Automated reduction and interpretation of

Over the last decade a variety of MS measurements, such as H͞D exchange, collision cross sections, and electron capture dissociation (ECD), have been used to characterize protein folding in the gas phase, in the absence of solvent. To the extensive data already available on ubiquitin, here photofragmentation of its ECDreduced (M ؉ nH) (n؊1)؉• ions shows that only the 6؉ to 9؉, not the 10؉ to 13؉ ions, have tertiary noncovalent bonding; this is indicated as hydrogen bonding by the 3,050 -3,775 cm ؊1 photofragment spectrum. ECD spectra and H͞D exchange of the 13؉ ions are consistent with an all ␣-helical secondary structure, with the 11؉ and 10؉ ions sufficiently destabilized to denature small bend regions near the helix termini. In the 8؉ and 9؉ ions these terminal helical regions are folded over to be antiparallel and noncovalently bonded to part of the central helix, whereas this overlap is extended in the 7؉, 6؉, and, presumably, 5؉ ions to form a highly stable three-helix bundle. Thermal denaturing of the 7؉ to 9؉ conformers both peels and slides back the outer helices from the central one, but for the 6؉ conformer, this instead extends the protein ends away to shrink the three-helix bundle. Thus removal of H 2O from a native protein negates hydrophobic interactions, preferentially stabilizes the ␣-helical secondary structure with direct solvation of additional protons, and increases tertiary interhelix dipole-dipole and hydrogen bonding.

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“…The 6 T Fourier transform ion cyclotron resonance mass spectrometer (Finnigan-MAT, San Jose, CA) has been described (11,24,26,27 (35).…”

Section: Methodsmentioning

confidence: 99%

Secondary and tertiary structures of gaseous protein ions characterized by electron capture dissociation mass spectrometry and photofragment spectroscopy

Breuker

Sze

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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240

297

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“…Initially, a set of m/z values were obtained from each spectrum and the charge states are determined from their respective 13 C-isotopic patterns. Because a theoretical 13 C-isotope pattern for a peptide can be adequately predicted using an averaging based model [29], most "non-peptide-like" analytes can be eliminated based on the lack of agreement between the observed and predicted isotopic pattern, given sufficient S/N. Once the list is filtered so as to contain only likely peptide peaks, the monoisotopic ion mass is generated (based on the assumption that the peptide detected is protonated) from the monoisotopic m/z and charge state, and 13 C enriched (isotopic) peaks are eliminated from further consideration.…”

Section: Using Ambiguous Mass Values To Obtain Amt Tag Identificationsmentioning

confidence: 99%

Proteome analyses using accurate mass and elution time peptide tags with capillary LC time-of-flight mass spectrometry

Strittmatter

Ferguson

Tang

et al. 2003

J. Am. Soc. Mass Spectrom.

148

113

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We describe the application of capillary liquid chromatography (LC) time-of-flight (TOF) mass spectrometric instrumentation for the rapid characterization of microbial proteomes. Previously (Lipton et al., Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 11049) the peptides from a series of growth conditions of Deinococcus radiodurans have been characterized using capillary LC MS/MS and accurate mass measurements which are captured as an accurate mass and time (AMT) tag database. Using this AMT tag database, detected peptides can be assigned using measurements obtained on a TOF due to the additional use of elution time data as a constraint. When peptide matches are obtained using AMT tags (i.e., using both constraints) unique matches of a mass spectral peak occurs 88% of the time. Not only are AMT tag matches unique in most cases, the coverage of the proteome is high; ϳ3500 unique peptide AMT tags are found on average per capillary LC run. From the results of the AMT tag database search, ϳ900 ORFs detected using LC-TOFMS, with ϳ500 ORFs covered by at least two AMT tags. These results indicate that AMT database searches with modest mass and elution time criteria can provide proteomic information for approximately one thousand proteins in a single run of Ͻ3 h. The advantage of this method over using MS/MS based techniques is the large number of identifications that occur in a single experiment as well as the basis for improved quantitation. For MS/MS experiments, the number of peptide identifications is severely restricted because of the time required to dissociate the peptides individually. These results demonstrate the utility of the AMT tag approach using capillary LC-TOF MS instruments, and also show that AMT tags developed using other instrumentation can be effectively utilized. (J Am Soc Mass Spectrom 2003, 14, 980 -991)

