Sample purification and preparation technique based on nano-scale reversed-phase columns for the sensitive analysis of complex peptide mixtures by matrix-assisted laser desorption/ionization mass spectrometry

A simple reversed-phase nano-column purification and sample preparation technique is described, which markedly improves the mass spectrometric analysis of complex and contaminated peptide mixtures by matrix-assisted laser desorption/ionization (MALDI). The method is simple, fast and utilizes only low-cost disposables. After loading the sample on the column and a subsequent washing step, the analyte molecules are eluted with 50-100 nl of matrix solution directly on to the MALDI/MS target. The washing step ensures removal of a wide range of contaminants. The small bed volume of the column allows efficient sample concentration and the elution process yields very small sample spots. This simplifies the analysis and minimizes discrimination effects due to sample heterogeneity, because the desorption/ionization laser simultaneously irradiates a large portion of the sample. Taken together, these features of the method significantly improve the sensitivity for MALDI/MS analysis of contaminated peptide samples compared with the commonly used sample preparation procedures. This is demonstrated with in-gel tryptic digests of proteins from human brain that were separated by 2D gel electrophoresis. Furthermore, it is shown that with this method 2,5-dihydroxybenzoic acid (DHB) acts as an efficient matrix for peptide mapping. Both detection sensitivity and sequence coverage are comparable to those obtained with the currently preferred matrix alpha-cyano-4-hydroxycinnamic acid (CHCA). The higher stability of peptide ions generated with DHB compared with CHCA is advantageous when analyzing fragile sample molecules. Therefore, the method described here is also of interest for the use of Fourier transform ion cyclotron resonance (FT-ICR) or ion-trap mass analyzers.     

Simple fabrication of hydrophobic surface target for increased sensitivity and homogeneity in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of peptides,phosphopeptides, carbohydrates and proteins

To enhance sample signals and improve homogeneity in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) analysis, a simple, rapid, and efficient sample preparation method was developed in this study. Polydimethylsiloxane (PDMS) was coated on a stainless steel MALDI plate, forming a transparent, hydrophobic surface that enhanced sample signals without producing observable background signals. Compared to the use of an unmodified commercial metal MALDI plate, peptide signals were enhanced by ~7.1–11.0-fold due to the reduced sample spot area of the PDMS-coated plate, and showed improved peptide mass fingerprinting (PMF) and MS/MS peptide sequencing results. In the analysis of phosphopeptides and carbohydrates with a 2,5-dihydroxybenzoic acid (DHB) matrix, the PDMS-coated plate showed improved sample homogeneity and signal enhancements of ~5.2–8.2-fold and ~2.8–3.2-fold, respectively. Improved sensitivity in the observation of more unique fragment ions by MS/MS analysis, to successfully distinguish isomeric carbohydrates, was also illustrated. In protein analysis with a sinapinic acid (SA) matrix, a ~3.4-fold signal enhancement was observed. The PDMS film coating was easily removed and refabricated to avoid sample carryover, and was applicable to diverse commercial MALDI plates. The PDMS-coated approach is a simple, practical, and attractive method for enhancing analyte signals and homogeneity.     

MALDI-TOF mass spectrometry of a combinatorial peptide library: effect of matrix composition on signal suppression

The effect of matrix composition on signal suppression caused by a dominant compound under MALDI ionization was studied using the combinatorial TQTXT pentapeptide library as a model system. The peptide library is composed of 19 components with all proteinogenic amino acids except cysteine in position X. From these compounds, only the Arg peptide (TQTRT) was detected with sufficient intensity in the MALDI-TOF mass spectrum under typical MALDI conditions (CCA matrix). The analysis of a set of compounds utilized as different matrix components, additives and a cationizing agent revealed that the composition of the matrix is a critical point in signal suppression. Highly improved ion yields were achieved by using a CCA/DHB mixture as a matrix. The addition of K(+) as a cationizing agent to the CCA matrix resulted in MALDI-TOF mass spectra with relative ion intensities very similar to those obtained by electrospray ionization.     

