144 research outputs found
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Editorial for āAdvances in Biological Mass Spectrometry and Proteomicsā
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Introduction of 4-chloro-alpha-cyanocinnamic acid liquid matrices for high sensitivity UV-MALDI MS
Matrix-assisted laser desorption/ionization (MALDI) is a key ionization technique in mass spectrometry (MS) for the analysis of labile macromolecules. An important area of study and improvements in relation to MALDI and its application in high-sensitivity MS is that of matrix design and sample preparation. Recently, 4-chloro-alpha-cyanocinnamic acid (ClCCA) has been introduced as a new rationally designed matrix and reported to provide an improved analytical performance as demonstrated by an increase in sequence coverage of protein digests obtained by peptide mass mapping (PMM) (Jaskolla, T. W.; et al. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 12200-12205). This new matrix shows the potential to be a superior alternative to the commonly used and highly successful alpha-cyano-4-hydroxycinnamic acid (CHCA). We have taken this design one step further by developing and optimizing an ionic liquid matrix (ILM) and liquid support matrix (LSM) using ClCCA as the principle chromophore and MALDI matrix compound. These new liquid matrices possess greater sample homogeneity and a simpler morphology. The data obtained from our studies show improved sequence coverage for BSA digests compared to the traditional CHCA crystalline matrix and for the ClCCA-containing ILM a similar performance to the ClCCA crystalline matrix down to 1 fmol of BSA digest prepared in a single MALDI sample droplet with current sensitivity levels in the attomole range. The LSMs show a high tolerance to contamination such as ammonium bicarbonate, a commonly used buffering agent
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Atmospheric pressure ultraviolet laser desorption and ionization from liquid samples for native mass spectrometry
Understanding protein structure is vital for evaluating protein interactions with drugs, proteins and other ligands. Native mass spectrometry (MS) is proving to be invaluable for this purpose, enabling analysis of ānative-likeā samples that mimic physiological conditions. Native MS is usually performed by electrospray ionization (ESI) with its soft ionization processes and the generation of multiply charged ions proving favourable for conformation retention and high mass analysis, respectively. There is scope to expand the currently available toolset, specifically to other soft ionization techniques such as soft laser desorption, for applications in areas like high-throughput screening and MS imaging. In this letter, observations made from native MS experiments using an ultraviolet (UV) laser-based ion source operating at atmospheric pressure are described. The ion source is capable of producing predominately multiply charged ions similar to ESI. Proteins and protein complexes were analyzed from a native-like sample droplet to investigate the technique. Ion mobility-mass spectrometry (IM-MS) measurements showed that folded protein conformations were detected for ions with low charge states. This observation indicates the source is suitable for native MS analysis and should be further developed for higher mass analysis in the future
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Collision-induced dissociation of doubly-charged barium-cationized lipids generated from liquid samples by atmospheric pressure matrix-assisted laser desorption/ionization provides structurally diagnostic product ions
Obtaining structural information for lipids such as phosphatidylcholines, in particular the location of double bonds in their fatty acid constituents, is an ongoing challenge for mass spectrometry (MS) analysis. Here, we present a novel method utilizing the doping of liquid matrix-assisted laser desorption/ionization (MALDI) samples with divalent metal chloride salts, producing ions with the formula [L+M]2+ (L = lipid, M = divalent metal cation). Multiply charged lipid ions were not detected with the investigated trivalent metal cations. Collision-induced dissociation (CID) product ions from these doubly-charged metal-cationized lipids include the singly charged intact fatty acids [snx+M-H]+, where āxā represents the position of the fatty acid on the glycerol backbone. The preference of the divalent metal cation to locate on the sn2 fatty acid during CID was found, enabling stereochemical assignment. Pseudo-MS3 experiments such as in-source decay (ISD)-CID and ion mobility-enabled time-aligned parallel (TAP) MS of [snx+M-H]+ provided diagnostic product ion spectra for determining the location of double bonds on the acyl chain and were applied to identify and characterize lipids extracted from soya milk. This novel method is applicable to lipid profiling in the positive mode, where structural information of lipids is often difficult to obtain
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Advancing liquid atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry toward ultra-high-throughput analysis
Label-free high-throughput screening using mass spectrometry has the potential to provide rapid large-scale sample analysis at a speed of more than one sample per second. Such speed is important for compound library, assay and future clinical screening of millions of samples within a reasonable time frame. Herein, we present a liquid atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) setup for high-throughput large-scale sample analysis (>5 samples per second) for three substance classes (peptides, antibiotics and lipids). Liquid support matrices (LSM) were used for the analysis of standard substances as well as complex biological fluids (milk). Throughput and analytical robustness were mainly dependent on the complexity of the sample composition and the current limitations of the commercial hardware. However, the ultimate limits of liquid AP-MALDI in sample throughput can be conservatively estimated to be beyond 10-20 samples per second. This level of analytical speed is highly competitive compared with other label-free MS methods, including electrospray ionization and solid state MALDI, as well as MS methods using multiplexing by labelling, which in principle can also be used in combination with liquid AP-MALDI MS
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Liquid AP-UV-MALDI enables stable ion yields of multiply charged peptide and protein ions for sensitive analysis by mass spectrometry
In biological mass spectrometry (MS), two ionization techniques are predominantly employed for the analysis of larger biomolecules, such as polypeptides. These are nano-electrospray ionization [1, 2] (nanoESI) and matrix-assisted laser desorption/ionization [3, 4] (MALDI). Both techniques are considered to be āsoftā, allowing the desorption and ionization of intact molecular analyte species and thus their successful mass-spectrometric analysis. One of the main differences between these two ionization techniques lies in their ability to produce multiply charged ions. MALDI typically generates singly charged peptide ions whereas nanoESI easily provides multiply charged ions, even for peptides as low as 1000 Da in mass. The production of highly charged ions is desirable as this allows the use of mass analyzers, such as ion traps (including orbitraps) and hybrid quadrupole instruments, which typically offer only a limited m/z range (< 2000ā4000). It also enables more informative fragmentation spectra using techniques such as collisioninduced dissociation (CID) and electron capture/transfer dissociation (ECD/ETD) in combination with tandem MS
(MS/MS). [5, 6] Thus, there is a clear advantage of using ESI in research areas where peptide sequencing, or in general, the structural elucidation of biomolecules by MS/MS is required. Nonetheless, MALDI with its higher tolerance to contaminants and additives, ease-of-operation, potential for highspeed and automated sample preparation and analysis as well as its MS imaging capabilities makes it an ionization technique that can cover bioanalytical areas for which ESI is less suitable. [7, 8]
If these strengths could be combined with the analytical power of multiply charged ions, new instrumental configurations and large-scale proteomic analyses based on MALDI MS(/MS) would become feasible
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Advances in mass spectrometry-based cancer research and analysis: from cancer proteomics to clinical diagnostics
Introduction: The last 20 years have seen significant improvements in the analytical capabilities of biological mass spectrometry. Studies using advanced mass spectrometry (MS) have resulted in new insights into cell biology and the aetiology of diseases as well as its use in clinical applications.