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“…Anderson@pnl.gov). Briefly, ICR-2LS converted the raw data into m͞z spectra that were subsequently transformed to generate a table of neutral masses by using an implementation of the THRASH algorithm originally developed by Horn et al (37). The frequency shift, obtained from the comparison of the theoretical and experimental mass of the internal standard, was then applied to all neutral masses and saved to a separate data file (PEK).…”

Section: Msmentioning

confidence: 99%

Direct mass spectrometric analysis of intact proteins of the yeast large ribosomal subunit using capillary LC/FTICR

Lee

Berger

Martinović

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

182

133

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Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry coupled with capillary reverse-phase liquid chromatography was used to characterize intact proteins from the large subunit of the yeast ribosome. High mass measurement accuracy, achieved by ''mass locking'' with an internal standard from a dual electrospray ionization source, allowed identification of ribosomal proteins. Analyses of the intact proteins revealed information on cotranslational and posttranslational modifications of the ribosomal proteins that included loss of the initiating methionine, acetylation, methylation, and proteolytic maturation. High-resolution separations permitted differentiation of protein isoforms having high structural similarity as well as proteins from their modified forms, facilitating unequivocal assignments. The study identified 42 of the 43 core large ribosomal subunit proteins and 58 (of 64 possible) core large subunit protein isoforms having unique masses in a single analysis. These results demonstrate the basis for the high-throughput analyses of complex mixtures of intact proteins, which we believe will be an important complement to other approaches for defining protein modifications and their changes resulting from physiological processes or environmental perturbations.M ass spectrometry has evolved into a powerful tool for analyzing biomolecules because of the development of matrix-assisted laser desorption ionization (1, 2) and electrospray ionization (ESI) (3) and advances in both mass analyzers and data processing capabilities. With these ionization methods, which are amenable to megadalton-size molecules, the broadly useful detection and characterization of biopolymers by mass spectrometric methods are feasible. For example, MS has become a preferred analytical tool for proteome analyses, in which dynamic populations of proteins are identified and quantified. Proteins are now routinely identified by mass spectrometric and tandem mass spectrometric analysis of proteolytic digests of individual protein spots on two-dimensional (2D) PAGE (4). However, the intact protein level analyses of complex protein mixtures, while potentially providing complementary and direct information for protein identification, have been a much greater experimental challenge and only rarely attempted (5, 6).Many pivotal cellular processes are not carried out by individual proteins, but rather by large protein-protein complexes and protein-nucleic acid complexes. The analyses of such complexes have been greatly expanded by improvements in both biological separations and MS. One of the more complicated protein complexes yet studied by MS is the cytosolic ribosome complex. Efforts to define the protein composition (7, 8), modifications (9), and subunit interactions (10) of ribosomes generally have mirrored the development of biomolecular analysis techniques and experimental approaches for the study of noncovalent protein complexes.Ribosomes are the canonical example of ribonucleoprotein complexes in both prokaryot...

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Automated reduction and interpretation of

Cited by 514 publications

References 44 publications

Secondary and tertiary structures of gaseous protein ions characterized by electron capture dissociation mass spectrometry and photofragment spectroscopy

Secondary and tertiary structures of gaseous protein ions characterized by electron capture dissociation mass spectrometry and photofragment spectroscopy

Proteome analyses using accurate mass and elution time peptide tags with capillary LC time-of-flight mass spectrometry

Direct mass spectrometric analysis of intact proteins of the yeast large ribosomal subunit using capillary LC/FTICR

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