Matrix-assisted Laser Desorption/ionization Sample Research Articles

RhizoMAP: a comprehensive, nondestructive, and sensitive platform for metabolic imaging of the rhizosphere

BackgroundElucidating the intricate structural organization and spatial gradients of biomolecular composition within the rhizosphere is critical to understanding important biogeochemical processes, which include the mechanisms of root-microbe interactions for maintaining sustainable plant ecosystem services. While various analytical methods have been developed to assess the spatial heterogeneity within the rhizosphere, a comprehensive view of the fine distribution of metabolites within the root-soil interface has remained a significant challenge. This is primarily due to the difficulty of maintaining the original spatial organization during sample preparation without compromising its molecular content.ResultsIn this study, we present a novel approach, RhizoMAP, in which the rhizosphere molecules are imprinted on selected polymer membranes and then spatially profiled using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI). We enhanced the performance of RhizoMAP by combining the use of two thin (< 20 μm) membranes (polyester and polycarbonate) with distinct MALDI sample preparations. This optimization allowed us to gain insight into the distribution of over 500 different molecules within the rhizosphere of poplar (Populus trichocarpa) grown in rhizoboxes filled with mycorrhizae soil. These two membranes, coupled with three different sample preparation conditions, enabled us to capture the distribution of a wide variety of molecules that included phytohormones, amino acids, sugars, sugar glycosides, polycarboxylic acids components of the Krebs cycle, fatty acids, short aldehydes and ketones, terpenes, volatile organic compounds, fertilizers from the soil, and others. Their spatial distribution varies greatly, with some following root traces, others showing diffusion from roots, some associated with soil particles, and many having distinct hot spots along the plant root or surrounding soil. Moreover, we showed how RhizoMAP can be used to localize the origin of the molecules and molecular transformation during root growth. Finally, we demonstrated the power of RhizoMAP to capture molecular distributions of key metabolites throughout a 20 cm deep rhizosphere.ConclusionsRhizoMAP is a method that provides nondestructive, untargeted, broad, and sensitive metabolite imaging of root-associated molecules, exudates, and soil organic matter throughout the rhizosphere, as demonstrated in a lab-controlled native soil environment.

Imaging Metabolites in Agar‐Based Bacterial Co‐Cultures with Minimal Sample Preparation using a DIUTHAME Membrane in Surface‐Assisted Laser Desorption/Ionization Mass Spectrometry**

AbstractWhile the imaging of bacteria, directly on agar‐based media, has been reported in matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI), such samples usually require laborious and time‐consuming preparation prior to analysis. Furthermore, the MALDI sample preparation may require desiccation, potentially causing sample deformation and/or degradation. Other issues may be associated with the use of a matrix in MALDI‐MSI. Here, we propose an alternative approach to image a co‐culture of Pseudomonas and Bacillus bacteria, grown on agar, in the positive and negative ionization modes, using surface‐assisted laser desorption/ionization (SALDI) MSI. A rapid and easy sample preparation was performed using a “desorption/ionization using through‐hole alumina membrane” (DIUTHAME) with a blotting method. The porous DIUTHAME membrane was used (1) for the transfer of the metabolites from the sample to the membrane, and (2) as assisting material in SALDI‐MSI. The advantages and limitations of this new sample preparation are discussed in this paper.

MALDI matrix cluster ions as internal references for ion mobility measurements

In the absence of high vacuum, the mobility of ions in an electric field is dependent on the charge, mass, shape and size of the ions. To achieve accurate calibration for ion mobility measurements, it is important to select calibrants with comparable physical properties to the molecular ions of interest. The size of an ion is often referred as its collision cross section (CCS). Currently, the information on the CCS values of various types of molecular ions are limited, thus representing a challenge to the calibration of ion mobility spectrometry. In this study, instead of finding a way to improve the calibration of ion mobility measurements, the intrinsic by-products of the conventional matrix-assisted laser desorption/ionization (MALDI) technique, namely MALDI matrix cluster (MAC) ions, are being used as internal references for ion mobility measurements. The standard MALDI matrix and sample preparation method are used. During the MALDI ionization process, MAC ions are generated and co-exist with the molecular ions of interest within the ion source. Our results indicate the MAC ions do possess suitable ion mobility characteristics, thus allowing the MAC ions to serve as internal reference for ion mobility measurements. The MAC ions cannot be used as internal standard or calibrants for ion mobility measurements, because their molecular structures as well as their CCS are unknown. However, the detection of MAC ions can allow us to determine whether the normal operation as well as the expected performance on the sensitivity and resolution of ion mobility spectrometry are achievable and reproducible. The MAC ions can also facilitate the transfer of specific experimental protocols between ion mobility instruments and/or laboratories. No extra materials, equipment or procedure are required for using MAC ions. For the proof of concept, all experimental work in this study was carried out on a traveling wave ion mobility mass spectrometry platform.