The improved performance of MALDI‐TOF MS on the analysis of herbal saponins by using DHB‐GO composite matrix

Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) is an excellent analytical technique for rapid analysis of a variety of molecules with straightforward sample pretreatment. The performance of MALDI‐TOF MS is largely dependent on matrix type, and the development of novel MALDI matrices has aroused wide interest. Herein, we devoted to seek more robust MALDI matrix for herbal saponins than previous reported, and ginsenoside Rb1, Re, and notoginsenoside R1 were used as model saponins. At the beginning of the present study, 2,5‐dihydroxybenzoic acid (DHB) was found to provide the highest intensity for saponins in four conventional MALDI matrices, yet the heterogeneous cocrystallization of DHB with analytes made signal acquisition somewhat “hit and miss.” Then, graphene oxide (GO) was proposed as an auxiliary matrix to improve the uniformity of DHB crystallization due to its monolayer structure and good dispersion, which could result in much better shot‐to‐shot and spot‐to‐spot reproducibility of saponin analysis. The satisfactory precision further demonstrated that minute quantities of GO (0.1 μg/spot) could greatly reduce the risk of instrument contamination caused by GO detachment from the MALDI target plate under vacuum. More importantly, the sensitivity and linearity of the standard curve for saponins were improved markedly by DHB‐GO composite matrix. Finally, the application of detecting the Rb1 in complex biological sample was exploited in rat plasma and proved it applicable for pharmacokinetic study quickly. This work not only opens a new field for applications of DHB‐GO in herbal saponin analysis but also offers new ideas for the development of composite matrices to improve MALDI MS performance.     

Improved peptide mass fingerprinting matches via optimized sample preparation in MALDI mass spectrometry

Peptide mass fingerprinting (PMF) is a powerful technique in which experimentally measured m/z values of peptides resulting from a protein digest form the basis for a characteristic fingerprint of the intact protein. Due to its propensity to generate singly charged ions, along with its relative insensitivity to salts and buffers, matrix-assisted laser desorption and ionization (MALDI)-time-of-flight mass spectrometry (TOFMS) is the MS method of choice for PMF. The qualitative features of the mass spectrum can be selectively tuned by employing different methods to prepare the protein digest and matrix for MALDI-TOFMS. The selective tuning of MALDI mass spectra in order to optimize PMF is addressed here. Bovine serum albumin, carbonic anhydrase, cytochrome c, hemoglobin alpha- and beta-chain, and myoglobin were digested with trypsin and then analyzed by MALDI-TOFMS. 2,5-dihydroxybenzoic acid (DHB) and alpha-cyano-4-hydroxycinnamic acid (CHCA) were prepared using six different sample preparation methods: dried droplet, application of protein digest on MALDI plate followed by addition of matrix, dried droplet with vacuum drying, overlayer, sandwich, and dried droplet with heating. Improved results were obtained for the matrix alpha-cyano-4-hydroxycinnamic acid using a modification of the died droplet method in which the MALDI plate was heated to 80 °C prior to matrix application, which is supported by observations from scanning electron microscopy. Although each protein was found to have a different optimum sample preparation method for PMF, in general higher sequence coverage for PMF was obtained using DHB. The best PMF results were obtained when all of the mass spectral data for a particular protein digest was convolved together.     

Quantitative analysis of microcystin variants by capillary electrophoresis mass spectrometry with dynamic pH barrage junction focusing

Dynamic pH junction is an online focusing method in CE based on the electrophoretic mobility difference of analytes in the sample matrix and the background electrolyte. An advantage of this method over the conventional CE is that the sensitivity can be significantly improved. By injecting a long sample plug in the capillary and focusing the analytes at the pH boundary between the background electrolyte and sample matrix, the LOD can be improved by 10–100 folds. The dynamic pH junction method can be easily coupled with ESI‐MS. In this work, we used this method for the analysis of microcystins (MCs). The detection limits and dynamic ranges were studied. The separation was optimized by adjusting the injection time, and concentrations and pH values of the background electrolyte. The optimization of analyte focusing leads to enhanced detection response compared to conventional injections, achieving 200–400 fold higher averaged peak heights for four microcystin (MC) variants. More importantly, this method was successfully used for the quantitative analysis of microcystins (MCs) in crude algae samples from natural water bodies, making it promising for practical applications.     

