Automated MALDI matrix deposition method with inkjet printing for imaging mass spectrometry

Careful matrix deposition on tissue samples for matrix-assisted laser desorption/ionization (MALDI) is critical for producing reproducible analyte ion signals. Traditional methods for matrix deposition are often considered an art rather than a science, with significant sample-to-sample variability. Here we report an automated method for matrix deposition, employing a desktop inkjet printer (<$200) with 5760 x 1440 dpi resolution and a six-channel piezoelectric head that delivers 3 pL/drop. The inkjet printer tray, designed to hold CDs and DVDs, was modified to hold microscope slides. Empty ink cartridges were filled with MALDI matrix solutions, including DHB in methanol/water (70:30) at concentrations up to 40 mg/mL. Various samples (including rat brain tissue sections and standards of small drug molecules) were prepared using three deposition methods (electrospray, airbrush, inkjet). A linear ion trap equipped with an intermediate-pressure MALDI source was used for analyses. Optical microscopic examination showed that matrix crystals were formed evenly across the sample. There was minimal background signal after storing the matrix in the cartridges over a 6-month period. Overall, the mass spectral images gathered from inkjet-printed tissue specimens were of better quality and more reproducible than from specimens prepared by the electrospray and airbrush methods.     

A MALDI sample preparation method suitable for insoluble polymers

Polyamides are insoluble or poorly soluble in common organic solvents, which makes normal sample preparation for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry very difficult. An new analytical protocol for MALDI analysis of polyamides or other insoluble samples is described. It consists of pressing a pellet from a solid mixture of the polymer and a matrix, both in the form of finely ground powder. This sample preparation is compared with the common dried droplet sample preparation method and found to perform much better, both in terms of robustness against variation of experimental parameters and high-mass capability.     

Massively parallel sample preparation for the MALDI MS analyses of tissues

Investigation of the peptidome of the nervous system containing large, often easily identifiable neurons has greatly benefited from single-cell matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and has led to the discovery of hundreds of novel cell-to-cell signaling peptides. By combining new sample preparation methods and established protocols for bioanalytical mass spectrometry, a high-throughput, small-volume approach is created that allows the study of the peptidome of a variety of nervous systems. Specifically, approximately single-cell-sized samples are rapidly prepared from thin tissue slices by adhering the tissue section to a glass bead array that is anchored to a stretchable membrane. Stretching the membrane fragments the tissue slice into thousands of individual samples, their dimensions predominately governed by the size of the individual glass beads. Application of MALDI matrix, followed by the repeated condensation of liquid microdroplets on the fragmented tissue, allows for maximal analyte extraction and incorporation into MALDI matrix crystals. During extraction, analyte migration between the pieces of tissue on separate beads is prevented by the underlying hydrophobic substrate and by controlling the size of the condensation droplets. The procedure, while general in nature, may be tailored to the needs of a variety of analyses, producing mass spectra equivalent to those acquired from single-cell samples.     

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.     

A microfluidic electrocapture device in sample preparation for protein analysis by MALDI mass spectrometry

The design and operation of a microfluidic device for sample preparation in MALDI mass spectrometry of peptides and proteins is described. It is particularly useful for proteomics applications and for mass determination of proteins in salt- and detergent-containing solutions. The system consists of a flow channel with two conductive areas or electrical junctions where proteins and peptides are retained by means of an electric field. The microfluidic device is made of PEEK tubing, and the junctions are covered with a conductive polymeric membrane. A syringe pump connected to the device produces a flow stream, and injection of sample is carried out manually via hydrodynamic pressure. Proteolytic peptides and intact proteins in salt- and detergent-containing acidic media were captured at the cathode junction followed by exchange of the original solution to a solvent suitable for subsequent mass spectrometry. Using this principle, a significant desalting effect was obtained for tryptic peptides in mass-mapping experiments. Protein sequence coverages were high (up to 40%) at subpicomole levels with results better than those obtained using reversed-phase solid-phase extraction. In contrast to the latter technique, the microfluidic device has the capacity to efficiently remove detergents such as CHAPS before peptide mapping and protein analysis.     

