402 research outputs found
Mapping the Human Plasma Proteome by SCX-LC-IMS-MS
The advent of on-line multidimensional liquid chromatography-mass spectrometry has significantly impacted proteomic analyses of complex biological fluids such as plasma. However, there is general agreement that additional advances to enhance the peak capacity of such platforms are required to enhance the accuracy and coverage of proteome maps of such fluids. Here, we describe the combination of strong-cation-exchange and reversed-phase liquid chromatographies with ion mobility and mass spectrometry as a means of characterizing the complex mixture of proteins associated with the human plasma proteome. The increase in separation capacity associated with inclusion of the ion mobility separation leads to generation of one of the most extensive proteome maps to date. The map is generated by analyzing plasma samples of five healthy humans; we report a preliminary identification of 9087 proteins from 37,842 unique peptide assignments. An analysis of expected false-positive rates leads to a high-confidence identification of 2928 proteins. The results are catalogued in a fashion that includes positions and intensities of assigned features observed in the datasets as well as pertinent identification information such as protein accession number, mass, and homology score/confidence indicators. Comparisons of the assigned features reported here with other datasets shows substantial agreement with respect to the first several hundred entries; there is far less agreement associated with detection of lower abundance components
Solvent-free MALDI-MS for the analysis of biological samples via a mini-ball mill approach
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AP-UV-MALDI mit flüssigen Matrizes: stabile Ionenausbeuten von mehrfach geladenen Peptid- und Proteinionen für die empfindliche Massenspektrometrie
In der biologischen Massenspektrometrie (MS) werden
überwiegend zwei Ionisationstechniken für die Analyse von
grçßeren Biomolekfürlen wie Polypeptiden eingesetzt. Dies
sind die Nano-Elektrospray-Ionisation[1,2] (nanoESI) und die
matrixunterstfürtzte Laserdesorption/-ionisation[3, 4] (MALDI).
Beide Techniken werden als „sanft“ bezeichnet, weil sie die
Desorption und Ionisation von intakten Analytmolekfürlen
und damit ihre erfolgreiche massenspektrometrische Analyse
erlauben. Einer der wichtigsten Unterschiede zwischen
diesen beiden Ionisationstechniken liegt in ihrer F�higkeit,
mehrfach geladene Ionen zu erzeugen. MALDI erzeugt typischerweise
einfach geladene Peptidionen, w�hrend nano-
ESI leicht mehrfach geladene Ionen produziert, sogar für
Peptide mit einer Masse von weniger als 1000 Da. Die Erzeugung
von hoch geladenen Ionen ist wünschenswert, da
dies die Verwendung von Massenanalysatoren wie Ionenfallen
(inkl. Orbitraps) und Hybrid-Quadrupolinstrumenten
ermçglicht, die typischerweise nur einen begrenzten m/z-
Bereich (<2000–4000) bieten. Hohe Ladungszust�nde ermçglichen
auch die Aufnahme von informativeren Fragmentionenspektren,
wenn Methoden wie die kollisionsinduzierte
Dissoziation (CID), die Elektroneneinfang-Dissoziation
(ECD) und die Elektronentransfer-Dissoziation (ETD) in
Kombination mit der Tandem-MS (MS/MS) verwendet
werden
Solvent-free MALDI-MS for the analysis of β-amyloid peptides via the mini-ball mill approach: Qualitative and quantitative advances
Test purchase, synthesis, and characterization of 2-methoxydiphenidine (MXP) and differentiation from its meta- and para-substituted isomers
The structurally diverse nature of the 1,2-diphenylethylamine template provides access to a range of substances for drug discovery work but some have attracted attention as ‘research chemicals’. The most recent examples include diphenidine, i.e. 1-(1,2-diphenylethyl)piperidine and 2-methoxydiphenidine, i.e. 1-[1-(2-methoxyphenyl)-2-phenylethyl]piperidine (MXP, methoxyphenidine, 2-MXP) that have been associated with uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist activity. Analytical challenges encountered during chemical analysis include the presence of positional isomers. Three powdered samples suspected to contain 2-MXP were obtained from three Internet retailers in the United Kingdom and subjected to analytical characterization by gas chromatography (GC) and high performance liquid chromatography (HPLC) coupled to various forms of mass spectrometry (MS). Nuclear magnetic resonance spectroscopy, infrared spectroscopy and thin layer chromatography were also employed. This was supported by the synthesis of all three isomers (2-, 3- and 4-MXP) by two different synthetic routes. The analytical data obtained for the three purchased samples were consistent with the synthesized 2-MXP standard and the differentiation between the isomers was possible. Distinct stability differences were observed for all three isomers during in-source collision-induced dissociation of the protonated molecule when employing detection under HPLC selected-ion monitoring detection, which added to the ability to differentiate between them. Furthermore, the analysis of a 2-MXP tablet by matrix assisted inlet ionization Orbitrap mass spectrometry confirmed that it was possible to detect the protonated molecule of 2-MXP directly from the tablet surface following addition of 3-nitrobenzonitrile as the matrix
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Protein identification using a nanoUHPLC-AP-MALDI MS/MS workflow with CID of multiply charged proteolytic peptides
Liquid AP-MALDI can produce predominantly multiply charged ESI-like ions and stable durable analyte ion yields with samples allowing good shot-to-shot reproducibility and exhibiting self-healing properties during laser irradiation. In this study, LC-MALDI MS/MS workflows that utilize multiply charged ions are reported for the first time and compared with standard LC-ESI MS/MS for bottom-up proteomic analysis. The proposed method is compatible with trifluoroacetic acid as an LC ion pairing reagent and allows multiple MS/MS acquisitions of the LC-separated samples without substantial sample consumption. In addition, the method facilitates the storage of fully spotted MALDI target plates for months without significant sample degradation
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Investigation and optimization of parameters affecting the multiply charged ion yield in AP-MALDI MS
Liquid matrix-assisted laser desorption/ionization (MALDI) allows the generation of predominantly multiply
charged ions in atmospheric pressure (AP) MALDI ion sources for mass spectrometry (MS) analysis.
The charge state distribution of the generated ions and the efficiency of the ion source in generating such
ions crucially depend on the desolvation regime of the MALDI plume after desorption in the AP-tovacuum
inlet. Both high temperature and a flow regime with increased residence time of the desorbed
plume in the desolvation region promote the generation of multiply charged ions. Without such measures
the application of an electric ion extraction field significantly increases the ion signal intensity of
singly charged species while the detection of multiply charged species is less dependent on the extraction
field. In general, optimization of high temperature application facilitates the predominant formation and
detection of multiply charged compared to singly charged ion species. In this study an experimental setup
and optimization strategy is described for liquid AP-MALDI MS which improves the ionization effi-
ciency of selected ion species up to 14 times. In combination with ion mobility separation, the method
allows the detection of multiply charged peptide and protein ions for analyte solution concentrations
as low as 2 fmol/lL (0.5 lL, i.e. 1 fmol, deposited on the target) with very low sample consumption in
the low nL-range
The analysis of polystyrene and polystyrene aggregates into the mega dalton mass range by cryodetection MALDI TOF MS
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