Sample preparation for high throughput accurate mass analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

An automated sample preparation for high throughput accurate mass determinations by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) has been developed. Sample preparation was performed with an automated workstation and automated mass analyses were performed with a commercial MALDI-TOF mass spectrometer. The method was tested with a 41-sample library. MALDI-TOFMS was found to give the needed sensitivity, accurate mass measurement, and soft ionization necessary for structure confirmation, even of mixtures. A mass accuracy of 5 ppm or less was obtained in over 80% of known compound measurements. A mass accuracy better than 10 ppm was obtained for all measurements of known compounds. Analyses of parallel synthesis products resulted in 77% of the measurements with a mass accuracy of 5 ppm or better.     

Short monolithic columns for purification and fractionation of peptide samples for matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry analysis in proteomics

This study records a novel application of methacrylate-based monolithic columns for MALDI-TOF/TOF MS analyses in proteomics for pre-concentration and separation of peptides derived from protein digestion. Reversed-phase monolithic capillary columns (30 mm × 0.32 mm i.d.) were created inside the fused silica capillary via thermal-initiated free-radical polymerization of ethylene glycol dimethacrylate and lauryl methacrylate monomers in the presence of 1-propanol and 1,4-butandiol as a porogen system. The elution of peptides was achieved using a linear gradient of acetonitrile from 0 to 60% in water with 0.1% trifluoroacetic acid formed in a microsyringe. Individual fractions of separated peptides were collected on the MALDI target spots covered with alpha-cyano-4-hydroxycinnamic acid used as a matrix and then they were analyzed using MALDI-TOF/TOF mass spectrometry. The developed method was tested with a mixture of tryptic peptides from bovine serum albumin and its applicability was also tested for tryptic in-gel digests from barley grain extracts of water soluble proteins separated using SDS gel electrophoresis. The number of detected peptides was approximately three to four times higher compared to the analysis without previous separation. These results show an improved quality of sample information with the higher amount of identified peptides which increased protein sequence coverage and improved sensitivity of mass spectrometry measurements.     

Evaluation of combined matrix-assisted laser desorption/ionization time-of-flight and matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry experiments for peptide mass fingerprinting analysis

Peptide Mass Fingerprinting (PMF) is still of significant interest in proteomics because it allows a large number of complex samples to be rapidly screened and characterized. The main part of post-translational modifications is generally preserved. In some specific cases, PMF suffers from ambiguous or unsuccessful identification. In order to improve its reliability, a combined approach using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICRMS) was evaluated. The study was carried out on bovine serum albumin (BSA) digest. The influence of several important parameters (the matrix, the sample preparation method, the amount of the analyte) on the MOWSE score and the protein sequence coverage were evaluated to allow the identification of specific effects. A careful investigation of the sequence coverage obtained by each kind of experiment ensured the detection of specific peptides for each experimental condition. Results highlighted that DHB-FTICRMS and DHB- or CHCA-TOFMS are the most suited combinations of experimental conditions to achieve PMF analysis. The association (convolution) of the data obtained by each of these techniques ensured a significant increase in the MOWSE score and the protein sequence coverage.     

Application of DNA-binding polymers for preparation of DNA for analysis by matrix-assisted laser desorption/ionization mass spectrometry

The susceptibility of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to the presence of salts in a sample, especially salts of alkali metals, requires careful and often tedious desalting procedures which complicate and slow the throughput of MS-based methods. A novel approach to sample preparation was developed based on the extraction of DNA out of solution onto a solid surface with an attached DNA-binding polymer, such as polyethyleneimine or polyvinylpyrrolidone. The observed binding is strong enough to sustain washing, and, as a result, desalting and concentration can be performed in a single fast step. After DNA has been immobilized on the surface and supernatant solution removed, subsequent addition of MALDI matrix releases material from the surface, which co-crystallizes with matrix. The mass spectrometric analysis is then performed directly from this support. Analysis of oligonucleotides and three-fold multiplexed SNP typing reactions performed by this method shows improved sensitivity and excellent resolution for various DNA fragments, together with high tolerance to various buffer components, such as alkali metals and surfactants. Simplicity and speed make it attractive for high-throughput sample preparation and analysis of oligonucleotide mixtures by MALDI-MS.     