Integrated sample preparation and MALDI mass spectrometry on a microfluidic compact disk

High-throughput microfluidic processing of protein digests integrated with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry on a compact disk (CD) is described. Centrifugal force moves liquid through multiple microstructures, each containing a 10-nL reversed-phase chromatography column. The CD enables parallel preparation of 96 samples with volumes ranging from one to several microliters. The peptides in the digests are concentrated, desalted, and subsequently eluted from the columns directly into MALDI target areas (200 x 400 microm) on the CD using a solvent containing the MALDI matrix. After crystallization, the CD is inserted into the MALDI instrument for peptide mass fingerprinting and database identification at a routine sensitivity down to the 200-amol level. Detection of proteolytic peptides down to the 50-amol level is demonstrated. The success rate of the CD technology in protein identification is about twice that of the C(18) ZipTips and standard MALDI steel targets. The CDs are operated using robotics to transfer samples and reagents from microcontainers to the processing inlets on the disposable CD and spinning to control the movement of liquid through the microstructures.     

Deamidation of -Asn-Gly- sequences during sample preparation for proteomics: Consequences for MALDI and HPLC-MALDI analysis

We find that peptides containing -Asn-Gly- sequences typically show approximately 70-80% degree of deamidation after standard overnight (approximately 12 h) tryptic digestion at 37 degrees C. This emphasizes the need for more detailed information about the deamidation reaction in -Asn-Gly- sequences, in which two deamidated species are produced, one containing an aspartic acid (-Asp-Gly-) residue and the other containing an isoaspartic acid (-betaAsp-Gly-) residue. For the peptide SLNGEWR (54-60 beta-galactosidase, E. coli), all three components of the reaction mixture were separated by HPLC on C18 300-A sorbent, with trifluoroacetic acid as an ion-pairing modifier. Their intensity ratios suggested the elution order -betaAsp-/-Asn-/-Asp-, which was subsequently confirmed by MALDI MS and MS/MS analysis. The kinetics of the deamidation was studied in detail for the synthetic SLNGEWR parent using RP HPLC with UV detection. The half-life of this peptide was found to be approximately 8 h under digestion conditions. Analysis of a large pool of peptide retention data shows that the -betaAsp-/-Asn-/ -Asp- retention order is normally observed under the above conditions, especially if the original -NG- sequence is surrounded by hydrophobic amino acids. However, changing chromatographic conditions to 100-A pore size sorbents, or using formic acid as a modifier, increases the retention time of -betaAsp- relative to the -Asn-/-Asp- pair, so the order can sometimes be different.     

Importance of solubility in the sample preparation of poly(ethylene terephthalate) for MALDI TOFMS

The role of solubility in the sample preparation process for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is demonstrated for oligomeric and medium molar mass poly(ethylene terephthalate) (PET). For low molar mass oligomers (PET-1), minor discrimination effects were observed when the sample was not completely in solution. MALDI spectra of medium molar mass PET, representative of the entire molar mass distribution, were obtained only when a good solvent for PET was used, such as 1,1,1,3,3,3-hexafluoro-2-propanol (commonly referred to as HFIP), as the sample preparation solvent and dithranol as the matrix. The azeotropic composition of 70:30 CH(2)Cl(2)/HFIP better solubilizes the more nonpolar matrixes, which enables more latitude in selecting sample preparation conditions than pure HFIP. Segregation effects were observed when the azeotrope mixture was diluted with tetrahydrofuran, resulting in large molar mass distribution discrimination effects in the MALDI spectra. Dilution with CH(2)Cl(2) resulted in a significant decrease in the overall signal intensity for the entire polymer distribution. With each attempt to dilute the azeotrope, the sample after solvent evaporation was visibly heterogeneous, which resulted in shot-to-shot variability. Both examples demonstrate the importance of constant solvent composition during solvent evaporation. The compatibility of matrix and polymer was explored using relative HPLC retention times. Consistent with previous work in our laboratories, it was found that the matrix/polymer combination that has the closest match of retention time resulted in the best MALDI signal intensity.     

Infrared laser ablation sample transfer for MALDI imaging

An infrared laser was used to ablate material from tissue sections under ambient conditions for direct collection on a matrix assisted laser desorption ionization (MALDI) target. A 10 μm thick tissue sample was placed on a microscope slide and was mounted tissue-side down between 70 and 450 μm from a second microscope slide. The two slides were mounted on a translation stage, and the tissue was scanned in two dimensions under a focused mid-infrared (IR) laser beam to transfer material to the target slide via ablation. After the material was transferred to the target slide, it was analyzed using MALDI imaging using a tandem time-of-flight mass spectrometer. Images were obtained from peptide standards for initial optimization of the system and from mouse brain tissue sections using deposition either onto a matrix precoated target or with matrix addition after sample transfer and compared with those from standard MALDI mass spectrometry imaging. The spatial resolution of the transferred material is approximately 400 μm. Laser ablation sample transfer provides several new capabilities not possible with conventional MALDI imaging including (1) ambient sampling for MALDI imaging, (2) area to spot concentration of ablated material, (3) collection of material for multiple imaging analyses, and (4) direct collection onto nanostructure assisted laser desorption ionization (NALDI) targets without blotting or ultrathin sections.     