MALDI‐TOF mass spectrometry of hordeins: rapid approach for identification of malting barley varieties

A procedure for identification of malting barley varieties using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) of ethanol‐soluble barley proteins (hordeins) is described. The hordeins were first extracted from milled barley grains by several extraction protocols (using different extraction agents and conditions). Hordein extracts were then analyzed directly via MALDI‐TOF MS without any preliminary purification or separation step, and the protein profiles of analyzed hordein extracts were compared in order to find out the most suitable extraction procedure for mass spectrometric analysis. The optimized procedure was successfully applied to identification of 13 malting barley varieties. Our results revealed that the proposed mass spectrometry‐based approach provides characteristic mass patterns of extracted hordeins, which can be advantageously used for barley variety identification. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of bupivacaine and metabolites in rat urine using capillary electrophoresis with mass spectrometric detection

A method using capillary electrophoresis-mass spectrometry (CE-MS) was developed for the structural elucidation of bupivacaine and metabolites in rat urine. Prior to CE-MS analysis, solid-phase extraction (SPE) was used for sample cleanup and preconcentration purposes. Exact mass and tandem mass spectrometric (MS/MS) experiments were performed to obtain structural information about the unknown metabolites. Two instruments with different mass analyzers were used for mass spectrometric detection. A quadrupole time-of-flight (Q-TOF) and a magnetic sector hybrid instrument were coupled to CE and used for the analysis of urine extracts. Hydroxybupivacaine as well as five other isomerically different metabolites were detected including methoxylated bupivacaine.     

Solid phase microextraction with matrix assisted laser desorption/ionization introduction to mass spectrometry and ion mobility spectrometry

Solid phase microextraction (SPME) with matrix assisted laser desorption/ionization (MALDI) introduction was coupled to mass spectrometry and ion mobility spectrometry. Nicotine and myoglobin in matrix 2,5-dihydroxybenzonic acid (DHB), enkephalin and substance P in alpha-cyano-4-hydroxy cinnaminic acid were investigated as the target compounds. The tip of an optical fiber was silanized for extraction of the analytes of interest from solution. The optical fiber thus served as the sample extraction surface, the support for the sample plus matrix, and the optical pipe to transfer the laser energy from the laser to the sample. The MALDI worked under atmospheric pressure, and both an ion mobility spectrometer and a quadrupole/time-of-flight mass spectrometer were used for the detection of the SPME/MALDI signal. The spectra obtained demonstrate the feasibility of the SPME with MALDI introduction to mass spectrometry instrumentation.     

Analysis of cellular release using capillary electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectrometry.

In order to increase our understanding of the mechanisms of learning and memory in the central nervous system, it is necessary to know the neurotransmitters and neuromodulators used in the specific neuronal circuits under study. Methods have been developed to identify the peptides released from single neurons and neuronal clusters from the common neuronal model Aplysia californica. Specifically, solid-phase extraction (SPE), capillary electrophoresis (CE) and matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) are combined for profiling neuropeptide releasates. A variety of combinations of SPE and CE were coupled off-line with MALDI-TOF-MS to reduce the high physiological salts, to concentrate the analytes, and to reduce the complexity of the mass spectra using separation. With these protocols, peptides and proteins up to 11000 Da were detected in releasates, offering a much wider mass range compared to direct MALDI analysis of the same releasates. A number of expected and unknown neuropeptides, including egg-laying hormone (ELH) and the partially processed delta/gamma-bag cell peptide were observed in the SPE-treated releasates from a single Aplysia-cultured bag cell neuron. However, by adding a CE separation after the SPE step preceding off-line MALDI-TOF-MS detection, the most complete neuropeptide profiles were obtained.     