Intact protein analysis by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry

Direct tandem mass spectrometric (MS/MS) analysis of small, singly charged protein ions by tandem time-of-flight mass spectrometry (TOFMS) is demonstrated for proteins up to a molecular mass of 12 kDa. The MALDI-generated singly charged precursor ions predominantly yield product ions resulting from metastable fragmentation at aspartyl and prolyl residues. Additional series of C-terminal sequence ions provide in some cases sufficient information for protein identification. The amount of sample required to obtain good quality spectra is in the high femtomolar to low picomolar range. Within this range, MALDI-MS/MS using TOF/TOF trade mark ion optics now provides the opportunity for direct protein identification and partial characterization without prior enzymatic hydrolysis.     

Investigations of matrix-assisted laser desorption/ionization sample preparation by time-of-flight secondary ion mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) analyses are compared to gain insight into some of the details of sample preparation for MALDI analysis of synthetic polymers. ToF-SIMS imaging of MALDI samples shows segregation of the cationization agent from the matrix crystals. The amount of observed segregation can be controlled by the sample preparation technique. Electrospray sample deposition minimizes segregation. Comparing ToF-SIMS and MALDI mass spectra from the same samples confirms that ToF-SIMS is significantly more surface sensitive than MALDI. This comparison shows that segregation of the oligomers of a polymer sample can occur during MALDI sample preparation. Our data indicate that MALDI is not as sensitive to those species dominating the sample surface as to species better incorporated into the matrix crystals. Finally, we show that matrix-enhanced SIMS can be an effective tool to analyze synthetic polymers, although the sample preparation conditions may be different than those optimized for MALDI.     

Improvement of in-gel digestion protocol for peptide mass fingerprinting by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

High-sensitivity, high-throughput analysis of proteins for proteomics studies is usually performed by polyacrylamide gel electrophoresis in combination with mass spectrometry. However, the quality of the data obtained depends on the in-gel digestion procedure employed. This work describes an improvement in the in-gel digestion efficiency for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis. A dramatic improvement in the coverage of tryptic peptides was observed when n-octyl glucoside was added to the buffer. Whole cell extracted proteins from S. cerevisiae were separated by two-dimensional gel electrophoresis and stained with silver. Protein spots were identified using our improved in-gel digestion method and MALDI-TOFMS. In addition, the mass spectra obtained by using the matrix alpha-cyano-4-hydroxycinnamic acid (CHCA) were compared with those obtained using 2,5-dihydroxybenzoic acid (DHB). The DHB matrix usually gave more peaks, which led to higher sequence coverage and, consequently, to higher confidence in protein identification. This improved in-gel digestion protocol is simple and useful for protein identification by MALDI-TOFMS.     

Miniaturizing sample spots for matrix-assisted laser desorption/ionization mass spectrometry

The trend of miniaturization in bioanalytical chemistry is shifting from technical development to practical application. In matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), progress in miniaturizing sample spots has been driven by the needs to increase sensitivity and speed, to interface with other analytical microtechnologies, and to develop miniaturized instrumentation.We review recent developments in miniaturizing sample spots for MALDI-MS. We cover both target modification and microdispensing technologies, and we emphasize the benefits with respect to sensitivity, throughput and automation.We hope that this review will encourage further method development and application of miniaturized sample spots for MALDI-MS, so as to expand applications in analytical chemistry, protein science and molecular biology.     

Effect of sodium dodecyl sulfate micelles on peptide mass fingerprinting by matrix-assisted laser desorption/ionization mass spectrometry

Here we have examined the effect of sodium dodecyl sulfate (SDS) at various concentrations on matrix-assisted laser desorption/ionization (MALDI) peptide mass fingerprinting experiments. Several model proteins were digested with trypsin and then various amounts of SDS were added prior to MALDI mass spectrometry. Evaluation of the data was made by calculating the amino acid sequence coverage within each analysis. It was found that addition of 0.1-0.3% w/v SDS prior to MALDI analysis results in an increase in the number of tryptic peptides detected thereby improving the total sequence coverage of the analysis. The use of SDS at concentrations near its critical micelle concentration can improve sequence coverage from MALDI peptide mass fingerprinting analyses allowing for increased confidence in protein identification or additional opportunities to identify putative regions of posttranslational modification.     

