Material-enhanced Laser Desorption/ionization Research Articles

Matrix Free Material Enhanced Laser Desorption Ionization Mass Spectrometric Analysis of Amino Acids in Althaea Officinalis, Matricaria Chamomilla, and Taraxacum Officinale

The standardization of natural product drugs needs reliable methods for the analysis of raw materials and final products. In this study, qualitative analysis of amino acids in herbal extracts was performed using mass spectrometric method, that is, matrix- free material enhanced laser desorption ionization time of flight mass spectrometry (mf-MELDI-MS). Samples employed in the study were standards and water extracts from Althaea officinalis, Taraxacum officinale, and Matricaria chamomilla. These analyses confirmed the presence of amino acids and clearly proved the adequate performance of mf-MELDI-MS for the qualitative analysis of complex mixtures, as targets do not need modification and analysis requires only a few minutes. Determination of amino acids in these plants were performed and reported for the first time using the modified silica gel (4,4′-azodianiline modified silica gel) and mf-MELDI-MS technique.

Ceria-based nanocomposites for the enrichment and identification of phosphopeptides

Nanocomposites are given preference over the individual materials due to the combined properties of the components involved. Ceria has a high efficiency in phosphopeptide enrichment as well as in dephosphorylation. Iron oxide and tin oxide are chosen as counter metal oxides to synthesize the ceria nanocomposites using a co-precipitation method. The nanocomposites are characterized by Fourier-transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM). Tryptic β-casein digest shows the feasibility of phosphopeptide enrichment by the two nanocomposites. Selectivity studies show their performance in comparison to ceria. Being more selective in the extended mass range, both nanocomposites are applied to spiked human serum and non-fat milk digest. The ceria nanocomposites are also capable of being used as material-enhanced laser desorption/ionization (MELDI) carrier/affinity materials for real biological samples with varying degrees of complexity. The enriched content is analyzed by MALDI-TOF MS. All the phosphopeptides in all variants of casein are identified. The sequence coverage of caseins is also interpreted. Nanocomposites thus offer a high selectivity and sensitivity, which make them promising materials for biomarker discoveries and the identification of phosphorylation pathways for new post translational modifications (PTMs).

New analytical tools for quality control of natural products

Phytopharmacy with its huge variety and complexity of plants, extraction procedures and ingredients is a permanently demanding challenge for the analytical chemist. The separation of active components from natural products has become increasingly important and new advancements in chromatography allow exploring inaccessible areas of natural product isolation. There is a need for highly efficient techniques to handle the problems associated with sample preparation, separation and identification. Novel enrichment and purification methods based on modern SPE technologies are applied to reduce the complexity of plant materials while µ-HPLC is used for separation, preconcentration and fractionation. Recent research in the area of solid-phase extraction led to the development of novel stationary phases based on organic polymers and fullerene-silica for enrichment and purification of natural products. Considerable progress has been made in the development of stationary phases which can be tailored to a specific application allowing endless possibilities in terms of selectivity tuning. An analytical platform which allows to profile and characterize plant materials and their extractives by vibrational spectroscopy, infrared (IR) based imaging, CE-MS and matrix-free material enhanced laser desorption ionization time of flight mass spectrometry (mf-MELDI-TOF/MS) was established. Thereby, vibrational spectroscopy offers the advantages of fast, non-invasive and simultaneous determination of chemical and physical properties being suitable for high-throughput analytical quality control.

Infrared Spectroscopy: A Novel Tool for the Physicochemical Characterization of Particulate, Monolithic and Coated Stationary Phases

