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Chemistry	100%

Selected Abstracts

Protein identification via ion-trap collision-induced dissociation and examination of low-mass product ions

JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 1 2008
Jeremiah J. Bowers
Abstract A whole-protein tandem mass spectrometry approach for protein identification based on precursor ion charge state concentration via ion/ion reactions, ion-trap collisional activation, ion/ion proton-transfer reactions involving the product ions, and mass analysis over a narrow m/z range (up to m/z 2000) is described and evaluated. The experiments were carried out with a commercially available electrospray ion-trap instrument that has been modified to allow for ion/ion reactions. Reaction conditions and the approach to searching protein databases were developed with the assumption that the resolving power of the mass analyzer is insufficient to distinguish charge states on the basis of the isotope spacings. Ions derived from several charge states of cytochrome c, myoglobin, ribonuclease A, and ubiquitin were used to evaluate the approach for protein identification and to develop a two-step procedure to database searching to optimize specificity. The approach developed with the model proteins was then applied to whole cell lysate fractions of Saccharomyces cerevisiae. The results are illustrated with examples of assignments made for three a priori unknown proteins, each selected randomly from a lysate fraction. Two of the three proteins were assigned to species present in the database, whereas one did not match well any database entry. The combination of the mass measurement and the product ion masses suggested the possibility for the oxidation of two methionine residues of a protein in the database. The examples show that this limited whole-protein characterization approach can provide insights that might otherwise be lacking with approaches based on complete enzymatic digestion. Copyright © 2007 John Wiley & Sons, Ltd. [source]

Balancing robust quantification and identification for iTRAQ: Application of UHR-ToF MS

PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 11 2010
Saw Yen Ow
Abstract iTRAQ reagents allow the simultaneous multiplex identification and quantification of a large number of proteins. Success depends on effective peptide fragmentation in order to generate both peptide sequence ions (higher mass region, 150,2200,m/z) and reporter ions (low mass region, 113,121,m/z) for protein identification and relative quantification, respectively. After collision-induced dissociation, the key requirements to achieve a good balance between the high and low m/z ions are effective ion transmission and detection across the MS/MS mass range, since the ion transmission of the higher m/z range competes with that of the low m/z range. This study describes an analytical strategy for the implementation of iTRAQ on maXis UHR-Qq-ToF instruments, and discusses the impact of adjusting the MS/MS ion transmission parameters on the quality of the overall data sets. A technical discussion highlights a number of maXis-specific parameters, their impact of quantification and identification, and their cross-interactions. [source]

Optimal pressure conditions for unbiased external ion accumulation in a two-dimensional radio-frequency quadrupole for Fourier transform ion cyclotron resonance mass spectrometry

RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 21 2001
Mikhail E. Belov
When combined with on-line separations (e.g., capillary liquid chromatography (LC)), Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) provides a powerful tool for biological applications, and particularly proteomic studies. The sensitivity, dynamic range, and duty cycle provided by FTICR-MS have been shown to be increased by ion trapping and accumulation in a two-dimensional (2D) radio-frequency (rf)-only multipole positioned externally to an FTICR cell. However, it is important that ions be detected across the desired m/z range without a significant bias. In this work we found that pressure inside the accumulation rf-quadrupole plays an important role in obtaining ,unbiased' ion accumulation. Pressure optimization was performed in both pulsed and continuous modes. It was found that unbiased accumulation in a 2D rf-only quadrupole could be achieved in the pressure range of 5,×,10,4 to 5,×,10,3 Torr. External ion accumulation performed at the optimal pressure resulted in an increase in both the spectrum acquisition rates and dynamic range. Copyright © 2001 John Wiley & Sons, Ltd. [source]