Improved PSD and CID on a MALDI TOFMS

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

Exciton mobility and trapping in a MALDI matrix

Energy transfer (ET) from excited matrix to fluorescent traps is used to probe the mobility of excitations in the matrix-assisted laser desorption/ionization (MALDI) matrix material 2,5-dihydroxybenzoic acid. The dependence of host and guest fluorescence on excitation density (laser intensity) and trap concentration gives clear evidence for long-range energy transport in this matrix. This conclusion is further supported by time-resolved emission data showing a 2 ns delay between matrix and trap emission. Rate equation and random walker models give good agreement with the data, allowing determination of hopping, collision, and trapping parameters. Long-range energy transfer contributes to the pooling reactions which can lead to primary ions in MALDI. The results validate the pooling aspect of the prior quantitative MALDI ionization model (J. Mass Spectrom. 2002, 37, 867-877). It is shown that exciton trapping can decrease MALDI ion yield, even at low trap concentration.     

Novel vacuum stable ketone‐based matrices for high spatial resolution MALDI imaging mass spectrometry

We describe the use of aromatic ketones and cinnamyl ketones that have high vacuum stability for analyzing tissue sections using matrix‐assisted laser desorption/ionization imaging mass spectrometry. Specifically, the matrix, (E)‐4‐(2,5‐dihydroxyphenyl)but‐3‐en‐2‐one (2,5‐cDHA) provides high sensitivity and high vacuum stability while producing small size crystals (1‐2 μm). A high throughput and highly reproducible sample preparation method was developed for these matrices that first involves using an organic spray solution for small matrix crystal seeding followed by spraying of the matrix in a 30% acetonitrile/70% water solution on the tissue surface to obtain a homogeneous coating of small crystals, suitable for high spatial resolution imaging.     

Direct detection of peptides and proteins on a microfluidic platform with MALDI mass spectrometry

The ability to detect and quantify proteins of individual cells in high throughput is of enormous biological and clinical relevance. Most methods currently in use either require the measurement of large cell populations or are limited to the investigation of few cells at a time. In this report, we present the combination of a polydimethylsiloxane-based microfluidic device to a matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS) that allows the detection of as few as 300 molecules at the peptide level and ～10(6) to 10(7) molecules at the protein level. Moreover, we performed an immunoassay with subsequent MALDI-TOF-MS to capture and detect insulin immobilized on a surface (～0.05?mm(2)) in this device with a detection limit of 10(6) insulin molecules. This microfluidic-based approach therefore begins to approach the sample handling and sensitivity requirements for MS-based single-cell analysis of proteins and peptides and holds the potential for easy parallelization of immunoassays and other highly sensitive protein analyses.     

延时提取正交发射MALDI技术研究

An orthogonal injection (OI) home-made reflectror type time-of-flight (TOF) mass spectrometer has been constructed with a matrix-assisted laser desorption/ionization (MALDI) source. Ions generated by MALDI are measured using a pulsed voltage delayed extraction method. The laser used is a frequency quadrupled Nd:YAG laser with output at wavelength of 266 nm, the matrix used here is 2,5-dihydroxybenzonic acid (DHB), and the analytes are Malachite green and peptides. Measurements of resolving power and statistical evaluation of the mass accuracy are reported here. The results indicate that resolving power in the range of 3400 to 4000 (full width at half maximum), the average error of the mass accuracy is below 0.0075%, A perfectly linear (m/z)1/2 versus t plot is found. Finally, the initial velocity distribution of analyte and matrix ions in the range of 400~1000 m/s is measured.     

Tryptamine: a Reactive Matrix for MALDI Mass Spectrometry

A possibility of using tryptamine as a reactive matrix for the analysis of non-polar carbonyl compounds by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has been shown. Presence of a terminal primary amine group in the tryptamine molecule predetermines the formation of Schiff bases from aliphatic and alicyclic carbonyl compounds. No additional matrix compounds are necessary to register MALDI mass spectra, because the excess of the derivatization agent plays the role of a matrix. MALDI mass spectra demonstrate high efficiency of desorption/ionization of the derivatives. To discover reactive matrices, a set of aromatic primary amines (mainly substituted anilines) has been tested, but they have not demonstrated matrix properties.     

Magnetic iron oxide nanoparticle enrichment of phosphopeptides on a radiate microstructure MALDI chip

Several methods can be used to improve the enrichment of phosphorylated proteins. In this paper, phosphopeptides were enriched using magnetic iron(II,III) oxide (magnetite, Fe(3)O(4)) nanoparticles (NPs) on a radiate microstructure silicon chip and then analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) without further purification processes. We have developed a radiate microstructure chip on which samples can be concentrated for analysis by MALDI-TOFMS. The phosphoprotein digests and magnetic iron oxide NPs aqueous solution were deposited onto the central zone of the radiate microstructure silicon chip and enabled the on-chip enrichment of phosphopeptides. Microscopic analysis confirmed that the applied samples were confined to the central zone. Sample spots focused on the chip were much smaller than those on an unmodified plate with the same total volume. Different additives were used and optimized processes were performed to minimize non-phosphopeptides interference. These data collectively demonstrate that our on-chip phosphopeptide enrichment protocol is a rapid and easy-to-use method for phosphoproteome analysis.     

