sample preparation protocol are also more reproducible than results obtained using a conventional DD sample preparation protocol.     

Capillary-scale frontal affinity chromatography/MALDI tandem mass spectrometry using protein-doped monolithic silica columns

Frontal affinity chromatography (FAC) interfaced with electrospray mass spectrometry (ESI-MS) has been reported as a potential method for screening of compound mixtures against immobilized target proteins. However, the interfacing of bioaffinity columns to ESI-MS requires that the eluent that passes through the protein-loaded column have a relatively low ionic strength to produce a stable spray. Such low ionic strength solvents can cause serious problems with protein stability and may also affect binding constants and lead to high nonspecific binding to the column. Herein, we report on the interfacing of bioaffinity columns to matrix-assisted laser desorption/ionization (MALDI) MS/MS as a new platform for FAC/MS studies. Capillary columns containing a monolithic silica material with entrapped dihydrofolate reductase were used for frontal affinity chromatography of small-molecule mixtures. The output from the column was combined with a second stream containing alpha-cyano-hydoxycinnamic acid in methanol and was deposited using a nebulizer-assisted electrospray method onto a conventional MALDI plate that moved relative to the column via a computer-controlled x-y stage, creating a semipermanent record of the FAC run. The use of MALDI MS/MS allowed for buffers with significantly higher ionic strength to be used for FAC studies, which reduced nonspecific binding of ionic compounds and allowed for better retention of protein activity over multiple runs. Following deposition, MALDI analysis required only a fraction of the chromatographic run time, and the deposited track could be rerun multiple times to optimize ionization parameters and allow signal averaging to improve the signal-to-noise ratio. Furthermore, high levels of potential inhibitors could be detected via MALDI with limited ion suppression effects. Both MALDI- and ESI-based analysis showed similar retention of inhibitors present in compound mixtures when using identical ionic strength conditions. The results show that FAC/MALDI-MS should provide advantages over FAC/ESI-MS for high-throughput screening of compound mixtures.     

Impulse-driven heated-droplet deposition interface for capillary and microbore LC-MALDI MS and MS/MS

An automated off-line liquid chromatography-matrix-assisted laser desorption ionization (LC-MALDI) interface capable of coupling both capillary and microbore LC separations with MALDI mass spectrometry (MS) and tandem mass spectrometry (MS/MS) has been developed. The interface is a combination of two concepts: analyte concentration from heated hanging droplets and impulse-driven droplet deposition of LC fractions onto a MALDI sample plate. At room temperature the interface allows the coupling of capillary LC separations (i.e., flow rate of <5 microL/min) with MALDI MS. With heating, it can be used to combine microbore LC operated at a relatively high flow rate of up to 50 microL/min with MALDI MS. The collected fractions can be analyzed by MALDI MS and MS/MS instruments, such as time-of-flight (TOF) and quadrupole-TOF MS. Performance of the interface was examined using several peptide and protein standards. It was shown that, using MALDI-TOF MS, [GLU1]-fibrinopeptide B could be detected with a total injection amount of 5 fmol to microbore LC. Chromatographic performance was also monitored. A peak width of 12 s at half-height for [GLU1]-fibrinopeptide B showed no evidence of band broadening due to the interface. The ability of the interface to mitigate ion suppression was studied using a mixture of 100 fmol of [GLU1]-fibrinopeptide B and 10 pmol of cytochrome c tryptic digest. Although fully suppressed under direct MALDI conditions, LC-MALDI analysis was able to detect the 100 fmol peptide with 10 s fraction collection. Finally, the ability to inject relatively large sample amounts to improve detectability of low-abundance peptides was illustrated in the analysis of phosphopeptides from alpha-casein tryptic digests. A digest loaded on column to 2.4 microg and analyzed by LC-MALDI MS/MS resulted in 82% sequence coverage and detection of all nine phosphoserine residues. It is concluded that, being able to handle both high- and low-flow LC separations, the impulse-driven heated-droplet interface provides the flexibility to carry out MALDI analysis of peptides and proteins depending on the information sought after, analysis speed, and sample size.