Combination of Aqueous and Non-Aqueous Capillary Electrophoresis with Electrospray Mass Spectrometry for the Determination of Drug Residues in Water

Summary.  The work presented in this paper deals with the combination of capillary electrophoresis (CE) with electrospray mass spectrometry (MS) for the determination of drug residues in water. CE/MS methods have been developed based on either aqueous or non-aqueous ammonium acetate solutions as the carrier electrolyte for the separation of selected drugs. The different separation conditions were compared in terms of selectivity and detection limits; both aqueous and non-aqueous CE proved to be suitable for the present analytical task, exhibiting detection limits between 3 and 93 μg/dm³ (injected standard concentration) corresponding to concentrations between 5 and 19 ng/dm³ in the sample. A combination of liquid-liquid extraction and solid-phase extraction was investigated for sample pretreatment, yielding enrichment factors of 10000. The applicability of CE/MS was demonstrated for the analysis of several river water samples. Received August 25, 2000. Accepted October 17, 2000     

On‐chip solid‐phase extraction pre‐concentration/focusing substrates coupled to atmospheric pressure matrix‐assisted laser desorption/ionization ion trap mass spectrometry for high sensitivity biomolecule analysis

Atmospheric pressure matrix‐assisted laser desorption/ionization (AP‐MALDI) has proven a convenient and rapid method for ion production in the mass spectrometric (MS) analysis of biomolecules. AP‐MALDI and electrospray ionization (ESI) sources are easily interchangeable in most mass spectrometers. However, AP‐MALDI suffers from less‐than‐optimal sensitivity due to ion losses during transport from the atmosphere into the vacuum of the mass spectrometer. Here, we study the signal‐to‐noise ratio (S/N) gains observed when an on‐chip dynamic pre‐concentration/focusing approach is coupled to AP‐MALDI for the MS analysis of neuropeptides and protein digests. It was found that, in comparison with conventional AP‐MALDI targets, focusing targets showed (1) a sensitivity enhancement of approximately two orders of magnitude with S/N gains of 200–900 for hydrophobic substrates, and 150–400 for weak cation‐exchange (WCX) substrates; (2) improved detection limits as low as 5 fmol/µL for standard peptides; (3) significantly reduced matrix background; and (4) higher inter‐day reproducibility. The improved sensitivity allowed successful tandem mass spectrometric (MS/MS) sequencing of dilute solutions of a derivatized tryptic digest of a protein standard, and enabled the first reported AP‐MALDI MS detection of neuropeptides from Aedes aegypti mosquito heads. Copyright © 2009 John Wiley & Sons, Ltd.     

Tandem Mass Spectrometric Characterization of Fetuin Sialylated Glycopeptides Enriched by TiO2 Microcolumn

Sialylation of glycoproteins is vital for the function or physicochemical properties of a protein. It becomes more and more important to develop approaches that can be used to efficiently isolate and identify sialylated glycopeptides or glycoproteins for monitoring changes in glycoproteome. In the present study, we analyze intact structures of the enriched sialylated glycopeptides of bovine fetuin by matrix‐assisted laser desorption/ionization‐tandem mass spectrometry (MALDI‐MS/MS), without any chemical derivation. The experimental data show that the optimal loading buffer for TiO₂ as matrix is 80% acetonitrile/2% TFA (trifluoroacetic acid)/100 mg/mL DHB (2,5‐dihydroxybenzoic acid) which is also compatible with MALDI‐mass spectrometric analysis. This study indicates that the improved enrichment approach combined with MALDI‐MS/MS may be a powerful tool to analyze intact structures and components of the sialylated glycopeptides from complex peptide mixture.     

