Analysis of Biomolecules by Atmospheric Pressure Visible-Wavelength MALDI-Ion Trap-MS in Transmission Geometry

We report the development of a new AP visible-wavelength MALDI-ion trap-MS instrument with significantly improved performance over our previously reported system (Int. J. Mass Spectrom. 315, 66–73 (2012)). A Nd:YAG pulsed laser emitting light at 532 nm was used to desorb and ionize oligosaccharides and peptides in transmission geometry through a glass slide. Limits of detection (LODs) achieved in MS mode correspond to picomole quantities of oligosaccharides and femtomole quantities of peptides. Tandem MS (MS/MS) experiments enabled identification of enzymatically digested proteins and oligosaccharides by comparison of MS/MS spectra with data found in protein and glycan databases. Moreover, the softness of ionization, LODs, and fragmentation spectra of biomolecules by AP visible-wavelength MALDI-MS were compared to those obtained by AP UV MALDI-MS using a Nd:YAG laser emitting light at 355 nm. AP visible-wavelength MALDI appears to be a softer ionization technique then AP UV MALDI for the analysis of sulfated peptides, while visible-wavelength MALDI-MS, MS/MS, and MS/MS/MS spectra of other biomolecules analyzed were mostly similar to those obtained by AP UV MALDI-MS. Therefore, the methodology presented will be useful for MS and MSⁿ analyses of biomolecules at atmospheric pressure. Additionally, the AP visible-wavelength MALDI developed can be readily used for soft ionization of analytes on various mass spectrometers.

Imaging of Noncovalent Complexes by MALDI-MS

Noncovalent interactions govern how molecules communicate. Mass spectrometry is an important and versatile tool for the analysis of noncovalent complexes (NCX). Electrospray mass spectrometry (ESI-MS) is the most widely used MS technique for the study of NCXs because of its softer ionization and easy compatibility with the solution phase of NCX mixtures. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has also been used to study NCXs. However, successful analysis depends upon several experimental factors, such as matrix selection, solution pH, and instrumental parameters. In this study, we employ MALDI imaging mass spectrometry to investigate the location and formation of NCXs, involving both peptides and proteins, in a MALDI sample spot.

Fluorometric Beam Profiling of UV MALDI Lasers

The photon distribution (beam profile) of the laser as projected onto the sample is an important variable in matrix assisted laser desorption ionization mass spectrometry (MALDI-MS). Measurement of the beam profile is, therefore, an important factor within MALDI-MS. In this study a simple, low-cost fluorometric laser beam profiling technique is presented and applied in conjunction with MALDI-MS experiments. A comparison of the beam profile information afforded by a commercial system and the fluorometric method is carried out to determine the variation of beam profile for an Nd:YVO₄ laser operated between 1 and 25 kHz. The beam profile information can be used, in conjunction with corresponding ion yields, to inform MALDI-MS experiments. The fluorometric beam profiling technique is used to obtain information about the beam dimensions as incident upon the MALDI-MS sample plate in-source. These values are compared with equivalent information obtained from ablation of thin film α-cyano-4-hydroxycinnamic acid (CHCA). In this study, area estimation by ablation provided a value 1.6 times smaller than that obtained by the fluorometric method, demonstrating the need for caution when measuring beam profile and, therefore, fluence, in MALDI-MS.

Recent methodological advances in MALDI mass spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is widely used for characterization of large, thermally labile biomolecules. Advantages of this analytical technique are high sensitivity, robustness, high-throughput capacity, and applicability to a wide range of compound classes. For some years, MALDI-MS has also been increasingly used for mass spectrometric imaging as well as in other areas of clinical research. Recently, several new concepts have been presented that have the potential to further advance the performance characteristics of MALDI. Among these innovations are novel matrices with low proton affinities for particularly efficient protonation of analyte molecules, use of wavelength-tunable lasers to achieve optimum excitation conditions, and use of liquid matrices for improved quantification. Instrumental modifications have also made possible MALDI-MS imaging with cellular resolution as well as an efficient generation of multiply charged MALDI ions by use of heated vacuum interfaces. This article reviews these recent innovations and gives the author’s personal outlook of possible future developments.

