Protein abundance quantification in embryonic stem cells using incomplete metabolic labelling with 15N amino acids, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry, and analysis of relative isotopologue abundances of peptides.

An isotope dilution method for protein quantification is presented in the context of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) and mass fingerprinting experiments, revealing an unappreciated high reproducibility and accuracy of relative peak intensity measurements. Labelled proteins were generated by growing cells in a medium containing (15)N-enriched amino acids, and were mixed with proteins of natural isotopic composition from control cells in ratios of approximately 0:1, 1:7, 1:2, 2:1, 7:1, and 1:0 (labelled/unlabelled). Mixtures were separated by two-dimensional gel electrophoresis and analysed by MALDI-TOFMS using typical experimental conditions. A linear relationship is demonstrated between the relative isotopologue abundances (RIA values) for particular peaks in the isotopic distribution of tryptic peptide fragments of the proteins, and the mole fractions of labelled proteins in the mixture. Analysis of RIA values (ARIA quantification) for peptides of six typical silver-stained protein spots for the various mixtures could reproduce the experimentally contrived ratios with approximate errors between 4% (2:1 mixture) and about 18% (1:7 mixture). A consideration of error and its propagation is discussed. ARIA does not require complete separation of the isotope patterns of labelled and unlabelled peptides, and is therefore advantageous in combination with all kinds of labelling experiments in biological systems, because it is compatible with minimal metabolic incorporation of labelling reagent. Simulations indicate that the minimum required (15)N enrichment of the total amino acid pool sufficient for ARIA is less than 4%. In an accompanying paper in this issue, we apply ARIA to proteins differentially labelled with isotope-coded alkylation reagents.

Analysis of relative isotopologue abundances for quantitative profiling of complex protein mixtures labelled with the acrylamide/D3-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 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 microg of total liver protein. The precision and limitations of ARIA quantification of peptides differentially alkylated with isotopomeric reagents are discussed.

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'-D3 ]-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.