Reflectron MALDI TOF and MALDI TOF/TOF mass spectrometry reveal novel structural details of native lipooligosaccharides

Lipooligosaccharides (LOS) are powerful Gram-negative glycolipids that evade the immune system and invade host animal and vegetal cells. The structural elucidation of LOS is pivotal to understanding the mechanisms of infection at the molecular level. The amphiphilic nature of LOS has been the main obstacle for structural analysis by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). Our approach has resolved this important issue and has permitted us to obtain reflectron MALDI mass spectra of LOS to reveal the fine chemical structure with minimal structural variations. The high-quality MALDI mass spectra show LOS species characteristic of molecular ions and defined fragments due to decay in the ion source. The in-source decay yields B-type ions, which correspond to core oligosaccharide(s), and Y-type ions, which are related to lipid A unit(s). MALDI tandem time-of-flight (TOF/TOF) MS of lipid A allowed for the elucidation of its structure directly from purified intact LOS without the need for any chemical manipulations. These findings constitute a significant advancement in the analysis of such an important biomolecule by MALDI MS.     

Magnetic carbon nanocomposites as a MALDI co-matrix enhancing MS-based glycomics

Analytical and Bioanalytical Chemistry - More than 50% of all known proteins are glycosylated, which is critical for many biological processes such as protein folding and signal transduction....     

Comparative TOF-SIMS and MALDI TOF-MS analysis on different chromatographic planar substrates

A comparison is made between two high resolution, surface-based, mass spectrometric methods: time-of-flight secondary ion mass spectrometry (TOF-SIMS) and matrix-assisted laser desorption/ionisation mass spectrometry (MALDI TOF-MS) in indication of abietic and gibberellic acids molecular profiles on different chromatographic thin layers. The analytes were applied to silica gel chromatographic thin layers with SIMS on-line interfacing channel, monolithic silica gel ultra-thin layers, and thin layers specifically designed for direct Raman spectroscopic analysis. Two MALDI matrices were used in this research: ferulic acid and 2,5-dihydroxybenzoic acid. The silica gel SIMS-interfacing channel strongly supported formation of numerous different MALDI MS fragments with abietic and gibberellic acids, and ferulic acid matrix. The most intense fragments belonged to [M-OH](+) and [M](+) ions from ferulic acid. Intense conjugates were detected with gibberellic acid. The MALDI MS spectrum from the monolithic silica gel surface showed very low analyte signal intensity and it was not possible to obtain MALDI spectra from a Raman spectroscopy treated chromatographic layer. The MALDI TOF MS gibberellic acid fragmentation profile was shielded by the matrix used and was accompanied by poor analyte identification. The most useful TOF-SIMS analytical signal response was obtained from analytes separated on monolithic silica gel and a SIMS-interfacing modified silica gel surface. New horizons with nanostructured surfaces call for high resolution MS methods (which cannot readily be miniaturised like many optical and electrochemical methods) to be integrated in chip and nanoscale detection systems.     

Quantitative transfer of polar analytes on a solid surface to a liquid matrix in MALDI profiling

In profiling of a specimen by matrix‐assisted laser desorption ionization (MALDI) using a solid matrix, the solvent of the matrix solution extracts an analyte(s). A quantitative profiling cannot be achieved if the solvent evaporates before the complete extraction of the analyte. The extraction can become more quantitative when a liquid matrix dissolved in a solvent is used, which remains a liquid even after the evaporation of the solvent. To check this, radii of an analyte circle (r_A), a matrix solution drop (r_D) and a liquid matrix (r_M) remaining after the solvent evaporation were controlled. Three types of samples were prepared, case A (r_A, r_D < r_M), case B (r_D ≤ r_M < r_A) and case C (r_M < r_D < r_A). In case A, the analyte amount in the matrix layer determined by MALDI was the same as the prepared amount inside the analyte circle. In case B, the analyte amount was the same as the amount inside the matrix circle. Only the analytes in contact with the liquid matrix layer, not more and not less, are transferred to the matrix layer. In case C, the analyte amount was greater than the amount inside the matrix circle, presumably because some of the analyte outside the matrix circle was dissolved by the solvent of the matrix solution. Copyright © 2016 John Wiley & Sons, Ltd.     