The design of novel stationary phases is a permanent demanding challenge in chromatographic and electrophoretic separation science to enable analysis with enhanced selectivity, specificity and speed. Therefore, the characterization of chemical and physical properties is next to calculation of chromatographic parameters essential. Conventionally, column performance is checked by a test run which only enables to prove efficiency column by column. Chemical parameters including surface coverage is normally analyzed by burning combustion or frontal analysis, physical parameters including particle size, pore size, pore volume and surface area by SEM, mercury intrusion porosimetry and Brunauer Emmett Teller. All these methods are extremely time consuming, invasive and require, beside special equipment, some specially trained lab staff. In order to enable a fast high-throughput control of chemical and physical parameters we introduced near-infrared spectroscopy (NIRS, 4,000–10,000 cm−1) as a non-invasive, easy-to-handle technology enabling simultaneous analysis within only a few seconds at higher precision than the conventional applied methods. The method is applicable to porous/non-porous silica gel, carbon-based nano-materials (e.g. fullerenes), polymer beads, monoliths for solid-phase extraction, liquid chromatography, micro-liquid chromatography, material-enhanced laser desorption ionization and coated capillaries for capillary electrophoresis and capillary electrochromatography. Different carriers themselves and their kind of chemical derivatization (reversed-phase, normal-phase, ion-exchanger, immobilized affinity chromatography, affinity, endcapping) can be determined by applying principal component analysis of spectra recorded. Partial least square regression enables the determination of physical parameters particle size, pore size, pore volume, porosity, total porosity and surface area with one single measurement in an established quantitative model. For a detailed investigation NIRS imaging, which benefits from the combination of a NIR spectrometer with a microscope, allows to analyze materials of interest with resolution down to 4 μm. For the optimized design of polymeric phases real-time in situ monitoring to control polymerization progress can be highly helpful. In this article, the advantages of this novel infrared-based method for the fast high-throughput quality control are introduced.

Protein profiling for cancer biomarker discovery using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and infrared imaging: A review

Cancer biomarker refers to a substance or process that is indicative of the presence of cancer in the body. A biomarker might be either a molecule secreted by a tumor or it can be a specific response of the body to the presence of cancer. Cancer biomarker-based diagnostics have applications for establishing disease predisposition, early detection, cancer staging, therapy selection, identifying whether or not a cancer is metastatic, therapy monitoring, assessing prognosis, and advances in the adjuvant setting. Full adoption of cancer biomarkers in the clinic has to date been slow, and only a limited number of cancer biomarker products are currently in routine use. Among proteomic technologies, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) is a technique that has allowed rapid progress in cancer biology. Different further developed methods including e.g. SELDI (surface-enhanced laser desorption/ionization) and MELDI (material-enhanced laser desorption/ionization) are simple and high-throughput techniques that analyze with high sensitivity and specificity intact proteins expressed in complex biological mixtures, such as serum, urine, and tissues. The combination of mass spectrometry (MS) with infrared (IR) spectroscopic imaging is an attempt to combine different technologies in systems analytics. Both MALDI-TOF and infrared tissue imaging enable studying proteins distribution in tissue samples with a resolution down to 50 and 5 μm, respectively. In this review, we summarize recent applications and the synergistic combination of these new technologies to proteomic profiling for cancer biomarker discovery.

Nanostructured Diamond-Like Carbon on Digital Versatile Disc as a Matrix-Free Target for Laser Desorption/Ionization Mass Spectrometry

A nanostructured diamond-like carbon (DLC) coated digital versatile disk (DVD) target is presented as a matrix-free sample support for application in laser desorption/ionization mass spectrometry (LDI-MS). A large number of vacancies, defects, relative sp(2) carbon content, and nanogrooves of DLC films support the LDI phenomenon. The observed absorptivity of DLC is in the range of 305-330 nm (nitrogen laser, 337 nm). The universal applicability is demonstrated through different analytes like amino acids, carbohydrates, lipids, peptides, and other metabolites. Carbohydrates and amino acids are analyzed as sodium and potassium adducts. Peptides are detectable in their protonated forms, which avoid the extra need of additives for ionization. A bovine serum albumin (BSA) digest is analyzed to demonstrate the performance for peptide mixtures, coupled with the material-enhanced laser desorption/ionization (MELDI) approach. The detection limit of the described matrix-free target is investigated to be 10 fmol/microL for [Glu(1)]-fibrinopeptide B (m/z 1570.6) and 1 fmol/microL for L-sorbose (Na(+) adduct). The device does not require any chemical functionalization in contrast to other matrix-free systems. The inertness of DLC provides longer lifetimes without any deterioration in the detection sensitivity. Broad applicability allows high performance analysis in metabolomics and peptidomics. Furthermore the DLC coated DVD (1.4 GB) sample support is used as a storage device for measured and processed data together with sampling on a single device.