Measuring salty samples without adducts with MALDI MS

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) has been used for the discovery of hundreds of novel cell to cell signaling peptides. Beyond its advantages of sensitivity and minimal sample preparation requirements, MALDI MS is attractive for biological analyses as high quality mass spectra may be obtained directly from specific locations within prepared tissue sections. However, due to the large quantity of salts present in physiological tissues, these mass spectra often contain many adducts of cationic salts such as sodium and potassium, in addition to the molecular ion [M + H]⁺. To reduce the presence of cation adducts in MALDI mass spectra obtained directly from tissues, we present a methodology that uses a slow condensation procedure to enable the formation of distinct regions of matrix/analyte crystals and cation (salt) crystals. Secondary ion mass spectrometric imaging suggests that the salts and MALDI matrix undergo a mutually exclusive crystallization process that results in the separation of the salts and matrix in the sample.     

Dynamic pH barrage junction focusing of amino acids,peptides, and digested monoclonal antibodies in capillary electrophoresis–mass spectrometry

Dynamic pH barrage junction focusing in CE enables effective signal enhancement, quantitative capture efficiencies, and straightforward optimization. The method is a technical variant of dynamic pH junction focusing. CE separation with dynamic pH barrage junction focusing is compatible with both optical and mass spectrometric detection. We developed a CE–MS/MS method using hydrophilic polyethyleneimine-coated capillaries and validated it for the qualitative analysis of amino acids, peptides, and tryptic peptides of digested monoclonal antibodies. The S/N of extracted ion electropherograms of zwitterionic analytes were enhanced by approximately two orders of magnitude with a tradeoff of a shortened separation window. Online focusing improved the MS signal intensity of a diluted antibody digest, enabling more precursor ions to be analyzed with subsequent tandem mass spectrometric identification. It also broadened the concentration range of protein digest samples for which adequate sequence coverage data can be obtained. With only 0.9 ng of digested infliximab sample loaded into the capillary, 76% and 100% sequence coverage was realized for antibody heavy and light chains, respectively, after online focusing. Full coverage was achieved with 9 ng of injected digest.     

Identification of proteins directly from tissue: in situ tryptic digestions coupled with imaging mass spectrometry

A novel method for on-tissue identification of proteins in spatially discrete regions is described using tryptic digestion followed by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) with MS/MS analysis. IMS is first used to reveal the protein and peptide spatial distribution in a tissue section and then a serial section is robotically spotted with small volumes of trypsin solution to carry out in situ protease digestion. After hydrolysis, 2,5-Dihydroxybenzoic acid (DHB) matrix solution is applied to the digested spots, with subsequent analysis by IMS to reveal the spatial distribution of the various tryptic fragments. Sequence determination of the tryptic fragments is performed using on-tissue MALDI MS/MS analysis directly from the individual digest spots. This protocol enables protein identification directly from tissue while preserving the spatial integrity of the tissue sample. The procedure is demonstrated with the identification of several proteins in the coronal sections of a rat brain.     

Flavonoid–matrix cluster ions in MALDI mass spectrometry

The behaviour of 2,5‐dihydroxybenzoic acid (2,5‐DHB) matrix under matrix‐assisted laser desorption/ionisation (MALDI) conditions was investigated, and the formation of 2,5‐DHB cluster ions, mainly dehydrated 2,5‐DHB ions, is reported. Interestingly, in the mass spectra of this compound, besides dimers and trimers, protonated tetramers, pentamers, hexamers and heptamers were also found with significant abundance. The MALDI behaviour of four flavonoids, quercetin, myricetin, luteolin and kaempferol, using 2,5‐DHB as matrix, was also investigated. The mass spectra of the flavonoids studied revealed a number of flavonoid–2,5‐DHB cluster ions (mainly with the dehydrated 2,5‐DHB). The number of clusters formed is dependent on the structure of the analyte. For luteolin and kaempferol, in particular, evidence was found for the formation of cluster ions involving retro Diels Alder fragments and intact flavonoids molecules, as well as the corresponding protonated retro Diels Alder fragments with dehydrated DHB molecules. All ion compositions were attributed taking into account high accuracy mass measurements and tandem mass spectrometry experiments. Copyright © 2009 John Wiley & Sons, Ltd.     

