Analysis of relative isotopologue abundances for quantitative profiling of complex protein mixtures labelled with the acrylamide/D₃‐acrylamide alkylation tag system

Abstract

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.

Metabolic Labeling of Proteins for Proteomics

“…Such approaches include ICAT and its modifications (4 -7), acrylamide labeling (8), 18 O-labeling during proteolysis (9,10), and guanidination of lysine residues (11)(12)(13). In all of these approaches, the degree of labeling is controlled by the isotope enrichment of the labeling reagent and the completeness of the modification chemistry and is applied to proteins or protein mixtures obtained after cell disruption.…”

What does it need to be a biomarker? Relationships between resolution, differential quantification and statistical validation of protein surrogate biomarkers

“…The experimental techniques used for protein separation, quantitative differential analysis of protein spots, and protein identification have been published elsewhere [12][13][14][16][17][18]. In brief, high-resolution IEF-SDS-PAGE was performed by 54-cm daisy chain serial IPG-IEF.…”

Protein Expression Profiling in Esophageal Adenocarcinoma Patients Indicates Association of Heat-Shock Protein 27 Expression and Chemotherapy Response

“…For all gels, the first dimension focusing was done over an effective separation distance of 18 cm. Each first dimension focusing was afterwards loaded onto a second dimension SDS-PAGE gel, and radioactive proteins were imaged by ProteoTope as described (16,22,23). Gels loaded with I-125 and I-131 labeled proteins were dried after electrophoresis, laminated in 0.07 mm plastic, and exposed to routine ProteoTope imaging for each isotope for between 20 and 24 h per exposition.…”

“…Protein identification by MS. For MS-based identification of proteins, preparative tracer-control enrichment gels (tracer gels) were used, in which a trace of radioactively labeled protein sample corresponding to the sample used for analytic two-color gels was coelectrophoresed with a vast excess of nonradioactively labeled protein from the pooled reference sample (f200 Ag) to provide preparative amounts of protein for identification (22,24). After electrophoresis gels are silver stained according to Shevchenko et al (25).…”

“…The resulting solution is analyzed first with a high throughput peptide mass fingerprint procedure based on MALDI-TOF-MS. Mass spectra were internally mass calibrated with the use of trypsin autodigestion peptide signals as reference values. Mass measurement accuracies were typically 50 parts per million (22,24). For the identification of the proteins, the peptide masses extracted from the mass spectra were searched against the National Center for Biotechnology Information nonredundant protein database 11 with the use of the MASCOT software version 1.9 (Matrix Science).…”