Improved MALDI-TOF Microbial Mass Spectrometry Imaging by Application of a Dispersed Solid Matrix

The key step in high quality microbial matrix-assisted laser desorption/ionization mass spectrometry imaging (microbial MALDI MSI) is the fabrication of a homogeneous matrix coating showing a fine-grained morphology. This application note addresses a novel method to apply solid MALDI matrices onto microbial cultures grown on thin agar media. A suspension of a mixture of 2,5-DHB and α-CHCA is sprayed onto the agar sample surface to form highly homogeneous matrix coatings. As a result, the signal intensities of metabolites secreted by the fungus Aspergillus fumigatus were found to be clearly enhanced.

Distribution study of atorvastatin and its metabolites in rat tissues using combined information from UHPLC/MS and MALDI-Orbitrap-MS imaging

The combination of ultrahigh-resolution mass spectrometry imaging (UHRMSI) and ultrahigh-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC/MS/MS) was used for the identification and the spatial localization of atorvastatin (AT) and its metabolites in rat tissues. Ultrahigh-resolution and high mass accuracy measurements on a matrix-assisted laser desorption/ionization (MALDI)-Orbitrap mass spectrometer allowed better detection of desired analytes in the background of matrix and endogenous compounds. Tandem mass spectra were also used to confirm the identification of detected metabolites in complex matrices. The optimization of sample preparation before imaging experiments included the tissue cryogenic sectioning (thickness 20 μm), the transfer to stainless steel or glass slide, and the selection of suitable matrix and its homogenous deposition on the tissue slice. Thirteen matrices typically used for small molecule analysis, e.g., 2,5-dihydroxybenzoic acid (DHB), 1,5-diaminonaphthalene (DAN), 9-aminoacridine (AA), etc., were investigated for the studied drug and its metabolite detection efficiency in both polarity modes. Particular matrices were scored based on the strength of extracted ion current (EIC), relative ratio of AT molecular adducts, and fragment ions. The matrix deposition on the tissue for the most suitable matrices was done by sublimation to obtain the small crystal size and to avoid local variations in the ionization efficiency. UHPLC/MS profiling of drug metabolites in adjacent tissue slices with the previously optimized extraction was performed in parallel to mass spectrometry imaging (MSI) measurements to obtain more detailed information on metabolites in addition to the spatial information from MSI. The quantitation of atorvastatin in rat liver, serum, and feces was also performed.

Data-Independent Microbial Metabolomics with Ambient Ionization Mass Spectrometry

Atmospheric ionization methods are ideally suited for prolonged MS/MS analysis. Data-independent MS/MS is a complementary technique for analysis of biological samples as compared to data-dependent analysis. Here, we pair data-independent MS/MS with the ambient ionization method nanospray desorption electrospray ionization (nanoDESI) for untargeted analysis of bacterial metabolites. Proof-of-principle data and analysis are illustrated by sampling Bacillus subtilis and Pseudomonas aeruginosa directly from Petri dishes. We found that this technique enables facile comparisons between strains via MS and MS/MS plots which can be translated to chemically informative molecular maps through MS/MS networking. The development of novel techniques to characterize microbial metabolites allows rapid and efficient analysis of metabolic exchange factors. This is motivated by our desire to develop novel techniques to explore the role of interspecies interactions in the environment, health, and disease. This is a contribution to honor Professor Catherine C. Fenselau in receiving the prestigious ASMS Award for a Distinguished Contribution in Mass Spectrometry for her pioneering work on microbial mass spectrometry.