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.     

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.     

Automated acoustic interferometer

Translated from Izmeritel'naya Tekhnika, No. 12, pp. 41–43, December, 1989.     

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.     

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.     

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.     

Alkylated dihydroxybenzoic acid as a MALDI matrix additive for hydrophobic peptide analysis

Hydrophobic peptides are generally difficult to detect using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) because the majority of MALDI matrixes are hydrophilic and therefore have a low affinity for hydrophobic peptides. Here, we report on a novel matrix additive, o-alkylated dihydroxybenzoic acid (ADHB), which is a 2,5-dihydroxybenzoic acid (DHB) derivative incorporating a hydrophobic alkyl chain on a hydroxyl group to improve its affinity for hydrophobic peptides, thereby improving MALDI-MS sensitivity. The addition of ADHB to the conventional matrix α-cyano-4-hydroxycinnamic acid (CHCA) improved the sensitivity of hydrophobic peptides 10- to 100-fold. The sequence coverage of phosphorylase b digest was increased using ADHB. MS imaging indicated that hydrophobic peptides were enriched in the rim of a matrix/analyte dried spot when ADHB was used. In conclusion, the addition of ADHB to the standard matrix led to improved sensitivity of hydrophobic peptides by MALDI-MS.     

Combination detergent/MALDI matrix: functional cleavable detergents for mass spectrometry

This study reports the synthesis of the first functional cleavable detergent designed specifically for applications in mass spectrometry. Upon cleavage, two inert compounds and the MALDI matrix are formed, eliminating sources of potential interference originating from traditional cleavable detergents. Analysis of peptides demonstrates that MALDI matrix generated in situ results in MALDI spectra equivalent to those prepared using established protocols. Analysis of the membrane protein diacylglycerol kinase was accomplished using the combination detergent/MALDI matrix. Applications of the functional cleavable detergents to the profiling of whole cell lysates results in increased signal-to-noise ratios of many ions and the detection of additional proteins previously not observed.     

Matrix solution fixation: histology-compatible tissue preparation for MALDI mass spectrometry imaging

Mass spectrometry imaging (MSI) acquires a grid of spatially resolved mass spectra and provides a molecular landscape of a tissue. This can have a myriad of uses: from basic tissue characterization to a comprehensive pathological diagnosis. We have developed a fast, inexpensive, histology-compatible tissue preparation method for matrix-assisted laser desorption/ionization (MALDI)-MSI, which overcomes current sample preparation-imposed limitations in image resolution. Tissue sections are prepared via simultaneous fixation and matrix deposition. This is accomplished by incorporating the MALDI matrix into solvents that preserve tissue integrity when applied according to standard histology procedures. This concept was expanded to include multiple histology protocols, thereby enabling analysis to be tailored to a variety of biomolecules and tissue types.     

Automated target preparation for microarray-based gene expression analysis

DNA microarrays have rapidly evolved toward a platform for massively paralleled gene expression analysis. Despite its widespread use, the technology has been criticized to be vulnerable to technical variability. Addressing this issue, recent comparative, interplatform, and interlaboratory studies have revealed that, given defined procedures for "wet lab" experiments and data processing, a satisfactory reproducibility and little experimental variability can be achieved. In view of these advances in standardization, the requirement for uniform sample preparation becomes evident, especially if a microarray platform is used as a facility, i.e., by different users working in the laboratory. While one option to reduce technical variability is to dedicate one laboratory technician to all microarray studies, we have decided to automate the entire RNA sample preparation implementing a liquid handling system coupled to a thermocycler and a microtiter plate reader. Indeed, automated RNA sample preparation prior to chip analysis enables (1) the reduction of experimentally caused result variability, (2) the separation of (important) biological variability from (undesired) experimental variation, and (3) interstudy comparison of gene expression results. Our robotic platform can process up to 24 samples in parallel, using an automated sample preparation method that produces high-quality biotin-labeled cRNA ready to be hybridized on Affymetrix GeneChips. The results show that the technical interexperiment variation is less pronounced than with manually prepared samples. Moreover, experiments using the same starting material showed that the automated process yields a good reproducibility between samples.