Identification of Microbial Strains via 2D Cross-Correlation of LC-MS Data.

Mass spectrometry is commonly used in the identification of species present in microbial samples, but the high similarity in the peptide composition between strains of a single species has made analysis at the subspecies level challenging. Prior research in this area has employed methods such as Principal Component Analysis (PCA), the k-Nearest Neighbors' (kNN) algorithm, and Pearson correlation. Previously, 1D cross-correlation of mass spectra has been shown to be useful in the classification of small molecule compounds as well as in the identification of peptide sequences via the SEQUEST algorithm and its variants. While direct application of cross-correlation to mass spectral data has been shown to aid in the identification of many other types of compounds, this type of analysis has not been demonstrated in the literature for the purpose of LC-MS based identification of microbial strains. A method of identifying microbial strains is presented here that applies the principle of 2D cross-correlation to LC-MS data. For a set of N = 30 yeast isolate samples representing 5 yeast strains (K-97, S-33, T-58, US-05, WB-06), high-resolution LC-MS-Orbitrap data were collected. Reference spectra were then generated for each strain from the combined data of each sample of that strain. Sample strains were then predicted by computing the 2D cross-correlation of each sample against the reference spectra, followed by application of correction factors measuring the asymmetry of the 2D correlation functions.

The cationization of synthetic polymers in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: Investigations of the salt-to-analyte ratio.

Matrix-assisted laser desorption/ionization (MALDI) is a soft ionization technique that when used to analyze synthetic polymer analytes often requires the addition of a metal cationization agent (herein termed the "salt"). The choice of both the matrix and the cationization agent needs to be taken into account when considering the polymer under study; different polymers have shown different affinities toward different cationization agents, and their selectivity can change as the matrix changes. Salt-to-analyte ratio (S/A) plots are used in this work to investigate the effect of the quantity of cationization agent employed in the analysis of a poly (methylmethacrylate) (PMMA) analyte with different MALDI matrices. The point at which analyte signal stops increasing with the added cationization agent is termed the "cation saturation point," and it was found to occur around a S/A of 1. When the analyte signal after this point remains constant, it is termed an "ideal case." The "non-ideal case" occurs when the analyte signal decreases after the cation saturation point. The amount of matrix present (measured as the matrix-to-analyte molar ratio, M/A) and the use of different counterions for the salt are also found to affect the intensity of the analyte signal. In non-ideal cases, changes in the counterion or an increase in the M/A are found to increase the analyte signal, often converting an initially observed non-ideal case into an ideal case. Several experiments attempting to uncover the reason for observation of the non-ideal S/A behavior are also described.

A Method for Defining the Position of Ion Formation in a MALDI TOFMS by Analysis of the Laser Image on the Sample Surface.

A method is developed to determine the position of ion formation along the flight axis of a MALDI TOFMS instrument using the image of the laser on the sample surface. Previous work (JASMS 2018, 29, 422-434) showed that misalignment of the sample stage in a Bruker Autoflex III MALDI TOFMS as well as multiple insertions/mountings of the target plate and differences in target plate shape itself produced reproducible changes in the measured ion time-of-flight which could be attributed to changes in the position of ion formation along the instrument flight axis. Here, a small but reproducible change in the position of the laser in the sample-viewing camera image was observed, with the movement depending on both the sample position and target plate used. Using the change in coordinates of the laser position in the camera image and the known angle of incidence of the laser on the sample surface, the initial z-axis position of the ion at different locations on the plate can be calculated, exactly defining changes in the ion flight path length and the distance between the sample plate and first extraction plate/grid with sample position on the target plate. A correction method is developed to correct the time-of-flight values collected from different locations on the sample plate using the laser images, with the relative standard deviation (RSD) being reduced from 23 ppm to below 6 ppm. The laser images, along with the measured target plate heights, are also used to calculate the misalignment of the sample stage. Graphical Abstract.

Cell-Free Identification of S. cerevisiae Strains by Analysis of Supernatant Using LC-MS.

Current literature shows a gap for methods which can identify yeast sub-species (strains or serovars) in samples where there are no viable cells remaining. Presented here is a technique for the analysis of yeast supernatant, including solid phase extraction, data-dependent acquisition liquid chromatography/mass spectrometry (LC-MS), and two chemometric methods to identify and classify yeast strains. Five strains of Saccharomyces cerevisiae were successfully identified in various stages of growth. In addition, peptide/protein identification was performed, without the need for additional data acquisition. Graphical Abstract ᅟ.