Effective analysis of degree of polymerization of polysialic acids in mass spectrometry by combining novel sample preparation and dynamic instrument optimization methods

This work demonstrates a mass spectrometry technique to improve data reliability when analyzing degree of polymerization (DP) of high-mass polysialic acids (PSAs). Matrix-assisted laser-desorption/ionization (MALDI) time-of-flight mass spectrometry is the technique of choice for analyzing large molecules due to its wide mass working range; however, the observed DP of PSAs using such an instrument is unreliable owing to sensitivity bias towards low-mass ions. A multi-layer MALDI sample preparation protocol is demonstrated in the current study to improve PSA sensitivity, and a dynamic instrument optimization method (DIOM) is employed to minimize detector saturation over a wide mass range. The DP information obtained in the DIOM combines a series of mass spectral data obtained with individually optimized instrument parameters to minimize the problem of sensitivity bias in respective mass ranges. The resultant mass spectra facilitate unambiguous determination of DP in the high-mass range due to significantly improved spectral quality. The main instrument parameters involved in the optimization process include extraction delay in MALDI ion source as well as the cutoff mass of the ion detector. In comparison to conventional methods, the DIOM doubles the maximum DP that can be unambiguously identified by mass spectrometry.

Large-Area Graphene Films as Target Surfaces for Highly Reproducible Matrix-Assisted Laser Desorption Ionization Suitable for Quantitative Mass Spectrometry

Due to the known sweet-spot issues that intrinsically arise from inhomogeneous formation of matrix-analyte crystals utilized as samples in matrix-assisted laser desorption ionization (MALDI) mass spectrometry, its reproducibility and thus its applications for quantification have been somewhat limited. In this paper, we report a simple strategy to improve the uniformity of matrix-analyte crystal spots, which we realized by adapting large-area graphene films, i.e., non-inert, interacting surfaces, as target surfaces. In this example, the graphitic surfaces of the graphene films interact with excess matrix molecules during the sample drying process, which induces spontaneous formation of optically uniform MALDI sample crystal layers on the film surfaces. Further, mass spectrometric imaging reveals that the visible uniformity is indeed accompanied by reproducible MALDI ionization over an entire sample spot, which greatly suppresses the appearance of sweet spots. The results of this study confirm that the proposed method achieves good linear responses of ion intensity to the analyte concentration (R2 > 0.99) with small relative standard deviations (σ < 10%), which is a range applicable for quantitative measurements using MALDI mass spectrometry. Graphical Abstract ᅟ.

Forced dried droplet method for MALDI sample preparation

A forced dried droplet method (FDD) is developed to overcome the drawbacks of the conventional dried-droplet (DD) method for matrix assisted laser desorption/ionization (MALDI) sample preparation. The crystals produced by the DD method are heterogeneous and irregularly distributed, and thus many methods have tried to solve the problems. However, most of them spend more time or need additional instruments to generate homogeneous microcrystals. The FDD sample preparation method can produce uniform microcrystals with homogeneous size distribution in few minutes without additional instruments. Stirring the sample spot solution (an agitation process) with a pipette tip can change the crystal size distribution which is observed by the microscope. Mass spectrometric analysis shows that the smaller the crystal size is, the better the ion signal intensity is. The formation of microcrystals can be explained with the effective rate of secondary nucleation. The relative standard deviation (RSD) of the FDD method is ∼16% which is comparable to the two-layer (TL) method and is better than the DD method.