Metal labeling for accurate multiplexed peptide quantification via matrix-assisted laser desorption/ionization mass spectrometry

Two peptide quantification strategies, the isobaric tags for relative or absolute quantitation (iTRAQ) labeling methodology and a metal-chelate labeling approach, were compared using matrix-assisted laser desorption/ionization-TOF/TOF MS and MS/MS analysis. Amino- and cysteine-directed labeling using the rare earth metal chelator 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid (DOTA) were applied for relative quantification of single peptides and a six-protein mixture. For analyte ratios close to one, iTRAQ and amino-directed DOTA labeling delivered overall comparable results regarding accuracy and reproducibility. In contrast, the MS-based quantification via amino-directed lanthanide-DOTA tags was more accurate for analyte ratios ≥5 and offered an extended dynamic range of three orders of magnitude. Our results show that the amino-directed DOTA labeling is an alternative relative quantification tool offering advantages like flexible multiplexing possibilities and, in particular, large dynamic ranges, which should be useful in sophisticated, targeted issues, where the accurate determination of extremely different protein or peptide concentration becomes relevant.     

On-line atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry

A new technique is presented for the coupling of atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) mass spectrometry with liquid delivery systems. Mass measurements of polymers and peptides are demonstrated using a co-dissolved matrix, e.g. alpha-cyano-4-hydroxycinnamic acid (HCCA). Improvements in terms of sensitivity are achieved by optimizing the shape und control of the exit capillary and by using a laser (355 nm) at a 1 kHz repetition rate. Two calibration experiments promise a good applicability of the presented coupling method for quantitative measurements. The limit of detection achieved so far is 500 nM for peptides in methanol solution containing 25 mM HCCA.     

Direct surface analysis of fungal species by matrix-assisted laser desorption/ionization mass spectrometry

In this study various methods of sample preparation and matrices were investigated to determine optimum collection and analysis criteria for fungal analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Intact spores and/or hyphae of Aspergillus niger, Rhizopus oryzae, Trichoderma reesei and Phanerochaete chrysosporium were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The fungal samples were applied to the MALDI sample target as untreated, sonicated, or acid/heat treated samples, or blotted directly from the fungal culture with double-stick tape. Ferulic acid or sinapinic acid matrix solution was layered over the dried samples and analyzed by MALDI-MS. Statistical analysis showed that simply using double-stick tape to collect and transfer to a MALDI sample plate typically worked as well as the other preparation methods, and required the least sample handling.     

Sequencing of a branched peptide using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Chemical degradation methods combined with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and post-source decay (PSD)-MALDI reflex TOF mass spectrometry (MS) were used to determine the sequence of a peptide branched on to a known peptide backbone. This study was applied to a branched peptide model (derivative of substance P). The branched peptide mimics a digest of a membrane receptor on to which a derivative of substance P was photochemically linked. Chemical degradation based on N-terminal ladder sequencing in combination with MALDI-TOF-MS gave only partial sequence information. Although single PSD mass spectra still remain difficult to interpret unambiguously, PSD-MALDI-TOF-MS was combined with on-target acetylation and H -- D exchange to give a better and successful approach to the unambiguous determination of the complete amino acid side-chain sequence. This study shows the capability of MALDI-TOF-MS to help in characterizing ligand-receptor interactions.     

Ganglioside analysis by thin-layer chromatography matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry

Metastable decomposition of ions generated in matrix-assisted laser desorption/ionization (MALDI) mass spectrometers complicates analysis of biological samples that have labile bonds. Recently, several academic laboratories and manufacturers of commercial instruments have designed instruments that introduce a cooling gas into the ion source during the MALDI event and have shown that the resulting vibrational cooling stabilizes these labile bonds. In this study, we compared stabilization and detection of desorbed gangliosides on a commercial orthogonal time-of-flight (oTOF) instrument with results we reported previously that had been obtained on a home-built Fourier transform mass spectrometer. Decoupling of the desorption/ionization from the detection steps resulted in an opportunity for desorbing thin-layer chromatography (TLC)-separated gangliosides directly from a TLC plate without compromising mass spectral accuracy and resolution of the ganglioside analysis, thus coupling TLC and oTOF mass spectrometry. The application of a declustering potential allowed control of the matrix cluster and matrix adduct formation, and, thus, enhanced the detection of the gangliosides.     

