Name: Addressing accuracy and precision issues in iTRAQ quantitation
Keywords: Not available

Dataset Information

Addressing accuracy and precision issues in iTRAQ quantitation

ABSTRACT: Not available

INSTRUMENT(S): QSTAR XL, instrument model, QSTAR

ORGANISM(S): Erwinia Carotovora

SUBMITTER: Phil Charles

PROVIDER: PRD000105 | Pride | 2010-11-10

REPOSITORIES: Pride

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Project description:The use of internal calibrants (the so called lock mass approach) provides much greater accuracy in mass spectrometry based proteomics. However, the polydimethylcyclosiloxane (PCM) peaks commonly used for this purpose are quite unreliable, leading to missing calibrant peaks in spectra and correspondingly lower mass measurement accuracy. Therefore, we here introduce a universally applicable and robust internal calibrant, the tripeptide Asn3. We show that Asn3 is a substantial improvement over PCM both in terms of consistent detection and resulting mass measurement accuracy. Asn3 is also very easy to adopt in the lab, as it requires only minor adjustments to the analytical setup. Data analysis: For mass measurement accuracy (MMA) calculations and comparisons, the following Mascot workflow was used. From the MS/MS data in each LC run, Mascot Generic Files were created using Distiller software (version 2.4.3.3, Matrix Science, London, UK, www.matrixscience.com/distiller.html). These peak lists were then searched with the Mascot search engine (Matrix Science) using the Mascot Daemon interface (version 2.4.0, Matrix Science). Spectra were searched against the Swiss-Prot database (version 13_04 of UniProtKB/Swiss-Prot protein database containing 20,232 sequence entries of human proteins) concatenated with its reversed sequence database. Variable modifications were set to pyro-glutamate formation of amino terminal glutamine and acetylation of the protein N-terminus, whereas fixed modifications only included oxidation of methionine. Mass tolerance on peptide ions was set to 10 ppm (with Mascot’s C13 option set to 1), and the mass tolerance on peptide fragment ions was set to 20 millimass units (mmu), except for the space-charge effect experiment(LMA5) where an extra search was done with a setting of 3 mmu. The peptide charge was set to 1+,2+,3+ and instrument setting was put on ESI-QUAD. Enzyme was set to trypsin allowing for one missed cleavage, and cleavage was allowed when arginine or lysine is followed by proline. Only peptides that were ranked one and scored above the threshold score, set at 99% confidence, were withheld. All data was processed and managed by ms_lims.

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Addressing accuracy and precision issues in iTRAQ quantitation

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