The composition of liquid atmospheric pressure matrix-assisted laser desorption/ionization matrices and its effect on ionization in mass spectrometry

New liquid atmospheric pressure (AP) matrix-assisted laser desorption/ionization (MALDI) matrices that produce predominantly multiply charged ions have been developed and evaluated with respect to their performance for peptide and protein analysis by mass spectrometry (MS). Both the chromophore and the viscous support liquid in these matrices were optimized for highest MS signal intensity, S/N values and maximum charge state. The best performance in both protein and peptide analysis was achieved employing light diols as matrix support liquids (e.g. ethylene glycol and propylene glycol). Investigating the influence of the chromophore, it was found that 2,5-dihydroxybenzoic acid resulted in a higher analyte ion signal intensity for the analysis of small peptides; however, larger molecules (>17 kDa) were undetectable. For larger molecules, a sample preparation based on α-cyano-4-hydroxycinnammic acid as the chromophore was developed and multiply protonated analytes with charge states of more than 50 were detected. Thus, for the first time it was possible to detect with MALDI MS proteins as large as ∼80 kDa with a high number of charge states, i.e. m/z values below 2000. Systematic investigations of various matrix support liquids have revealed a linear dependency between laser threshold energy and surface tension of the liquid MALDI sample.

Use of MALDI-MS Combined with Differential Hydrogen-Deuterium Exchange for Semiautomated Protein Global Conformational Screening.

Matrix-assisted laser desorption/ionization (MALDI) coupled with a time-of-flight (TOF) mass-spectrometry (MS) detector is acknowledged to be very useful for analysis of biological molecules. At the same time, hydrogen-deuterium exchange (HDX) is a well-known technique for studying protein higher-order structure. However, coupling MALDI with HDX has been challenging because of undesired back-exchange reactions during analysis. In this report, we survey an approach that utilizes MALDI coupled with an automated sample preparation to compare global conformational changes of proteins under different solution conditions using differential HDX. A nonaqueous matrix was proposed for MALDI sample preparation to minimize undesirable back-exchange. An automated experimental setup based on the use of a liquid-handling robot and automated data acquisition allowed for tracking protein conformational changes as a difference in the number of protons exchanged to deuterons at specified solution conditions. Experimental time points to study the deuteration-labeling kinetics were obtained in a fully automated manner. The use of a nonaqueous matrix solution allowed experimental error to be minimized to within 1% RSD. We applied this newly developed MALDI-HDX workflow to study the effect of several common excipients on insulin folding stability. The observed results were corroborated by literature data and were obtained in a high-throughput and automated manner. The proposed MALDI-HDX approach can also be applied in a high-throughput manner for batch-to-batch higher-order structure comparison, as well as for the optimization of protein chemical modification reactions.

Preparation of Homogeneous MALDI Samples for Quantitative Applications.

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The heterogeneity of ion signals is a severe problem in MALDI, which results in poor data reproducibility and makes MALDI unsuitable for quantitative analysis. By regulating sample plate temperature during sample preparation, thermal-induced hydrodynamic flows inside droplets of sample solution are able to reduce the heterogeneity problem. A room-temperature sample preparation chamber equipped with a temperature-regulated copper base block that holds MALDI sample plates facilitates precise control of the sample drying condition. After drying of sample droplets, the temperature of sample plates is returned to room temperature and removed from the chamber for subsequent mass spectrometric analysis. The areas of samples are examined with MALDI-imaging mass spectrometry to obtain the spatial distribution of all components in the sample. In comparison with the conventional dried-droplet method that prepares samples under ambient conditions without temperature control, the samples prepared with the method demonstrated herein show significantly better spatial distribution and signal intensity. According to observations using carbohydrate and peptide samples, decreasing substrate temperature while maintaining the surroundings at ambient temperature during the drying process can effectively reduce the heterogeneity of ion signals. This method is generally applicable to various combinations of samples and matrices.

Liquid MALDI MS Analysis of Complex Peptide and Proteome Samples.

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) is well-known to be a powerful technique for the analysis of biological samples. By using glycerol-based liquid support matrices (LSMs) instead of conventional MALDI matrices the power of this technique can be extended further. In this study, we exploited LSMs for the identification of complex samples, that is, the Lactobacillus proteome and a bovine serum albumin (BSA) digest. Liquid and solid MALDI samples were manually and robotically prepared by coupling a nanoflow high-performance liquid chromatography (nanoHPLC) system to an automated MALDI sample spotting device. MS and MS/MS data were successfully acquired at the femtomole level using TOF/TOF as well as Q-TOF instrumentation and used for protein identification searching sequence databases. For the BSA digest analysis, liquid MALDI samples resulted in peptide mass fingerprints, which led to a higher confidence in protein identification compared with solid (crystalline) MALDI samples; however, postsource decay (PSD) MS/MS analysis of both the proteome of Lactobacillus plantarum WCFS1 cells and BSA digest showed that further optimization of the formation and detection of peptide fragment ions is still needed for liquid MALDI samples, as the MS/MS ion search score was lower than that for the solid MALDI samples, reflecting the poorer quality of the liquid MALDI-PSD spectra, which can be attributed to the differences in PSD parameters and their optimization that is currently achievable.