Screening of Complicated Matrixes with Paper Assisted Ultrasonic Spray Ionization Mass Spectrometry

To analyze compounds in complicated matrixes using mass spectrometry, we describe a novel ambient ionization approach, termed paper assisted ultrasonic spray ionization (PAUSI). The ionization process is based on the ultrasonic vibration of the piezoelectric ceramic disk, on which the samples are placed. Porous materials are utilized to generate fine initial droplet, which could alleviate matrix effect during ionization process for complicated matrix. PAUSI was evaluated as an attractive tool to screen analytes from complicated matrixes, such as (1) bovine serum with NaCl 150 g/L, (2) viscous samples, and (3) biological fluid, without any sample preparation. Moreover, it provides great advantage in simplifying the mass spectrometry analysis process, and the ionization device is inexpensive and easy to operate.

High-Mass MALDI-MS Using Ion Conversion Dynode Detectors: Influence of the Conversion Voltage on Sensitivity and Spectral Quality

With the development of special ion conversion dynode (ICD) detectors for high-mass matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), the mass-to-charge ratio is no longer a limiting factor. Although these detectors have been successfully used in the past, there is lack of understanding of the basic processes in the detector. We present a systematic study to investigate the performance of such an ICD detector and separate the contributions of the MALDI process from the ones of the ion-to-secondary ion and the secondary ion-to-electron conversions. The performance was evaluated as a function of the voltages applied to the conversion dynodes and the sample amount utilized, and we found that the detector reflects the MALDI process correctly: limitations such as sensitivity or deviations from the expected signal intensity ratios originate from the MALDI process itself and not from the detector.

Overcoming Selectivity and Sensitivity Issues of Direct Inject Electrospray Mass Spectrometry via DAPNe-NSI-MS

Direct inject electrospray mass spectrometry offers minimal sample preparation and a “shotgun” approach to analyzing samples. However, complex matrix effects often make direct inject an undesirable sample introduction technique, particularly for trace level analytes. Highlighted here is our solution to the pitfalls of direct inject mass spectrometry and other ambient ionization methods with a focus on trace explosives. Direct analyte-probed nanoextraction coupled to nanospray ionization mass spectrometry solves selectivity issues and reduces matrix effects while maintaining minimal sample preparation requirements. With appropriate solvent conditions, most explosive residues can be analyzed with this technique regardless of the nature of the substance (i.e., nitroaromatic, oxidizing salt, or peroxide).

Rapid and sensitive analysis of phthalate metabolites, bisphenol A, and endogenous steroid hormones in human urine by mixed-mode solid-phase extraction, dansylation, and ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry

Steroid hormone levels in human urine are convenient and sensitive indicators for the impact of phthalates and/or bisphenol A (BPA) exposure on the human steroid hormone endocrine system. In this study, a rapid and sensitive method for determination of 14 phthalate metabolites, BPA, and ten endogenous steroid hormones in urine was developed and validated on the basis of ultra-performance liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry. The optimized mixed-mode solid phase-extraction separated the weakly acidic or neutral BPA and steroid hormones from acidic phthalate metabolites in urine: the former were determined in positive ion mode with a methanol/water mobile phase containing 10 mM ammonium formate; the latter were determined in negative ion mode with a acetonitrile/water mobile phase containing 0.1 % acetic acid, which significantly alleviated matrix effects for the analysis of BPA and steroid hormones. Dansylation of estrogens and BPA realized simultaneous and sensitive analysis of the endogenous steroid hormones and BPA in a single chromatographic run. The limits of detection were less than 0.84 ng/mL for phthalate metabolites and less than 0.22 ng/mL for endogenous steroid hormones and BPA. This proposed method had satisfactory precision and accuracy, and was successfully applied to the analyses of human urine samples. This method could be valuable when investigating the associations among endocrine-disrupting chemicals, endogenous steroid hormones, and relevant adverse outcomes in epidemiological studies.

Plasma-Assisted Reaction Chemical Ionization for Elemental Mass Spectrometry of Organohalogens

We present plasma-assisted reaction chemical ionization (PARCI) for elemental analysis of halogens in organic compounds. Organohalogens are broken down to simple halogen-containing molecules (e.g., HBr) in a helium microwave-induced plasma followed by negative mode chemical ionization (CI) in the afterglow region. The reagent ions for CI originate from penning ionization of gases (e.g., N₂) introduced into the afterglow region. The performance of PARCI-mass spectrometry (MS) is evaluated using flow injection analyses of organobromines, demonstrating 5–8 times better sensitivities compared with inductively coupled plasma MS. We show that compound-dependent sensitivities in PARCI-MS mainly arise from sample introduction biases.