Resolving the composition of protein complexes using a MALDI LTQ orbitrap

Current biological studies have been advanced by the continuous development of robust, accurate, and sensitive mass spectrometric technologies. The MALDI LTQ Orbitrap is a new addition to the Orbitrap configurations, known for their high resolving power and accuracy. This configuration provides features inherent to the MALDI source, such as reduced spectra complexity, forgiveness to contaminants, and sample retention for follow-up analyses with targeted or hypothesis-driven questions. Here we investigate its performance for characterizing the composition of isolated protein complexes. To facilitate the assessment, we selected two well characterized complexes from Saccharomyces cerevisiae, Apl1 and Nup84. Manual and automatic MS and MS/MS analyses readily resolved their compositions, with increased confidence of protein identification compared with our previous reports using MALDI QqTOF and MALDI IT. CID fragmentation of singly-charged peptides provided sufficient information for conclusive identification of the isolated proteins. We then assessed the resolution, accuracy, and sensitivity provided by this instrument in the context of analyzing the isolated protein assemblies. Our analysis of complex mixtures of singly-charged ions up to m/z 4000 showed that (1) the resolving power, inversely proportional to the square root of m/z, had over four orders of magnitude dynamic range; (2) internal calibration led to improved accuracy, with an average absolute mass error of 0. 5 ppm and a distribution centered at 0 ppm; and (3) subfemtomole sensitivity was achieved using both CHCA and DHB matrices. Additionally, our analyses of a synthetic phosphorylated peptide in mixtures showed subfemtomole level of detection using neutral loss scanning.     

Optimization of a MALDI TOF-TOF mass spectrometer for intact protein analysis

A MALDI TOF-TOF instrument was optimized and evaluated for intact protein analysis by tandem mass spectrometry. Ion source voltages and delay times were adjusted to affect an up to a 10-fold improvement in fragment ion yield compared to data obtained using default settings employed in peptide analysis. For large peptides (3-4.5 kDa), up to 90% of all possible b- and y-fragment ions were observed, which provides sufficient information for de novo sequencing and unambiguous protein identification. Product ion signals associated with preferential cleavages C-terminal to aspartic acid and glutamic acid residues and N-terminal to proline residues became dominant with increased protein molecular weight. Matrix effects were also evaluated and, among the eight matrices examined, alpha-cyano-4-hydroxycinnamic acid (CHCA) was found to produce the best intact protein tandem mass spectra for proteins up to 12 kDa. Optimized performance yielded detection limits of 50-125 fmol for proteins of 4 and 12 kDa, respectively. This improved performance has yielded an instrument with potential to be a useful tool in proteomic investigations via analysis of intact proteins.     

Pseudo-MS3 in a MALDI orthogonal quadrupole-time of flight mass spectrometer

Both the matrix selected and the laser fluence play important roles in MALDI-quadrupole/time of flight (QqTOF) fragmentation processes. "Hot" matrices, such as alpha-cyano4-hydroxycinnamic acid (HCCA), can increase fragmentation in MS spectra. Higher laser fluence also increases fragmentation. Typical peptide fragment ions observed in the QqTOF are a, b, and y ion series, which resemble low-energy CID product ions. This fragmentation may occur in the high-pressure region before the first mass-analyzing quadrupole. Fragment ions can be selected by the first quadrupole (Q1), and further sequenced by conventional MS/MS. This allows pseudo-MS3 experiments to be performed. For peptides of higher molecular weight, pseudo-MS3 can extend the mass range beyond what is usually accessible for sequencing, by allowing one to sequence a fragment ion of lower molecular weight instead of the full-length peptide. Peptides that predominantly show a single product ion after MS/MS yield improved sequence information when this technique is applied. This method was applied to the analysis of an in vitro phosphorylated peptide, where the intact enzymatically-generated peptide showed poor dissociation via MS/MS. Sequencing a fragment ion from the phosphopeptide enabled the phosphorylation site to be unambiguously determined.     

Using fluorescence dyes as a tool for analyzing the MALDI process

In a recent paper (Setz, P. D.; Knochenmuss, R. Phys. Chem. A2005, 109, 4030-4037) energy-transfer from excited matrix molecules to fluorescent traps was used to study the role of pooling reactions for the ionization processes in matrix-assisted laser desorption ionization (MALDI) using 2,5-dihydroxybenzoic acid as matrix. Exciton trapping was shown to interfere with matrix ionization. These investigations were extended to analyze the influence of fluorescent traps on both matrix and analyte ions for alpha-cyano-4-hydroxycinnamic acid and further matrices. A strong influence of the fluorescent traps on both matrix and analyte ionization was revealed, depending on the matrix:trap ratio, and manifested itself differently for low and high mass analytes. The observations are rationalized by the intermediate formation of a "hot spot" due to an efficient conversion of electronic excitation energy to vibronic energy within the fluorescent traps. This process favors the desorption and ionization of small vaporizable analytes and compromises the cluster ablation process needed for larger analyte ions.     

MALDI Imaging Assisted Discovery of a Di-O-glycosyltransferase from Platycodon grandiflorum Root

A matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) assisted genome mining strategy was developed for the discovery of glycosyltransferase (GT) from the root of Platycodon grandiflorum. A di-O-glycosyltransferase PgGT1 was discovered and characterized that is capable of catalyzing platycoside E (PE) synthesis through the attachment of two β-1,6-linked glucosyl residues sequentially to the glucosyl residue at the C3 position of platycodin D (PD). Although UDP-glucose is the preferred sugar donor for PgGT1, it could also utilize UDP-xylose and UDP-N-acetylglucosamine as weak donors. Residues S273, E274, and H350 played important roles in stabilizing the glucose donor and positioning the glucose in the optimal orientation for the glycosylation reaction. This study clarified two key steps involved in the biosynthetic pathway of PE and could greatly contribute to improving its industrial biotransformation.