Reducing Spatial Heterogeneity of MALDI Samples with Marangoni Flows During Sample Preparation.

This work demonstrates a method to prepare homogeneous distributions of analytes to improve data reproducibility in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). Natural-air drying processes normally result in unwanted heterogeneous spatial distributions of analytes in MALDI crystals and make quantitative analysis difficult. This study demonstrates that inducing Marangoni flows within drying droplets can significantly reduce the heterogeneity problem. The Marangoni flows are accelerated by changing substrate temperatures to create temperature gradients across droplets. Such hydrodynamic flows are analyzed semi-empirically. Using imaging mass spectrometry, changes of heterogeneity of molecules with the change of substrate temperature during drying processes are demonstrated. The observed heterogeneities of the biomolecules reduce as predicted Marangoni velocities increase. In comparison to conventional methods, drying droplets on a 5 °C substrate while keeping the surroundings at ambient conditions typically reduces the heterogeneity of biomolecular ions by 65%-80%. The observation suggests that decreasing substrate temperature during droplet drying processes is a simple and effective means to reduce analyte heterogeneity for quantitative applications. Graphical Abstract ᅟ.

Preparation of Homogeneous Solid Samples for Reproducible and Quantitative MALDI

Preparing samples with good homogeneity is one of the requirements for generating reproducible matrix‐assisted laser desorption ionization (MALDI) spectra and for using them in analyte quantification. Thus far, we have not been successful in producing homogeneous solid samples from popular matrixes apart from α‐cyano‐4‐hydroxycinnamic acid (CHCA). In this work, we demonstrate the production of an outwardly homogeneous solid sample by loading a methanol solution of a matrix into a shallow reservoir on a coated MALDI sample plate and then vacuum‐drying it. Out of ten popular matrixes tested, seven yielded homogeneous samples. These were 9‐aminoacridine, 6‐aza‐2‐thiothymine, CHCA, 2,5‐dihydroxybenzoic acid, ferulic acid, sinapinic acid, and 2,4,6‐trihydroxyacetophenone. For MALDI with these matrixes, linear calibration curves covering wide dynamic ranges were acquired. Performances of these matrixes in various aspects of analyte quantification have been investigated.

Semiquantitation of diphenidine in tissue sections obtained from a human cadaver in a poisoning case by direct MALDI-QTOF mass spectrometry

Diphenidine [1-(1,2-diphenyl-ethyl)piperidine] in the tissue sections of the adipose, brain, heart, kidney, and liver obtained from a human cadaver in a poisoning case was directly analyzed as the first trial by matrix-assisted laser desorption ionization (MALDI)-quadrupole time-of-flight mass spectrometry (QTOF-MS). The frozen tissue block of approximately 5 × 5 × 5 mm3 was sectioned to a thickness of 10 or 20 μm at −20 or −35 °C. For quantitation, the reference standard solutions of diphenidine were placed at 0, 0.1, 1, and 10 ng/well on the wells of a MALDI sample plate firstly, and about 20 tissue sections (ca. 5 × 5 mm2 each) were placed on the sample plate secondly, followed by detection by MALDI-MS/MS after desiccation of the tissue sections and spraying α-cyano-4-hydroxycinnamic acid as the matrix. The quantitation was performed by the standard addition method without the use of blank human tissues. The correlation coefficients between the concentrations of diphenidine and the relative signal intensities were from 0.937 to 0.994 for five kinds of tissue. The suppression of signals by tissue was increased in the order of adipose tissue < brain < heart, kidney, and liver; hence, the adipose tissue was most suitable tissue among them for the detection of diphenidine. In the present method, the concentration linearity and repeatability were not satisfactory for quantitative analysis, but allowable for semiquantitation. Therefore, the present method can be used for a semiquantitative analysis of diphenidine in tissue sections because of the simplicity of the method.

Ghost peaks observed after atmospheric pressure matrix-assisted laser desorption/ionization experiments may disclose new ionization mechanism of matrix-assisted hypersonic velocity impact ionization.