Derivatized graphitic nanofibres (GNF) as a new support material for mass spectrometric analysis of peptides and proteins

Graphitic nanofibres (GNFs), 100-200 nm in diameter and 5-20 microm in length have been modified in order to yield different affinities (Cu2+ and Fe3+ loaded immobilized metal affinity chromatography (IMAC) as well as cation and anion exchange materials) for the extraction of a range of biomolecules by their inherited hydrophobicity and the hydrophilic chemical functionalities, obtained by derivatization. Modified GNFs have for the first time been employed as carrier materials for protein profiling in material-enhanced laser desorption/ionization (MELDI) for the enrichment and screening of biofluids. For that purpose, the derivatized GNF materials have comprehensively been characterized regarding surface area, structural changes during derivatization, IMAC, as well as ion exchange and protein-loading capacity and recovery. GNF derivatives revealed high protein-binding capacity (2,000 microg ml(-1) for insulin) and ideal sensitivities, resulting in a detection limit of 50 fmol microl(-1) (for insulin), which is crucial for the detection of low abundant species in biological samples. Compared to other MELDI carrier materials, sensitivity was enhanced on GNF derivatives, which might be ascribed to the fact that GNFs support desorption and ionization mechanisms and by absorbing laser energy in addition to matrix.

Determination of carbohydrates in medicinal plants – comparison of different techniques

Determination of carbohydrates is a challenging and therefore a complex analysis field in phytochemistry. Owing to the high degree of isomerization and epimerization, to the low volatility and to the lack of UV absorbing moieties several analytical techniques cannot be employed without prior modification of target molecules. Additionally within medicinal plants beside carbohydrates several other substances and analytes are present in the produced extract. Aim of this study was the evaluation of different analytical approaches in order to recognize the most suitable technique for qualitative and quantitative analysis of carbohydrates. Focus within the study was placed on thin layer chromatography (TLC), gas chromatography (GC) and a newly developed mass spectrometric method, i.e. matrix free material enhanced laser desorption ionization time of flight mass spectrometry (mf-MELDI-MS) [1]. Samples employed within the study were standards and microwave assisted water extracts from Quercus cortex. TLC was developed using a solvent mixture of butanol:acetic acid:H2O (8:3:2). The visualization of different carbohydrates was performed after reaction with a mixture of acetone – aniline -diphenylamine – phosphoric acid. Mono-, di- and trisaccharides were detected in reference to the carbohydrate standards. Gas chromatography with MS detection has been employed after derivatisation of targets. For di- and tri-saccharides single peaks were obtained, which was not the case for monosaccharides: Glucose for example delivered two signals, but at a constant ratio. Within mf-MELDI-MS from mono- to deca-saccharides clear potassium and sodium adduct signals could be obtained. Evaluation of all three employed techniques clearly proved the performance of mf-MELDI-MS for the qualitative analysis of complex mixtures, as target don't need modification and analysis needs only a few minutes. Beside that GC-MS is suitable for quantitative analysis.

A New Analytical Material-Enhanced Laser Desorption Ionization (MELDI) Based Approach for the Determination of Low-Mass Serum Constituents Using Fullerene Derivatives for Selective Enrichment

60]fullerene derivatives (dioctadecyl methano[60]fullerene, [60]fullerenoacetic acid, and IDA-[60]fullerene) were prepared and subjected to a comprehensive characterization study including protein binding properties and capacity. These fullerene derivatives were successfully applied as material-enhanced laser desorption/ionization (MELDI) carrier materials. It is shown that diverse functionalities result in characteristic human serum peak patterns (m/z 2000-20 000) in terms of signal intensity as well as the number of detectable masses. In addition, the fullerene derivatives clearly provided differences in the low molecular weight mass region (m/z 1000-4000) after elution of the adsorbed serum constituents, and [60]fullerenoacetic acid was the most effective carrier material. Novel high-speed, monolithic, high-resolution capillary columns, prepared by thermally initiated copolymerization of methylstyrene (MSt) and 1,2-bis(p-vinylphenyl)ethane (BVPE) were employed for eluate separation and target spotting. Thus, serum compounds in the low-mass range were successfully fractionated and subjected to MALDI-MS/MS analysis. This contribution, hence, proposes a new "top-down" strategy for proteome research enabling protein profiling as well as biomarker identification in the low-mass range using selective enrichment, high-resolution separation, and offline MALDI-MS/MS evaluation.