Introducing a Cell-Free Approach for the Identification of Brewing Yeast (Saccharomyces cerevisiae) Strains Using MALDI-TOF MS.

Matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry (TOF MS) is now accepted as a quick, easy-to-use, cost-effective, and accurate technique for the identification of microorganisms. However, the successful identification of microorganisms is dependent upon careful attention to factors such as growth conditions, extraction methods, mass spectral data collection, and data analysis procedures. Currently, most microorganism identification has been limited to the species level, and only a limited number of publications have been successful in achieving strain-level identification. In this work, a "cell-free" approach is introduced where peptide analytes secreted by several Saccharomyces cerevisiae strains during their growth period are analyzed. The analysis of the cell supernatant generates mass spectral patterns that are specific to each strain. The patterns generated in combination with a robust data analysis workflow using the open-source programs MALDIquant and Mass-Up allows for strain-level identification of S. cerevisiae. The cell-free approach using the yeast supernatant to accurately identify yeast strains is presented here as a proof of concept. Graphical Abstract.

Diagnosing and Correcting Mass Accuracy and Signal Intensity Error Due to Initial Ion Position Variations in a MALDI TOFMS.

Frustrated by worse than expected error for both peak area and time-of-flight (TOF) in matrix assisted laser desorption ionization (MALDI) experiments using samples prepared by electrospray deposition, it was finally determined that there was a correlation between sample location on the target plate and the measured TOF/peak area. Variations in both TOF and peak area were found to be due to small differences in the initial position of ions formed in the source region of the TOF mass spectrometer. These differences arise largely from misalignment of the instrument sample stage, with a smaller contribution arising from the non-ideal shape of the target plates used. By physically measuring the target plates used and comparing TOF data collected from three different instruments, an estimate of the magnitude and direction of the sample stage misalignment was determined for each of the instruments. A correction method was developed to correct the TOFs and peak areas obtained for a given combination of target plate and instrument. Two correction factors are determined, one by initially collecting spectra from each sample position used and another by using spectra from a single position for each set of samples on a target plate. For TOF and mass values, use of the correction factor reduced the error by a factor of 4, with the relative standard deviation (RSD) of the corrected masses being reduced to 12-24 ppm. For the peak areas, the RSD was reduced from 28% to 16% for samples deposited twice onto two target plates over two days. Graphical Abstract.

Improving the analyte ion signal in matrix-assisted laser desorption/ionization imaging mass spectrometry via electrospray deposition by enhancing incorporation of the analyte in the matrix.

RATIONALE: Matrix-assisted laser desorption/ionization (MALDI) is widely used as the ionization method in high-resolution chemical imaging studies that seek to visualize the distribution of analytes within sectioned biological tissues. This work extends the use of electrospray deposition (ESD) to apply matrix with an additional solvent spray to incorporate and homogenize analyte within the matrix overlayer. METHODS: Analytes and matrix are sequentially and independently applied by ESD to create a sample from which spectra are collected, mimicking a MALDI imaging mass spectrometry (IMS) experiment. Subsequently, an incorporation spray consisting of methanol is applied by ESD to the sample and another set of spectra are collected. The spectra prior to and after the incorporation spray are compared to evaluate the improvement in the analyte signal. RESULTS: Prior to the incorporation spray, samples prepared using α-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB) as the matrix showed low signal while the sample using sinapinic acid (SA) initially exhibited good signal. Following the incorporation spray, the sample using SA did not show an increase in signal; the sample using DHB showed moderate gain factors of 2-5 (full ablation spectra) and 12-336 (raster spectra), while CHCA samples saw large increases in signal, with gain factors of 14-172 (full ablation spectra) and 148-1139 (raster spectra). CONCLUSIONS: The use of an incorporation spray to apply solvent by ESD to a matrix layer already deposited by ESD provides an increase in signal by both promoting incorporation of the analyte within and homogenizing the distribution of the incorporated analyte throughout the matrix layer. Copyright © 2017 John Wiley & Sons, Ltd.

Microscale Solubility Measurements of Matrix-Assisted Laser Desorption-Ionization (MALDI) Matrices Using Attenuated Total Reflection (ATR) Fourier Transform Infrared Spectroscopy (FT-IR) Coupled with Partial Least Squares (PLS) Analysis.