Pharmaceutical Analysis by Solid-Substrate Electrospray Ionization Mass Spectrometry with Wooden Tips

Electrospray ionization (ESI) using wooden tips as solid substrates allows direct ionization of various samples and their simple and efficient analyses by mass spectrometry (MS). In this study, wooden-tip ESI-MS was used for pharmaceutical analysis. A wide variety of active components present in pharmaceuticals with forms of tablets, capsules, granules, dry suspensions, suspensions, drops, and oral liquids, etc., were all successfully ionized directly for mass spectrometric analysis. Trace degradation products were also sensitively detected using wooden-tip ESI-MS. This strategy was extended to construct chemical fingerprints of herbal products containing complex and unknown components, and the fingerprints provided valuable information for their quality assessment and origin tracing. Our experimental data demonstrated that wooden-tip ESI-MS is a useful tool for rapid pharmaceutical analysis, with high sensitivity and wide applicability, showing promising perspectives for quality assessment and control, authentication, and origin tracing of pharmaceuticals.

Microchip-based strategy for enrichment of acetylated proteins

We have developed a simple microchip-based method for the separation and enrichment of acetylated proteins and peptides using a microchip technique. Poly (dimethylsiloxane) (PDMS) microfluidic channels were modified by passing an acidic solution of hydrogen peroxide through them. This resulted in hydrophilic silanol-covered surfaces onto which poly (diallyldimethylammonium chloride) (PDDA) can be coated. Protein A/G beads were then captured by the PDDA layer and antibodies can then be immobilized via the protein A/G. This technique enables efficient capture of antigens due to the optimal spacing and orientation of surface molecules. Two solutions, one containing 72.5 fmol?μL^?1 of acetylated bovine serum albumin (BSA-Ac), the other 72.5 fmol?μL^?1 of tryptic BSA-Ac digest were then enriched. High selectivities were obtained, and a 82.4 % recovery of the acetylated proteins was attained. This on-chip platform was then coupled to MALDI-MS to provide information on the acetylation sites of proteins and peptides. Additional peaks were observed in the mass spectra after enrichment and were assigned to acetylated peptides. This is significant with respect to understanding the mechanism and function of acetylation. In our opinion, this microchip-based technique has a large potential for detecting acetylated proteins and peptides in complex biological mixtures, and in acetylomics in general.

Impact of homogeneous and filamentary discharge modes on the efficiency of dielectric barrier discharge ionization mass spectrometry

The present study contributes to the evaluation of dielectric barrier discharge-based ambient ionization for mass spectrometric analysis (DBDI-MS) by providing a further step towards an understanding of underlying ionization processes. This examination highlights the effect of physical discharge modes on the ionization efficiency of the DBDI source. A distinction is made between the homogeneous and filamentary discharge mode due to different plasma gases in barrier configurations. Therefore, we first report on discharge modes of DBDI by demonstrating a universally applicable method to classify the predominant modes. Then, the ionization efficiency of these two modes is evaluated by a laser desorption-DBDI-MS with different molecular analytes. Here, the laser desorption is used to deliver neutral analytes which will be ionized by the plasma jet applied as dielectric barrier discharge ionization. With a clear increase of signal intensities in the homogeneous mode in contrast to the filamentary one, the present study indicates a pronounced dependence of the ionization efficiency on the discharge mode allowing further insight into the mechanisms of the ionization process.

Rapid enrichment of phosphopeptides by BaTiO3 nanoparticles after microwave-assisted tryptic digest of phosphoproteins, and their identification by MALDI-MS

We show that BaTiO₃ nanoparticles (NPs) can be used as a novel substrate for the rapid enrichment of phosphopeptides from microwave tryptic digests of α-casein and non-fat milk prior to their identification by MALDI-MS. Protein digestion is achieved by microwave tryptic digest for 50?s, and the resulting phosphopeptides can be effectively adsorbed on the surfaces of the NPs. The phosphopeptides were selectively detected via MALDI-MS. Digestion, enrichment and detection are accomplished within ～60?min. The method was applied to the indentification of 24 phosphopeptides from α-casein and of 21 phosphopeptides (of the α-casein type) from nonfat milk.