Mass Spectrometric Identification of Serum Peptides Employing Derivatized Poly(glycidyl methacrylate/divinyl benzene) Particles and μ-HPLC

Biomarkers play a key role in preclinical screening and diagnosis of a disease. Various support materials are utilized for this task, in combination with MALDI-TOF-MS. The way to effectively bind serum contents and their profiling is well-elaborated by the material-enhanced laser desorption ionization (MELDI) approach. In this particular work, focus is placed on the development of a strategy to identify low molecular weight serum peptides. Poly(GMA/DVB) is derivatized in a way to achieve an affinity termed as immobilized metal ion affinity chromatography (IMAC). Iminodiacetic acid (IDA) is used as a chelating ligand, whereas copper (Cu2+) acts as a metal ion for complexing peptides and proteins out of blood serum. Polymer binds the serum compounds over a broad mass range, which includes low mass peptides and high mass albumin (66 kDa). Bound contents are eluted from material by an acetonitrile/trifluoroacetic acid mixture, which proves the reversible nature of metal and amino acid linkage. Polystyrene/divinyl benzene (PS/DVB) monolithic capillary column is used for fractionation through RP-HPLC, prior to the target spotting. The tandem TOF fragment ion mass spectra of each fraction is acquired and used to search against the Swiss-Prot database, using the Mascot search engine for the identification of peptides.

Ultra‐fast mass fingerprinting by high‐affinity capture of peptides and proteins on derivatized poly(glycidyl methacrylate/divinylbenzene) for the analysis of serum and cell lysates

The development of support materials in mass fingerprinting is an important task required for diagnostic markers in conjunction with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The material-based approach, which we introduced as material-enhanced laser desorption/ionization (MELDI), focuses not only on different functionalities, but also emphasizes the morphology, i.e. porosity or particle size of the carrier material. As a result, it provides a quick and sensitive platform for effective binding of peptides and proteins out of different biofluids, e.g. serum, spinal fluid, urine or cell lysates, and to subsequently analyze them with MALDI-TOF MS. This approach includes a built-in desalting step for serum protein profiling and is sensitive enough to detect proteins and peptides down to 100 fmol/microL. Here we co-polymerized glycidyl methacrylate (GMA) with divinylbenzene (DVB) using thermal polymerization to yield a GMA/DVB polymer for further modifications. Different affinities have been created, such as immobilized metal ion affinity (IDA-Cu2+), reversed-phase (RP) and anion-exchanger (AX) chromatography. The diverse derivatizations and the dispersity of the particles created by different chemical synthetic approaches were confirmed by characteristic infrared (IR) peaks. The polymerization carried out by non-stirring yielded an average pore radius of 6.1 microm (macro-pores) that enhanced the binding capacity enormously by offering enlarged surface areas. Moreover, atomic absorption spectrometry (AAS) provided the metal content loaded on iminodiacetic acid (IDA) in the case of poly(GMA/DVB)-IDA-Cu2+. To summarize, the optimized MELDI approach is sensitive in its performance, extremely fast and can be adapted to robotic systems for routine analysis, allowing sample preparation in less than 5 min in contrast to the conventional surface-enhanced laser desorption/ionization (SELDI) methods.

Material-Enhanced Laser Desorption/Ionization (MELDI)—A New Protein Profiling Tool Utilizing Specific Carrier Materials for Time of Flight Mass Spectrometric Analysis

Over the past couple of years, proteomics pattern analysis has emerged as an effective method for the early diagnosis of diseases such as ovarian, breast, or prostate cancer, without identification of single biomarkers. MALDI-TOF MS, for example, offers a simple approach for fast and reliable protein profiling, especially by using carrier materials with various physical and chemical properties, in combination with a MALDI matrix. This approach is referred to as material-enhanced laser desorption/ionization (MELDI). In this paper, we report the development and application of derivatized carrier materials [cellulose, silica, poly(glycidyl methacrylate/divinylbenzene) (GMA/DVB) particles, and diamond powder] for fast and direct MALDI-TOF MS protein profiling. The applicability of MELDI for rapid protein profiling was evaluated with human serum samples. These carriers, having various hydrophobicities, resulted in characteristic mass fingerprints, even if all materials were derivatized with iminodiacetic acid (IDA) to yield an immobilized metal affinity chromatography (IMAC) functionality. Our study demonstrates that analyzing complex biological samples, such as human serum, by employing different MELDI carrier materials yielded type- and size-dependent performance variation.