Characterization of polyether mixtures using thin-layer chromatography and matrix-assisted laser desorption/ionization mass spectrometry

Thin-layer chromatography (TLC) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) were combined to achieve characterization of polyether mixtures. Three polyethers, polyethylene glycol (PEG), polypropylene glycol (PPG) and polytetramethylene glycol (PTMG), or mixtures of these compounds, were studied. One shortcoming of mixture analysis of synthetic polymers using MALDI-MS is that individual polymers in the mixture may display different detection sensitivities. For example, the MALDI mass spectrum of an equimolar mixture of PEG, PPG and PTMG displayed a high intensity of PPG ions, while no PTMG ions were detectable; however, PTMG ions were detected after the mixture had been separated by TLC. This combined TLC and MALDI-MS analysis of a PPG polymer bearing reactive epoxy groups showed that the polymer contained byproducts with different end-groups. These byproducts were identified as chloro-substituted polymers formed during polymer synthesis. Our study shows TLC to be a rapid and low-cost separation technique, and that it can be combined with MALDI-MS to achieve effective analysis of synthetic polymers.     

Compositional analysis of isobutylene/p-methylstyrene copolymers by matrix-assisted laser desorption/ionization mass spectrometry

A low molecular weight predominantly polyolefin copolymer of isobutylene and para methylstyrene (IMS) was studied using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Average composition information derived from the spectra was skewed to higher para methylstyrene (pMS) content as compared to that obtained using multiple NMR techniques, and drifted towards lower pMS incorporation at higher oligomer lengths. Although both observations were initially attributed in total to an inability to ionize the isobutylene component, comparison with subsequent field desorption (FD) mass spectrometry results gave similar values to that obtained via MALDI, even though FD ionizes oligomers not detected by MALDI. Instead, the compositional drift observed with MALDI roughly mirrored the mass distribution, and was determined to arise from a mass bias effect in oligomer ionization and detection. Composition with respect to oligomer mass was found to be relatively constant, although similarly higher in pMS content. Comparison of experimental peaks with a Bernoullian statistical model revealed severe overrepresentation of higher pMS composition oligomers with regard to the calculated distribution. This discrepancy is attributed to preferential ionization of oligomers with greater pMS content, and likely results in the observed difference between MALDI and NMR compositions.     

Application of an integrated matrix-assisted laser desorption/ionization time-of-flight, electrospray ionization mass spectrometry and tandem mass spectrometry approach to characterizing complex polyol mixtures

Polyols are being used in a wide range of industrial applications including surfactants and precursors for grafted polymers. The characterization of polyols is of significance in correlating compositions and structures with their properties. We illustrate two real world examples where traditional analytical methods including GPC and NMR failed to reveal compositional differences, but the combination of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), electrospray ionization mass spectrometry (ESI MS), and MS/MS can produce compositional information required for problem solving. The first example involves failure analysis of four ethylene oxide and propylene oxide (EO/PO) copolymer products. The results from the mass spectrometry analysis unequivocally demonstrate that one of the samples has a small variation in copolymer composition, leading to its abnormal activity. The second example is in the area of deformulation of complex polyol mixtures. Two samples displaying similar properties and activities were found to be two different polyol blends. One of the samples is a more cost-effective product. These examples demonstrate that MALDI, ESI MS, and MS/MS should be seriously considered as an integrated component of an overall polyol characterization program in product failure analysis and deformulation.     

Detection of immune complexes by matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used to detect an immune complex formed between beta-lactoglobulin and polyclonal anti-beta-lactoglobulin antibody in the gas phase. The most important experimental parameters to detect such a specific antibody-antigen complex by MALDI were the use of solutions at near-neutral pH and of sinapinic acid matrix prepared by the dried-droplet method. Under such conditions, predominantly one but also two molecules of antigen protein were complexed by the antibody. Specific formation of the antibody-antigen complex was confirmed by performing competitive reactions. Addition of antibody to a 1:1 mixture of beta-lactoglobulin and one control protein resulted not only in the appearance of the expected antibody-antigen complex, but also in a strong decrease in the free beta-lactoglobulin signal, while the abundance of the control protein was not influenced.