Device for the reversed-phase separation and on-target deposition of peptides incorporating a hydrophobic sample barrier for matrix-assisted laser desorption/ionization mass spectrometry

The separation of peptide mixtures from proteolytic cleavage is often necessary prior to mass spectrometry (MS) to enhance sensitivity and peptide mapping coverage. When buffers, salts, and other higher abundance peptides/contaminants are present, competition for charge during the electrospray ionization and matrix-assisted laser desorption/ionization (MALDI) processes can lead to ion suppression for the targeted analyte(s). In this note, a simple reversed-phase microcolumn sample separation and deposition device (Sep-Dep) is described. The use of this device improves or renders possible the analysis of complex or contaminated peptide mixtures by MALDI-MS. The method is simple and inexpensive and utilizes single-use low-cost Geloader-type columns packed with reversed-phase material. The device described utilizes an open column, allowing for a gradient or narrow-step gradient to be applied by any solvent delivery system or manually with a pipet. A key feature of the device is a deposition chamber that can be custom-built to hold any MALDI target. The Sep-Dep device is attached directly to an in-house vacuum line and draws solvent from the open-ended LC column. The elution of separated peptides is performed directly onto a target that has been treated with a hydrophobic barrier. This barrier effectively isolates fractions and improves the quality and morphology of the matrix crystals. The method produces efficient separations of proteolytic peptides, significantly reducing signal suppression effects in MALDI.     

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.     

Coupling of protein HPLC to MALDI-TOF MS using an on-target device for fraction collection, concentration, digestion, desalting, and matrix/analyte cocrystallization

Multidimensional protein chromatography offers an alternative to gel-based separations for large-scale proteomic analyses of highly complex mixtures. However, these liquid separations divide the original mixtures into multitudes of discrete samples, each of which may require numerous steps of sample manipulation, such as fraction collection, buffer exchange, protease digestion, peptide desalting, and, in the case of MALDI-MS, matrix and analyte cocrystallization on target. When traditional high-flow liquid chromatography is used, large volumes of solvent must also be removed from fractions to maximize MS sensitivity. Although robotic liquid-handling devices can facilitate these steps and reduce analyst/sample contact, they remain prototypic and expensive. Here, we explore the use of a novel, one-piece elastomeric device, the BD MALDI sample concentrator, which affixes to a MALDI target to create a prestructured 96-well sample array on the target surface. We have developed methodologies to process high-flow HPLC fractions by collecting them directly into the elastomeric device and then subjecting them to sequential on-target sample concentration, buffer exchange, digestion, desalting, and matrix/analyte cocrystallization for MALDI-MS analyses. We demonstrate that this methodology enables the rapid digestion and analysis of low amounts of proteins and that it is effective in the characterization of an HPLC-fractionated protein mixture by MALDI-TOF MS followed by peptide mass fingerprinting.     

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.     

Functional microfabricated sample targets for matrix-assisted laser desorption/ionization mass spectrometry analysis of ribonucleic acids

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a powerful analytical tool for the structural characterization of proteins and nucleic acids. However, many proteomics or genomics methodologies that employ MALDI-MS require external sample manipulation, which limits the overall throughput of analysis. We have focused on fabricating functional MALDI sample plates that would permit the on-probe characterization of nucleic acids. Here, we present results arising from the fabrication of functional sample plates composed of poly(methyl methacrylate) (PMMA). The PMMA sample plates were fabricated by a CNC milling technique. The key structural feature of our microfabricated samples plates is the presence of individual cylindrical posts (360 microm x 360 microm), which serve as individual sample targets within the overall PMMA-based MALDI sample plate. Functionality is added to these microposts via the covalent attachment of enzymes. As an example of the applicability of these microfabricated sample plates, enzymatic digestion of ribonucleic acids was performed on probe (i.e., on the micropost) with subsequent analysis by MALDI-MS. Advantages to such an approach include a reduction in sample handling (and concomitant sample losses) and a reduction in the amount of sample required for analysis due to the small surface area of the microposts.     