Laser-Induced Azomethine Ylide Formation and Its Covalent Entrapment by Fulleropyrrolidine Derivatives During MALDI Analysis

Two novel monofunctionalized fulleropyrrolidine derivatives (Prato adducts) were prepared and characterized by matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). MALDI experiments conducted in the positive-ion mode on pure and mixed samples of both monofunctionalized fullerene derivatives revealed the efficient formation of bisadducts (in the case of the pure samples) and mixed bisadducts (in the case of a mixed sample). Bisadducts were not observed in the ESI experiments and thus not present in the sample. A mechanism for the MALDI formation of these bisadduct ions is proposed in which an azomethine ylide fragment is formed in situ from the monofunctionalized fulleropyrrolidine species upon laser irradiation. This fragment, which can survive as an intact moiety in the gas phase in the special environment provided by the MALDI experiment, is then able to attach to a fulleropyrrolidine monoadduct which acts as a dipolarophile, thus leading to the formation of a bisadduct fullerene derivative. The unprecedented re-attachment of the azomethine ylide implies that the establishment of the ligand attainment of Prato adducts based on MALDI analysis alone can lead to wrong assignments.

Why do the Abundances of Ions Generated by MALDI Look Thermally Determined?

In a previous study (J. Mass Spectrom. 48, 299–305, 2013), we observed that the abundance of each ion in a matrix-assisted laser desorption ionization (MALDI) spectrum looked thermally determined. To find out the explanation for the phenomenon, we estimated the ionization efficiency and the reaction quotient (Q_A) for the autoprotolysis of matrix, M + M → [M + H]⁺ + [M ? H]^?, from the temperature-controlled laser desorption ionization spectra of α-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB). We also evaluated the equilibrium constants (K_A) for the autoprotolysis at various temperatures by quantum chemical calculation. Primary ion formation via various thermal models followed by autoprotolysis-recombination was compatible with the observations. The upper limit of the effective temperature of the plume where autoprotolysis-recombination occurs was estimated by equating Q_A with the calculated equilibrium constant.

Comprehensive and simultaneous coverage of lipid and polar metabolites for endogenous cellular metabolomics using HILIC-TOF-MS

The comprehensive metabolomic analyses using eukaryotic and prokaryotic cells are an effective way to identify biomarkers or biochemical pathways which can then be used to characterize disease states, differences between cell lines or inducers of cellular stress responses. One of the most commonly used extraction methods for comprehensive metabolomics is the Bligh and Dyer method (BD) which separates the metabolome into polar and nonpolar fractions. These fractions are then typically analysed separately using hydrophilic interaction liquid chromatography (HILIC) and reversed-phase (RP) liquid chromatography (LC), respectively. However, this method has low sample throughput and can also be biased to either polar or nonpolar metabolites. Here, we introduce a MeOH/EtOH/H₂O extraction paired with HILIC-time-of-flight (TOF)-mass spectrometry (MS) for comprehensive and simultaneous detection of both polar and nonpolar metabolites that is compatible for a wide array of cellular species cultured in different growth media. This method has been shown to be capable of separating polar metabolites by a HILIC mechanism and classes of lipids by an adsorption-like mechanism. Furthermore, this method is scalable and offers a substantial increase in sample throughput compared to BD with comparable extraction efficiency. This method was able to cover 92.2 % of the detectable metabolome of Gram-negative bacterium Sinorhizobium meliloti, as compared to 91.6 % of the metabolome by a combination of BD polar (59.4 %) and BD nonpolar (53.9 %) fractions. This single-extraction HILIC approach was successfully used to characterize the endometabolism of Gram-negative and Gram-positive bacteria as well as mammalian macrophages.