In this work an attenuated total reflection Fourier transform infrared (FT-IR) absorption based method is used to measure the solubility of two matrix-assisted laser desorption-ionization (MALDI) matrices in a few pure solvents and mixtures of acetonitrile and water using low microliter amounts of solution. Results from a method that averages the values obtained from multiple calibration curves created by manual peak picking are compared to those predicted using a partial least squares (PLS) chemometrics approach. The PLS method provided solubility values that were in good agreement with the manual method with significantly greater ease of analysis. As a test, the solubility of adipic acid in acetone was measured using the two methods of analysis, and the values are in good agreement with solubility values reported in literature. The solubilities of the MALDI matrices α-cyano-4-hydroxy cinnamic acid (CHCA) and sinapinic acid (SA) were measured in a series of mixtures made from acetonitrile (ACN) and water; surprisingly, the results show a highly nonlinear trend. While both CHCA and SA show solubility values of less than 10 mg/mL in the pure solvents, the solubility value for SA increases to 56.3 mg/mL in a 75:25 v/v ACN:water mixture. This can have a significant effect on the matrix-to-analyte ratios in the MALDI experiment when sample protocols call for preparation of a saturated solution of the matrix in the chosen solvent system.

Development of a combined standard additions/internal standards method to quantify residual PEG in ethoxylated surfactants by MALDI TOFMS.

Measuring the residual polyethylene glycol (PEG) in polyethylene oxide (PEO)-based surfactants is important to fully understanding the performance of these materials. Traditional methods of quantitating PEG in PEO-based surfactants can be time-consuming and struggle with low amounts or overlapping molecular mass distributions. This paper describes a matrix-assisted laser desorption/ionization (MALDI) mass spectrometry method developed to quantitate residual PEG in a series of ethoxylated surfactants. The technique addresses the difficulties faced in doing quantitative MALDI experiments by utilizing both internal standard and standard additions protocols. The method produces excellent straight line standard addition plots, and the quantitative results are verified using both a constructed standard and an independent traditional chromatographic separation.

Optimization of a modified aerospray deposition device for the preparation of samples for quantitative analysis by MALDI-TOFMS.

A modified aerospray apparatus was used to prepare a thin layer sample of matrix and analyte for quantitative analysis by MALDI-TOFMS. The apparatus consists of a set of coaxial tubing; the liquid sample is forced by a syringe pump through the inner capillary and it is nebulized by a flow of gas through the outer capillary. The small droplets of sample exiting the device are deposited onto a rotating plate, which serves as the sample surface for a time-of-flight mass spectrometer. An optimization was carried out after initial experiments with the device resulted in poorer than expected reproducibility of analyte signal. A two-level plus center point factorial experiment was performed investigating several factors, including the inner capillary internal diameter, gas pressure, liquid flow, spray distance, and time. After optimization the within-sample reproducibility of the analyte signal improved 3-fold, while the sample-to-sample reproducibility improved 4.5-fold.

Development of a dual-spray electrospray deposition system for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

A new method of matrix-assisted laser desorption/ionization (MALDI) sample preparation using a dual-spray electrospray deposition system is demonstrated and employed for the investigation of gas-phase cationization reactions in the MALDI plume. The dual-spray electrospray system is found to increase the homogeneity of the sample similarly to that of a conventional single-spray electrospray system. The dual-spray electrospray system allows for intimate mixing of separately prepared sample components and results in improved quantitative results. The development of this device also leads to the possibility of mixing sample components prepared in different solvents without the need to be concerned with solvent miscibility.

A study of gas-phase cationization in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

A specially constructed split sample probe was used to unequivocally demonstrate that gas-phase cationization occurs within the desorption plume during a matrix-assisted laser desorption/ionization experiment. Two separate samples were prepared for analysis: on side A, a mixture of poly(ethylene glycol) (PEG) 1500 analyte and 2,5-dihydroxybenzoic acid (DHB) matrix, and on side B a mixture of DHB matrix and lithium hydroxide (LiOH), the cationization reagent. Analysis of the data showed that when the ionization laser was focused on the split (so that both sides were illuminated), Li(+)-cationized PEG peaks were observed. Since the PEG analyte did not come into contact with Li(+) in either the solution or solid phase, the only possibility for the observed cationization was a reaction in the gas phase. Due to the difficulty in completely removing the adventitious cations (Na(+) and K(+)) present in DHB and on sample surfaces, gas-phase cationization could not be demonstrated to be either the only or most important mechanism operating in the MALDI experiment.

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.

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.

Improved PSD and CID on a MALDI TOFMS.