A CE-MALDI interface based on the use of prestructured sample supports

We have developed an off-line coupling of capillary electrophoresis (CE) to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS) based on CE fraction collection onto prestructured MALDI sample supports. Analyte carryover and detection sensitivity were investigated using a standard peptide mixture. Low femtomole amounts were detected, and no noticeable carryover was discovered. The performance of the method was evaluated with a mixture of tryptic digests of proteins from a human fetal brain cDNA expression library. The total number of identified peptides was increased from 47 to 211 when the CE-MALDI interface was used compared to direct MALDI-MS analysis. Sequence coverage with CE-MALDI was in the 25-60% range for the different proteins, corresponding to an increase of 1.3-4.9 times relative to that obtained with MALDI-MS of the crude mixture. Fractionation of sample components also facilitated protein identification by MALDI postsource decay analysis. Our initial results suggest this CE-MALDI interface can be used for the analysis of complex peptide mixtures isolated from biological tissues.     

Microdeposition device interfacing capillary electrochromatography and microcolumn liquid chromatography with matrix-assisted laser desorption/ionization mass spectrometry

A sample deposition device has been constructed and optimized for interfacing CEC and capillary LC columns to MALDI mass spectrometry. For CEC analysis, the device is composed of an inlet buffer reservoir and an outlet buffer reservoir connected to a matrix reservoir through a connection sleeve. The matrix reservoir is connected to a deposition capillary via another connection sleeve. CEC eluent is transported to the matrix reservoir via a capillary that is connected to the deposition capillary by the connection sleeve inside the matrix reservoir. This connection sleeve also acts as a mixing chamber, allowing the CEC eluent to be mixed with matrix prior to deposition. Complex glycan mixtures can be separated by CEC using hydrophilic-phase monolithic columns, with capillary eluent being deposited on a standard MALDI plate along with a suitable matrix solution. Thousands of discrete, highly homogeneous dots can be generated for a subsequent mass spectrometric analysis. With minor modifications, this device is also applicable to capillary LC of peptides using gradient elution. In this configuration, the outlet of the LC column is connected to a deposition capillary inside a matrix reservoir through a connection sleeve that allows mixing of the LC effluent with an appropriate matrix. The device has been evaluated with the tryptic digests of proteins.     

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.     

Microscale MALDI imaging of outer-layer lipids in intact egg chambers from Drosophila melanogaster

Fruit fly (Drosophila melanogaster) is a standard model organism used in genetics and molecular biology. Phospholipids are building blocks of cellular membranes, and components of a complex signaling network. Here, we present a facile method, based on matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS), for molecular imaging of phospholipid distributions in submillimeter-sized components of the fruit fly reproductive system. Individual egg chambers were deposited on a specially prepared MALDI target comprising an aluminum slide with a rough surface created by ablation with a microsecond-laser: this helped to immobilize biological specimens, remove excess of saline solution by adhesive forces, carry out microscopic observations, and facilitated distribution of the MALDI matrix. A continuous-flow ultrasound-assisted spray was used for the deposition of MALDI matrix (9-aminoacridine) onto the sample. The upper surface of the specimen was then scanned with a 355-nm solid-state laser with a preset beam focus of 10 μm to obtain negative-ion mode MALDI-MS images. Overall, this provided sufficient spatial resolution to reveal micrometer-scale gradient-like patterns of phospholipids along the anterior/posterior axis of egg chambers. Several phosphatidylinositols are seen to be segregated according to the number of unsaturated bonds, with an elevated abundance of polyunsaturated phosphatidylinositols within the oocyte compartment.     

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.     

Increasing surface capacity for on-probe affinity capture MALDI-MS via gold particle attachment to allyl amine plasma polymers

In this study, we demonstrate that the protein binding capacity of a surface modified matrix-assisted laser desorption/ionization (MALDI) target can be increased significantly by architecturing the surface of the MALDI probe using gold microparticles. In the present approach, a MALDI target, initially modified via pulsed rf plasma deposition of an allyl amine polymer thin film, is subsequently architectured via reaction with 2-iminothiolane and surface attachment of gold microparticles. The modified probe is then exposed to thiolated biotin to introduce an avidin binding element on the surface of the gold beads. The protein binding capacity of this architectured target is compared with a similarly plasma polymer modified MALDI target that is directly biotinylated. Application of various surface concentrations of avidin to the two probes and MALDI-MS analysis of avidin contained in the solution removed from the probe reveals that saturation of the gold-particle architectured target occurs at a factor of 15-30 higher applied surface concentration, as compared with the unarchitectured target. Furthermore, MALDI-MS analysis of the avidin retained on the two probes reveals that the limit of detection is lowered by a factor of 15-20 on the gold-particle architectured target as compared with the unarchitectured target.     

