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

Abstract

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.

Stem cell proteomes: A profile of human mesenchymal stem cells derived from umbilical cord blood

“…For PMF, protein spots were automatically located using the HTAnalyzer software (Genomic Solutions, Ann Arbor, MI, USA), excised using an automated spot-picker Flexys (Genomic Solutions), and destained as described in [36]. In-gel digestion with trypsin (Promega, Madison, WI, USA) was employed using a modified protocol as described in [37]. Samples were loaded onto prespotted AnchorChip targets which are stainless steel supports coated with hydrophobic material equipped with an array of 384 circular interruptions (anchors) of 600 mm diameter (Bruker-Daltonics, Bremen, Germany).…”

Current chemical tagging strategies for proteome analysis by mass spectrometry

“…Isotope coding can already be performed during cell culture, either by growth on isotope-labeled media [29][30][31][32][33][34] or by supplementing growth media with labeled amino acids [35][36][37][38][39][40][41][42][43]. This strategy has the advantage that all further sample preparation steps can be performed after combining the differentially labeled samples, thereby minimizing effects of parallel sample processing.…”

An Introduction to Proteomics Technologies for the Genomics Scientist

“…Stable isotopes have been used in gel-based proteomics, whereby different proteomes have been separately labeled with different stable isotopes (e.g. growing cells using 14 N-versus 15 N-labeled medium) prior to mixing and running together through the same 2DE separation [15]. In this case, abundance changes are monitored during the MS stage, which must be performed on each resolved protein, and are typically limited to comparisons made within a single gel.…”