The influence of several instrument-operating parameters on the product-ion resolution and mass accuracy in matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) post-source decay (PSD) and collision-induced dissociation (CID) experiments is reported. Voltages commonly applied to the reflectron for PSD and CID experiments were found to be non-ideal; optimization of these voltages resulted in better resolution across each segment of the measured PSD spectrum. Mass resolution, calculated as M/DeltaM (FWHM) for the product-ion peaks, was as high as 2500. Additionally, precursor-ion selection and segment mass range setup were each found to have dramatic influences on product-ion mass accuracy. An understanding of the influence of these variables aided in the interpretation of (a-NH3) and (b - NH3) ions observed in the PSD/CID spectra of a number of peptides. In addition, product ions resulting from coincidence peaks in the precursor-ion selection window were found to be a general problem. With the improvements to resolution and optimization of these mass accuracy variables, the mass accuracy of product ions from MALDI TOF PSD and CID experiments was tested with several reference materials, including the peptides Substance P, bradykinin, angiotensin I, and angiotensin II and the synthetic polymers poly(methyl methacrylate) and polystyrene. The absolute error (Da) for each test material was, on average, below 0.1 Da, demonstrating a significant improvement in mass accuracy using the improved operational parameters and an extension of the use of poly(ethylene glycol) (PEG) as a mass calibrant for the PSD/CID spectra.

Investigations of electrospray sample deposition for polymer MALDI mass spectrometry.

In the interest of a more thorough understanding of the relationship between sample deposition technique and the quality of data obtained using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, details of the electrospray (ES) process of sample deposition are investigated using a number of techniques. Sample morphology was observed with scanning electron microscopy (SEM) and atomic force microscopy (AFM), while matrix-enhanced secondary ion mass spectrometry (MESIMS) monitored surface coverage. Electrospray deposition reduces the analyte segregation that can occur during traditional dried droplet deposition for MALDI. We attribute statistically significant improvements in the reproducibility of signal intensity and MALDI average molecular mass measurements to the ES sample deposition technique.

Optimization of matrix-assisted laser desorption/ionization time-of-flight collision-induced dissociation using poly(ethylene glycol).

Details of the optimization of the collision-induced dissociation (CID) process, using a collision cell on a matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometer, are described using poly(ethylene glycol) 1000 (PEG 1000) as a model analyte. The effects of collision gas identity (helium, air, and argon), as well as collision gas pressure, on the resulting MS/MS data were investigated. With PEG 1000, helium was found to give the best results with respect to signal-to-noise (S/N) ratio. The optimum pressure for each gas was found to be in the range where the precursor ion signal was attenuated to approximately 30-50% for helium and 40-60% for argon. The effect of cation choice (Li, Na, and K) on the CID of PEG was also studied. CID spectra were produced for each, but PEG cationized with lithium was found to produce the spectra with the highest S/N ratio. The MALDI-TOF CID spectra that were generated for PEG were compared with the high-energy and low-energy MS/MS spectra obtained from a sector mass spectrometer and from a triple quadrupole mass spectrometer, respectively. The results observed for PEG confirm that CID on a MALDI-TOF mass spectrometer is a high-energy MS/MS technique.

Atmospheric pressure matrix-assisted laser desorption/ionisation ion trap mass spectrometry of synthetic polymers: a comparison with vacuum matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.

Atmospheric pressure matrix-assisted laser desorption/ionisation quadrupole ion trap (AP-MALDI/QIT) mass spectrometry has been investigated for the analysis of polyethylene glycol (PEG 1500) and a hyperbranched polymer (polyglycidol) in the presence of alkali-metal salts. Mass spectra of PEG 1500 obtained at atmospheric pressure showed dimetallated matrix/analyte adducts, in addition to the expected alkali-metal/PEG ions, for all matrix/alkali-metal salt combinations. The relative intensities of the desorbed ions were dependent on the matrix, the alkali-metal salt added to aid cationisation and the ion trap interface conditions [capillary temperature, in-source collisionally-induced dissociation (CID)]. These data indicate that the adducts are rapidly stabilised by collisional cooling enabling them to be transferred into the ion trap. Experiments using identical sample preparation conditions were carried out on a vacuum MALDI time-of-flight (ToF) mass spectrometer. In all cases, vacuum MALDI-ToF spectra showed only alkali-metal/PEG ions and no matrix/analyte adducts. The tandem mass spectrometry (MS/MS) capability of the ion trap has been demonstrated for a lithiated polyglycol yielding a rich fragment-ion spectrum. Analysis of the hyperbranched polymer polyglycidol by AP-MALDI/QIT reveals the characteristic ion series for these polymers as also observed under vacuum MALDI-ToF conditions.