Fractionation and solvent-free MALDI-MS analysis of polymers using liquid adsorption chromatography at critical conditions in combination with a multisample on-target homogenization/transfer sample preparation method

A solvent-free homogenization/transfer matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) method is described for the preparation and precise transfer of up to 100 samples simultaneously on a single MALDI plate. This method is demonstrated using a poly(ethylene oxide) (PEO) mixture consisting of different molecular weights (500-6000) and end groups (PEO, dimethoxy-PEO, monomethoxy monomethacrylate-PEO, and dimethacrylate-PEO) that was fractionated using liquid adsorption chromatography at critical conditions. Off-line fractionation is performed prior to the on-target homogenization/transfer solvent-free sample preparation and MALDI mass analysis. The miniaturization of the solvent-free MALDI approach allowed analysis of less than 2 microg per PEO component per fraction corresponding to approximately 200 pmol for PEO 6000. The amounts of polymer sample used for LC separation and the quality of the MS results are equivalent to the "dry spray" method; however, three times more fractions were collected and analyzed with the newly developed hyphenated approach. The off-line method eliminates optimization of, for example, spray conditions or spreading of organic solvents on the MALDI plate that occurs with droplet deposition methods. The widespread applications of MALDI make this solvent-free, multisample method particularly important as it expands the capabilities for obtaining mass measurements with great efficiencies in areas with increased sample numbers. In addition, the solvent-free method is well suited for automated MALDI analysis as it virtually eliminates the "dead-spot" phenomenon.     

Oil-assisted sample preparation: a simple method for analysis of solid samples using matrix-assisted laser desorption/ionization mass spectrometry

Common mass spectrometric techniques, e.g., electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), require samples to be soluble in suitable solvents. Samples with solubility problems have difficulties for their mass spectrometric characterization. In this paper, an oil-assisted sample preparation (OASP) method was introduced for the analysis of solid samples using MALDI-MS. The novel method involves the use of a droplet of oil (i.e., paraffin oil) as the mixing and loading media for solid analyte and solid matrix. Using this method, rapid on-target sample preparation can be easily achieved, and only a transferable minimal amount of analyte and matrix is required. This method was demonstrated to be applicable for a wide range of analytes, including poorly soluble organic compounds, polymers, organometallic compounds, membrane peptides, and biological solid samples. The novel method can also be used for the analysis of "wet" and solution samples. The limit of detection of the OASP MALDI-MS was determined to be 1 ng with reserpine.     

Integrated microfluidic system enabling (bio)chemical reactions with on-line MALDI-TOF mass spectrometry

A continuous flow micro total analysis system (micro-TAS) consisting of an on-chip microfluidic device connected to a matrix assisted laser desorption ionization [MALDI] time-of-flight [TOF] mass spectrometer (MS) as an analytical screening system is presented. Reaction microchannels and inlet/outlet reservoirs were fabricated by powderblasting on glass wafers that were then bonded to silicon substrates. The novel lab-on-a-chip was realized by integrating the microdevice with a MALDI-TOFMS standard sample plate used as carrier to get the microfluidic device in the MALDI instrument. A novel pressure-driven pumping mechanism using the vacuum of the instrument as a driving force induces flow in the reaction microchannel in a self-activating way. Organic syntheses as well as biochemical reactions are carried out entirely inside the MALDI-MS ionization vacuum chamber and analyzed on-line by MALDI-TOFMS in real time. The effectiveness of the micro-TAS system has been successfully demonstrated with several examples of (bio)chemical reactions.     

High-speed, high-resolution monolithic capillary LC-MALDI MS using an off-line continuous deposition interface for proteomic analysis

High-speed, high-resolution LC separations, using a poly(styrene-divinylbenzene) monolithic column, have been coupled to MALDI MS and MS/MS through an off-line continuous deposition interface. The LC eluent was mixed with alpha-cyano-4-hydroxycinnamic acid matrix solution and deposited on a MALDI plate that had been precoated with nitrocellulose. Deposition at subatmospheric pressure (80 Torr) formed a 250-microm-wide serpentine trace with uniform width and microcrystalline morphology. The deposited trace was then analyzed in the MS mode using a MALDI-TOF/TOF MS instrument. Continuous deposition allowed interrogation of the separation with a high data sampling rate in the chromatographic dimensions, thus preserving the high resolution of narrow peaks (3-5-s peak width at half-height) of the fast monolithic LC. No extracolumn band broadening due to the deposition process was observed. Over 2000 components were resolved in a 10-min linear gradient separation of the model sample, and 386 unique peptides were identified in the subsequent MS/MS analysis. The continuous deposition interface allows the coupling of high-resolution separations to MALDI MS without degradation in separation efficiency, thus enabling high-throughput proteome analysis.     