Uncovering matrix effects on lipid analyses in MALDI imaging mass spectrometry experiments

The specific matrix used in matrix‐assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) can have an effect on the molecules ionized from a tissue sample. The sensitivity for distinct classes of biomolecules can vary when employing different MALDI matrices. Here, we compare the intensities of various lipid subclasses measured by Fourier transform ion cyclotron resonance (FT‐ICR) IMS of murine liver tissue when using 9‐aminoacridine (9AA), 5‐chloro‐2‐mercaptobenzothiazole (CMBT), 1,5‐diaminonaphthalene (DAN), 2,5‐Dihydroxyacetophenone (DHA), and 2,5‐dihydroxybenzoic acid (DHB). Principal component analysis and receiver operating characteristic curve analysis revealed significant matrix effects on the relative signal intensities observed for different lipid subclasses and adducts. Comparison of spectral profiles and quantitative assessment of the number and intensity of species from each lipid subclass showed that each matrix produces unique lipid signals. In positive ion mode, matrix application methods played a role in the MALDI analysis for different cationic species. Comparisons of different methods for the application of DHA showed a significant increase in the intensity of sodiated and potassiated analytes when using an aerosol sprayer. In negative ion mode, lipid profiles generated using DAN were significantly different than all other matrices tested. This difference was found to be driven by modification of phosphatidylcholines during ionization that enables them to be detected in negative ion mode. These modified phosphatidylcholines are isomeric with common phosphatidylethanolamines confounding MALDI IMS analysis when using DAN. These results show an experimental basis of MALDI analyses when analyzing lipids from tissue and allow for more informed selection of MALDI matrices when performing lipid IMS experiments.     

A 2,5-dihydroxybenzoic acid/N,N-dimethylaniline matrix for the analysis of oligosaccharides by matrix-assisted laser desorption/ionization mass spectrometry

The use of a novel 2,5-dihydroxybenzoic acid/N,N-dimethylaniline (DHB/DMA) matrix-assisted laser desorption/ionization (MALDI) matrix for detection and quantitative analysis of native N-linked oligosaccharides was investigated in this study. Substantial improvements in sensitivity were observed relative to the signals obtained with a traditional DHB matrix. Moreover, the morphology of the matrix crystal layer was very uniform, unlike that of DHB. This resulted in highly homogeneous sample distribution throughout the spot, allowing reproducible and consistent mass spectra to be obtained without spot-to-spot variations in signal. Here, we also demonstrate an approach for performing sensitive and accurate quantitative analysis of native N-linked glycans with this novel matrix using an internal standard method.     

Three-layer matrix/sample preparation method for MALDI MS analysis of low nanomolar protein samples

A robust and sensitive sample preparation method is presented for matrix-assisted laser desorption ionization (MALDI) mass spectrometric analysis of low nanomolar concentrations of proteins containing high amounts of common salts and buffers. This method involves the production of densely packed sub-micrometer matrix crystals by depositing a matrix solution on top of a matrix seed-layer prepared on a MALDI target. A sub-microliter aliquot of analyte solution is then directly added to the top of the matrix crystals to form a thin-layer. alpha-Cyano-4-hydroxycinnamic acid (4-HCCA) is used as matrix and demonstrated to give better performance than other commonly used matrices, such as 2,5-dihydroxybenzoic acid (DHB), 2-(4-hydroxy-phenylazo) benzoic acid (HABA), or sinapinic acid. This three-layer method is shown to be superior to the other MALDI sample preparation methods, particularly for handling low nanomolar protein solutions containing salts and buffers.