Analysis of relative isotopologue abundances for quantitative profiling of complex protein mixtures labelled with the acrylamide/D 3 ‐acrylamide alkylation tag system

The new method of analysis of relative isotopologue abundances (ARIA) applied here is based on the evaluation of total isotope patterns of tryptic protein fragments measured by matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry (MALDI‐TOFMS) to calculate the mixing ratios o...

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Veröffentlicht in:Rapid communications in mass spectrometry 2003-06, Vol.17 (12), p.1283-1290
Hauptverfasser: Cahill, Michael A., Wozny, Wojciech, Schwall, Gerhard, Schroer, Klaus, Hölzer, Kerstin, Poznanovic, Slobodan, Hunzinger, Christian, Vogt, Josef A., Stegmann, Werner, Matthies, Helmut, Schrattenholz, André
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Sprache:eng
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Zusammenfassung:The new method of analysis of relative isotopologue abundances (ARIA) applied here is based on the evaluation of total isotope patterns of tryptic protein fragments measured by matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry (MALDI‐TOFMS) to calculate the mixing ratios of composites consisting of stable isotope labelled and isotopically natural (unlabelled) proteins, as described in an accompanying paper in this issue. Recently, Sechi (Rapid Commun. Mass Spectrom. 2002; 16: 1416–1424) and Gehanne et al. (Rapid Commun. Mass Spectrom. 2002; 16: 1692–1698) introduced the use of differential quantitative mass analysis by MALDI‐TOFMS using mixtures of standard proteins alkylated prior to two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) with either acrylamide (AA) or deuterium‐labelled [2,3,3′‐D 3 ]‐acrylamide (D3AA). In the present study we validate the AA/D3AA system, firstly by measuring the yield of proteins alkylated with AA, and secondly by using differential radioactive labels ( 125 I and 131 I) to quantitatively establish that non‐comigration in 2D‐PAGE is negligible. ARIA is then applied to quantitatively estimate the relative proportions of peptides labelled with AA or D3AA in the validated system, using typical silver‐stained 2D‐PAGE protein spots from 2D gels loaded with 150 μg of total liver protein. The precision and limitations of ARIA quantification of peptides differentially alkylated with isotopomeric reagents are discussed. Copyright © 2003 John Wiley & Sons, Ltd.
ISSN:0951-4198
1097-0231
DOI:10.1002/rcm.1046