Understanding the mechanisms of matrix-assisted laser desorption/ionization (MALDI) promises improvements in the sensitivity and specificity of many established applications in the field of mass spectrometry. This paper reports a serendipitous observation of a significant ion yield in a post-ionization experiment conducted after the sample had been removed from a standard atmospheric pressure (AP)-MALDI source. This post-ionization is interpreted in terms of collisions of microparticles moving with a hypersonic velocity into a solid surface. Calculations show that the thermal energy released during such collisions is close to that absorbed by the top matrix layer in traditional MALDI. The microparticles, containing both the matrix and analytes, could be detached from a film produced inside the inlet capillary during the sample ablation and accelerated by the flow rushing through the capillary. These observations contribute some new perspective to ion formation in both laser and laser-less matrix-assisted ionization. An AP-MALDI ion source hyphenated with a three-stage high-pressure ion funnel system was utilized for peptide mass analysis. After the laser had been turned off and the MALDI sample removed, ions were detected during a gradual reduction of the background pressure in the first funnel. The constant-rate pressure reduction led to the reproducible appearance of different singly and doubly charged peptide peaks in mass spectra taken a few seconds after the end of the MALDI analysis of a dried-droplet spot. The ion yield as well as the mass range of ions observed with a significant delay after a completion of the primary MALDI analysis depended primarily on the background pressure inside the first funnel. The production of ions in this post-ionization step was exclusively observed during the pressure drop. A lower matrix background and significant increase in relative yield of double-protonated ions are reported. The observations were partially consistent with a model of the supersonic jet from the inlet capillary accelerating detached particles to kinetic energies suitable for matrix-assisted hypersonic-velocity impact ionization.

A simple MALDI plate functionalization by Vmh2 hydrophobin for serial multi-enzymatic protein digestions.

The development of efficient and rapid methods for the identification with high sequence coverage of proteins is one of the most important goals of proteomic strategies today. The on-plate digestion of proteins is a very attractive approach, due to the possibility of coupling immobilized-enzymatic digestion with direct matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF)-mass spectrometry (MS) analysis. The crucial step in the development of on-plate immobilization is however the functionalization of the solid surface. Fungal self-assembling proteins, the hydrophobins, are able to efficiently functionalize surfaces. We have recently shown that such modified plates are able to absorb either peptides or proteins and are amenable to MALDI-TOF-MS analysis. In this paper, the hydrophobin-coated MALDI sample plates were exploited as a lab-on-plate for noncovalent immobilization of enzymes commonly used in protein identification/characterization, such as trypsin, V8 protease, PNGaseF, and alkaline phosphatase. Rapid and efficient on-plate reactions were performed to achieve high sequence coverage of model proteins, particularly when performing multiple enzyme digestions. The possibility of exploiting this direct on-plate MALDI-TOF/TOF analysis has been investigated on model proteins and, as proof of concept, on entire whey milk proteome.

A derivatization and validation strategy for determining the spatial localization of endogenous amine metabolites in tissues using MALDI imaging mass spectrometry.

Imaging mass spectrometry (IMS) studies increasingly focus on endogenous small molecular weight metabolites and consequently bring special analytical challenges. Since analytical tissue blanks do not exist for endogenous metabolites, careful consideration must be given to confirm molecular identity. Here, we present approaches for the improvement in detection of endogenous amine metabolites such as amino acids and neurotransmitters in tissues through chemical derivatization and matrix-assisted laser desorption/ionization (MALDI) IMS. Chemical derivatization with 4-hydroxy-3-methoxycinnamaldehyde (CA) was used to improve sensitivity and specificity. CA was applied to the tissue via MALDI sample targets precoated with a mixture of derivatization reagent and ferulic acid as a MALDI matrix. Spatial distributions of chemically derivatized endogenous metabolites in tissue were determined by high-mass resolution and MS(n) IMS. We highlight an analytical strategy for metabolite validation whereby tissue extracts are analyzed by high-performance liquid chromatography (HPLC)-MS/MS to unambiguously identify metabolites and distinguish them from isobaric compounds.

