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.
Cahill
Michael A
MA
ProteoSys AG, Carl-Zeiss-Str. 51, 55129 Mainz, Germany.
Wozny
Wojciech
W
ProteoSys AG, Carl-Zeiss-Str. 51, 55129 Mainz, Germany.
Schwall
Gerhard
G
ProteoSys AG, Carl-Zeiss-Str. 51, 55129 Mainz, Germany.
Schroer
Klaus
K
ProteoSys AG, Carl-Zeiss-Str. 51, 55129 Mainz, Germany.
Hölzer
Kerstin
K
ProteoSys AG, Carl-Zeiss-Str. 51, 55129 Mainz, Germany.
Poznanovic
Slobodan
S
ProteoSys AG, Carl-Zeiss-Str. 51, 55129 Mainz, Germany.
Hunzinger
Christian
C
ProteoSys AG, Carl-Zeiss-Str. 51, 55129 Mainz, Germany.
Vogt
Josef A
JA
University Hospital Ulm, Department of Anaesthesia, Parkstrasse 11, 89073 Ulm, Germany.
Stegmann
Werner
W
ProteoSys AG, Carl-Zeiss-Str. 51, 55129 Mainz, Germany.
Matthies
Helmut
H
ProteoSys AG, Carl-Zeiss-Str. 51, 55129 Mainz, Germany.
Schrattenholz
André
A
ProteoSys AG, Carl-Zeiss-Str. 51, 55129 Mainz, Germany.
D020106:Acrylamide / Q000737:chemistry*; D000478:Alkylation; D000595:Amino Acid Sequence; D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional; D007460:Iodoacetamide; D007553:Isotope Labeling; D007554:Isotopes / Q000032:analysis; D008099:Liver / Q000737:chemistry; D008969:Molecular Sequence Data; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D013552:Swine
D007554:Isotopes; D011506:Proteins; D020106:Acrylamide; D007460:Iodoacetamide