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.     

Ozone in ambient air as a source of adventitious oxidation. A mass spectrometric study

This paper reports on an oxidation of biomolecules that can occur during sample preparation for analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). In measurements performed over several years, the oxidation artifacts were detected on methionine and tryptophan side chains in peptides containing these residues, as well as at the carbon-carbon double bonds of unsaturated phospholipids. A distinct hallmark of the artifacts is their seasonal nature: the highest levels of oxidation are typically seen in samples prepared for MALDI analysis during daytime hours and warmer months. The artifacts appear strongest when samples are allowed to air-dry in the absence of MALDI matrix and can be minimized when sample preparation is carried out in a nitrogen atmosphere or under vacuum. It is proposed that ambient ozone (originating from outdoor air taken into the laboratory through the air handling system) is acting during the obligate MALDI sample dry-down and matrix crystallization steps. To confirm the hypothesis, ozonolysis experiments as well as direct measurements of ambient ozone levels were performed. The hypothesis is reminiscent of the recent finding that ozone is the source of degradation of self-assembled alkanethiol monolayers that are exposed to ambient air (Schoenfisch, M. H.; Pemberton, J. E. J. Am. Chem. Soc. 1998, 120, 4502-4513). Thus, caution should be taken in handling ozone-reactive substances that are placed thinly on surfaces and exposed to ambient air, whether for MALDI analysis or for other increasingly popular applications involving the microdeposition of liquids.     

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.     

On-target exoglycosidase digestions/MALDI-MS for determining the primary structures of carbohydrate chains

One method used to determine the primary sequence of oligosaccharides is to digest them with exoglycosidases and analyze the resulting digestion products by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Previous research has demonstrated that these digestions can be performed on the MALDI target. However, the procedure requires the sample to be incubated at elevated temperatures, and complete digestion requires a few hours. We demonstrate new conditions that permit exoglycosidase digestions to be performed on the MALDI target at room temperature within 30 min. Oligosaccharide standards were digested with one or more exoglycosidases to show that the enzymes retain their activity and specificity under these new reaction conditions. Using this method, the primary sequences of carbohydrate chains can be determined in a relatively short amount of time.     

2,5-Dihydroxybenzoic acid butylamine and other ionic liquid matrixes for enhanced MALDI-MS analysis of biomolecules

The performance of the new ionic liquid MALDI-MS matrix 2,5-dihydroxybenzoic acid butylamine (DHBB) was assessed and compared to results obtained with the ionic liquid MALDI-MS matrixes alpha-cyano-4-hydroxycinnamic acid butylamine (CHCAB), 3,5-dimethoxycinnamic acid triethylamine (SinTri), and the frequently used solid MALDI matrixes 2,5-dihydroxybenzoic acid (DHB) and alpha-cyano-4-hydroxycinnamic acid (CHCA). The vacuum-stable, liquid consistency of ionic liquid matrix sample preparations considerably enhanced MALDI-MS analysis in terms of shot-to-shot reproducibility. Consequently, relative standard deviations serving as a measure for reproducibility of intensity-values acquired from 90 different spots on one MALDI-MS preparation were approximately one-half as high when solid DHB was replaced by the ionic liquid DHBB and eight times lower after exchange of solid CHCA by ionic liquid CHCAB. Interestingly, the ionic liquid MALDI matrix DHBB conserved the broad applicability of its solid analogue DHB, reduced MALDI induced fragmentation of monosialylated glycans and gangliosides, and was the superior ionic liquid matrix for MALDI-MS analysis of oligosaccharides and polymers, such as poly(ethylene glycol). It also worked well with glycoconjugates, peptides, and proteins; however, the tendency of DHBB to form multiple alkali adduct ions with peptides and proteins made CHCAB the ionic liquid matrix of choice for peptides. SinTri was the best ionic liquid matrix for proteins of high molecular weight, such as IgG. Furthermore, it was demonstrated for the first time that solvent properties and MALDI matrix properties of ionic liquids, such as DHBB, can be combined to enable fast, direct screening of an enzymatic reaction. This was proven by the desialylation of sialylactose with sialidase from Clostridium perfringens in the presence of diluted aqueous DHBB and subsequent direct MALDI-MS analysis of the reaction mixture.