Scrambling of autoinducing precursor peptides investigated by infrared multiphoton dissociation with electrospray ionization and Fourier transform ion cyclotron resonance mass spectrometry
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Infrared multiphoton dissociation
Ion cyclotron resonance
Fragmentation
Pentapeptide repeat
Top-down proteomics
An efficient approach in combinatorial chemistry is the synthesis of one-bead-one-compound peptide libraries. In contrast to synthesis and functional screening, which is performed in a largely automated manner, structure determination has been frequently laborious and time-consuming. Here we report an approach for de novo sequencing of peptides on single beads by matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance (MALDI-FTICR) tandem mass spectrometry, using a resin with a photolinker for solid-phase peptide synthesis. Upon sorting out single beads, an efficient sample preparation on the MALDI target was developed that enables fragmentation upon irradiation of the bead-matrix mixture with the ultraviolet (UV)-MALDI laser, with enhanced yield of sequence-specific fragment ions at increased laser energy. This approach is illustrated by sequence determinations of two peptides from a library with sequences varying in a single amino acid; the feasibility with tandem-MS procedures and fragment ion assignment was ascertained by sustained off-resonance irradiation/collision induced dissociation (SORI/CID) and infrared multiphoton dissociation (IRMPD) fragmentation.
Infrared multiphoton dissociation
Fragmentation
Top-down proteomics
MALDI imaging
Collision-induced dissociation
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The characterization of cellular proteomes is important for understanding biochemical processes ranging from cell differentiation to cancer development. In one highly promising approach, whole protein extracts or fractions are digested (e.g., with trypsin) and injected into a packed capillary column for subsequent separation. The separated peptides are then introduced on-line to an electrospray ionization source of a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer for the detection of peptide accurate mass tags that serve as biomarkers for their parent proteins. In this work, we report the use of data-dependent selective external ion ejection in conjunction with FTICR and on-line capillary LC separations for the enhanced characterization of peptide mixtures and a yeast extract proteome. The number of peptides identified in an LC-FTICR analysis of a yeast proteome digest employing data-dependent rf-only dipolar ejection of the most abundant ion species prior to ion accumulation was 40% higher than that detected in a separate LC-FTICR analysis using conventional nonselective ion accumulation.
Ion cyclotron resonance
Top-down proteomics
Proteome
Bottom-up proteomics
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Abstract A microelectrospray ionization tandem Fourier transform ion cyclotron resonance mass spectrometry (ESI FT‐ICR MS n ) approach for structural characterization of protein phosphorylation is described. Identification of proteolytic peptides is based solely upon mass measurement by high field (9.4 Tesla) FT‐ICR MS. The location of the modification within any phosphopeptide is then established by FT‐ICR MS 2 and MS 3 experiments. Structural information is maximized by use of electron capture dissociation (ECD) and/or infrared multiphoton dissociation (IRMPD). The analytical utility of the method is demonstrated by characterization of protein kinase A (PKA) phosphorylation. In a single FT‐ICR MS experiment, 30 PKA tryptic peptides (including three phosphopeptides) were mass measured by internal calibration to within an absolute mean error of |0.7 ppm|. The location of each of the three sites of phosphorylation was then determined by MS 2 and MS 3 experiments, in which ECD and IRMPD provide complementary peptide sequence information. In two out of three cases, electron irradiation of a phosphopeptide [M + nH] n+ ion produced an abundant charge‐reduced [M + nH] (n−1)+• ion, but few sequence‐specific c and z • fragment ions. Subsequent IRMPD (MS 3 ) of the charge‐reduced radical ion resulted in the detection of a large number of ECD‐type ion products ( c and z ions), but no b or y type ions. The utility of activated ion ECD for the characterization of tryptic phosphopeptides was then demonstrated.
Infrared multiphoton dissociation
Phosphopeptide
Top-down proteomics
Ion cyclotron resonance
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We have developed a method for protein identification with peptide mass fingerprinting and sequence tagging using nano liquid chromatography (LC)/Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). To achieve greater sensitivity, a nanoelectrospray (nano-ES) needle packed with reversed-phase medium was used and connected to the nano-ES ion source of the FTICR mass spectrometer. To obtain peptide sequence tag information, infrared multiphoton dissociation (IRMPD) was carried out in nano-LC/FTICR-MS analysis. The analysis involves alternating nano-ES/FTICR-MS and nano-ES/IRMPD-FTICR-MS scans during a single LC run, which provides sets of parent and fragment ion masses of the proteolytic digest. The utility of this alternating-scan nano-LC/IRMPD-FTICR-MS approach was evaluated by using bovine serum albumin as a standard protein. We applied this approach to the protein identification of rat liver diacetyl-reducing enzyme. It was demonstrated that this enzyme was correctly identified as 3-alpha-hydroxysteroid dehydrogenase by the alternating-scan nano-LC/IRMPD-FTICR-MS approach with accurate peptide mass fingerprinting and peptide sequence tagging.