Areas Covered: This review will discuss recent developments in MS-based technologies and their cancer-related applications with a focus on proteomics. It will also discuss the issues around translating the research findings to the clinic and provide an outline of where the field is moving.
Expert Opinion: Proteomics has been problematic to adapt for the clinical setting. However, MS-based techniques continue to demonstrate potential in novel clinical uses beyond classical cancer proteomics
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Hydroponic isotope labeling of entire plants (HILEP) for quantitative plant proteomics
Quantitative analysis by mass spectrometry (MS) is a major challenge in
proteomics as the correlation between analyte concentration and signal
intensity is often poor due to varying ionisation efficiencies in the
presence of molecular competitors. However, relative quantitation
methods that utilise differential stable isotope labelling and mass
spectrometric detection are available. Many drawbacks inherent to
chemical labelling methods (ICAT, iTRAQ) can be overcome by
metabolic labelling with amino acids containing stable isotopes (e.g. 13C
and/or 15N) in methods such as Stable Isotope Labelling with Amino
acids in Cell culture (SILAC). SILAC has also been used for labelling of
proteins in plant cell cultures (1) but is not suitable for whole plant
labelling. Plants are usually autotrophic (fixing carbon from atmospheric
CO2) and, thus, labelling with carbon isotopes becomes impractical. In
addition, SILAC is expensive.
Recently, Arabidopsis cell cultures were labelled with 15N in a medium
containing nitrate as sole nitrogen source. This was shown to be suitable
for quantifying proteins and nitrogen-containing metabolites from this cell
culture (2,3).
Labelling whole plants, however, offers the advantage of studying
quantitatively the response to stimulation or disease of a whole multicellular
organism or multi-organism systems at the molecular level.
Furthermore, plant metabolism enables the use of inexpensive labelling
media without introducing additional stress to the organism. And finally,
hydroponics is ideal to undertake metabolic labelling under extremely
well-controlled conditions.
We demonstrate the suitability of metabolic 15N hydroponic isotope
labelling of entire plants (HILEP) for relative quantitative proteomic
analysis by mass spectrometry. To evaluate this methodology,
Arabidopsis plants were grown hydroponically in 14N and 15N media and subjected to oxidative stress
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Bacterial identification by lipid profiling using liquid atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry
Background In recent years, mass spectrometry (MS) has been applied to clinical microbial biotyping, exploiting the speed of matrix-assisted laser desorption/ionization (MALDI) in recording microbe-specific MS profiles. More recently, liquid atmospheric pressure (AP) MALDI has been shown to produce extremely stable ion flux from homogenous samples and 'electrospray ionization (ESI)-like' multiply charged ions for larger biomolecules, whilst maintaining the benefits of traditional MALDI including high tolerance to contaminants, low analyte consumption and rapid analysis. These and other advantages of liquid AP-MALDI MS have been explored in this study to investigate its potential in microbial biotyping. Methods Genetically diverse bacterial strains were analyzed using liquid AP-MALDI MS, including clinically relevant species such as Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae. Bacterial cultures were subjected to a simple and fast extraction protocol using ethanol and formic acid. Extracts were spotted with a liquid support matrix (LSM) and analyzed using a Synapt G2-Si mass spectrometer with an in-house built AP-MALDI source. Results Each species produces a unique lipid profile in the m/z range of 400-1100, allowing species discrimination. Traditional (solid) MALDI MS produced spectra containing a high abundance of matrix-related clusters and an absence of lipid peaks. The MS profiles of the bacterial species tested form distinct clusters using principle component analysis (PCA) with a classification accuracy of 98.63% using a PCA-based prediction model. Conclusions Liquid AP-MALDI MS profiles can be sufficient to distinguish clinically relevant bacterial pathogens and other bacteria, based on their unique lipid profiles. The analysis of the lipid MS profiles is typically excluded from commercial instruments approved for clinical diagnostics
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