Compressed matrix thin film (CMTF)-assisted laser desorption ionization mass spectrometric analysis

The inhomogeneous re-crystallization process of matrix materials is the major concerns associated with matrix assisted laser desorption/ionization (MALDI) analysis. We describe here the approach termed compressed matrix thin film (CMTF) in order to make a uniform matrix deposition. In this approach, solid matrix particles are compressed under 10MPa of pressure by a compressor that is regularly used in infrared spectroscopic analysis. Then aqueous samples can be deposited on the surface of the matrix film. Major advantages of the CMTF approach are summarized as follows. (1) Reproducible sample preparation procedure. Size and thickness of matrix thin films can be controlled by using a fixed mold.force and known amount of matrix materials. (2) Significantly decreased shot-to-shot variations and enhanced reproducibility. (3) Tolerance for in situ salt washing. Because matrix materials are hydrophobic, salts can be washed away while proteins or peptides are retained on the surface of matrix thin films through hydrophobic interactions. (4) Improved sensitivity. The hydrophobic coating of MALDI sample plate by matrix thin films prevents the spreading of samples across the plate and confines analytes to a small area, leading to increased local concentration. (5) A new means for tissue analysis. Tissue sections can be directly transferred to the uniform surface of matrix materials for reproducible and quantitative comparison of different molecules in different localization. The proposed CMTF should be an enabling technique for mass spectrometric analysis with improved correlations between signal intensities and sample quantities.

Selective Enhancement of Carbohydrate Ion Abundances by Diamond Nanoparticles for Mass Spectrometric Analysis

Diamond nanoparticles (DNPs) were incorporated into matrix-assisted laser desorption/ionization (MALDI) samples to enhance the sensitivity of the mass spectrometer to carbohydrates. The DNPs optimize the MALDI sample morphology and thermalize the samples for thermally labile compounds because they have a high thermal conductivity, a low extinction coefficient in UV-vis spectral range, and stable chemical properties. The best enhancement effect was achieved when matrix, DNP, and carbohydrate solutions were deposited and vacuum-dried consecutively to form a trilayer sample morphology. It allows the direct identification of underivatized carbohydrates mixed with equal amount of proteins because no increase in the ion abundance of proteins was achieved. For dextran with an average molecular weight of 1500, the trilayer method typically improves the sensitivity by 79- and 7-fold in comparison to the conventional dried-droplet and thin-layer methods, respectively.

Matrix Assisted Ionization Vacuum (MAIV), a New Ionization Method for Biological Materials Analysis Using Mass Spectrometry

The introduction of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) for the mass spectrometric analysis of peptides and proteins had a dramatic impact on biological science. We now report that a wide variety of compounds, including peptides, proteins, and protein complexes, are transported directly from a solid-state small molecule matrix to gas-phase ions when placed into the vacuum of a mass spectrometer without the use of high voltage, a laser, or added heat. This ionization process produces ions having charge states similar to ESI, making the method applicable for high performance mass spectrometers designed for atmospheric pressure ionization. We demonstrate highly sensitive ionization using intermediate pressure MALDI and modified ESI sources. This matrix and vacuum assisted soft ionization method is suitable for the direct surface analysis of biological materials, including tissue, via mass spectrometry.

Comparison of Sample Preparation Methods and Evaluation of Intra- and Intersample Reproducibility in Bacteria MALDI-MS Profiling

A study is presented on the reproducibility of mass spectral profiles of the whole bacterium E. coli resulting from laser sampling at different regions within and between matrix-assisted laser desorption ionization (MALDI) samples deposited onto the plate. Samples were prepared with different deposition methods and using different MALDI matrices. The three most common matrices used in MALDI-mass spectrometry (MS) bacteria profiling, α-cyano-4-hydroxycinnamic acid (CHCA), sinapinic acid (SA), and ferulic acid (FA), were compared in this study along with two pipet-based sample deposition methods, dried-droplet and premix. Sample variability was determined by analysis of variances (ANOVA), principal component analysis (PCA), and multivariate ANOVA (MANOVA). For the two pipet-based sample deposition methods tested in this study, the intrasample variability (most commonly referred to as "spot-to-spot" reproducibility) was of the same magnitude as the intersample variability for all MALDI matrices tested. By incorporating a spray nebulizer sample deposition method to produce uniform sample/matrix mixtures onto the MALDI plate, we demonstrate that the crystalline morphology of the MALDI sample greatly influences the intrasample reproducibility (i.e., spot-to-spot) of the resulting whole cell MALDI-MS profiles. Overall, for the pipet-based deposition methods, results showed that the smallest variability in bacteria MALDI mass spectral profiles was obtained from samples deposited using the premix method, regardless of the MALDI matrix used, with the best reproducibility obtained with the CHCA matrix. It is concluded that a sample preparation strategy that reduces or eliminates the MALDI matrix morphology heterogeneity can reduce variability (i.e., spot-to-spot) of the bacteria mass spectral profiles by up to 90%.