Infrared multiphoton dissociation
Top-down proteomics
Ion cyclotron resonance
Electron-capture dissociation
Orbitrap
Peptide mass fingerprinting
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Ion Structure and Energetics in the Gas Phase Characterized Using Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Chad A. Jones Department of Chemistry and Biochemistry, BYU Doctor of Philosophy In this dissertation, I use Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) to study the structure and energetics of gas phase ions. Infrared multiphoton dissociation spectroscopy (IRMPD) is a technique for measuring the IR spectrum of gas phase ions in a Penning trap. I use this technique to investigate the conformation of cucurbituril complexes, terminal diamines, and protonated amino acids. Cross sectional areas by Fourier transform ion cyclotron resonance mass spectrometry (CRAFTI) is a technique developed by the Dearden lab to measure the cross section of gas phase ions. In this work, I further develop a fundamental understanding of this technique. I investigate the role that dissociation plays in this and other FTICR-MS techniques. I also show that the principles of the CRAFTI technique can be used to measure the pressure inside the cell of an FTICR-MS. This technique, linewidth pressure measurement (LIPS), allows for a quantitative measurement and comparison of CRAFTI cross sections. To demonstrate the improvements to the technique, I measure the CRAFTI cross sections for the 20 standard amino acids and compare these to literature values measured by ion mobility measurements.
Infrared multiphoton dissociation
Ion cyclotron resonance
Selected ion monitoring
Top-down proteomics
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Electron-capture dissociation
Infrared multiphoton dissociation
Ion cyclotron resonance
Posttranslational modification
Top-down proteomics
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Abstract Electrospray ionization with Fourier transform ion cyclotron resonance mass spectrometry (ESI‐FTICRMS) was used for screening and structural elucidation of core oligosaccharides isolated from lipopolysaccharides of bacteria of the genus Proteus . Mass spectra allowed the determination of the molecular masses with high accuracy and the estimation of the chemical heterogeneity of the samples. They did not, however, provide sufficient information to identify structural details of the branched oligosaccharides. Therefore, various fragmentation techniques for determining such details were examined. Infrared multiphoton dissociation tandem mass spectrometry (IRMPD‐MS/MS) experiments in negative ion mode resulted in cleavage between the structurally conserved inner core region and the variable outer core region. Positive ion capillary skimmer dissociation mass spectra showed numerous fragment ion peaks, including those corresponding to the subsequent cleavage of the glycosidic linkages starting from the non‐reducing end of the oligosaccharide. Despite their complexity, these mass spectrometric studies allowed confirmation of previously determined Proteus lipopolysaccharide core structures, and identification of new related structures in other strains of these bacteria. Copyright © 2005 John Wiley & Sons, Ltd.
Infrared multiphoton dissociation
Top-down proteomics
Fragmentation
Glycosidic bond
Ion cyclotron resonance
Collision-induced dissociation
Oligosaccharide
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We report the first coupling of a desorption electrospray ionization (DESI) ion source to Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT-ICR-MS) for high-resolution protein analysis. The DESI FT-ICR-MS source design is described in detail along with preliminary data obtained on peptides and proteins ranging from 1 to 5.7 kDa.
Ion cyclotron resonance
Desorption electrospray ionization
Top-down proteomics
MALDI imaging
Selected ion monitoring
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Electron-capture dissociation
Ion cyclotron resonance
Infrared multiphoton dissociation
Top-down proteomics
Selected ion monitoring
Cyclotron resonance
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Chemical cross-linking has proved successful in combination with mass spectrometry as a tool for low-resolution structure determination of proteins. The integration of chemical cross-linking with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to determine protein interfaces was tested on the calcium-dependent complex between calmodulin (CaM) and a 26-amino acid peptide derived from the skeletal muscle myosin light chain kinase (M13). Different amine-reactive, homobifunctional cross-linkers and a "zero-length" cross-linker were employed. The covalently attached complexes were separated from nonreacted proteins by one-dimensional gel electrophoresis, and the bands of interest were excised and in-gel digested with trypsin. Digestion of the cross-linked complexes resulted in complicated peptide mixtures, which were analyzed by nano-HPLC/nano-ESI-FTICR mass spectrometry. The distance constraints obtained by chemical cross-linking were in agreement with the published NMR structure of the CaM/M13 complex, pointing to residues Lys-18 and Lys-19 of M13 being cross-linked with the central α-helix of CaM. Thus, the integrated approach described herein has proven to be an efficient tool for mapping the topology of the CaM/M13 complex. As such it is applicable as a general strategy for the investigation of the spatial organization of protein complexes and complements existing techniques, such as X-ray crystallography and NMR spectroscopy.
Ion cyclotron resonance
Top-down proteomics
Posttranslational modification
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