Femtomol sensitivity post-digest 18O labeling for relative quantification of differential protein complex composition

Marcus Bantscheff, Birgit Dümpelfeld, Bernhard Kuster

Research output: Contribution to journalArticlepeer-review

55 Scopus citations

Abstract

Stable isotope labeling (SIL) has emerged as a powerful tool to measure the relative quantitative differences between samples in many differential display-type proteomic applications. However, current SIL procedures tend to suffer from the fact that one needs to decide very early in a biochemical strategy whether or not a sample will be subjected to relative quantification. Typically, the entire strategy has to be adapted to the needs of the particular quantification method chosen which might limit the range of biochemical experiments amenable to quantification. Metabolic labeling approaches, albeit very sensitive, can only be applied to studies using appropriate cell culture systems which might not necessarily be compatible with the biological system under investigation. Chemical labeling of complex protein mixtures by, e.g., isotope-coded affinity tags (ICAT), can offer great simplification of protein mixtures but is restricted by the accessibility of the often few suitable peptides (i.e. cysteine containing peptides) for both protein identification and quantification. Here, we describe a post-digest 18O-labeling method that can circumvent some of the above limitations by separating protein identification from quantification. An aliquot of all samples in a set can be used for rapid protein ID using, e.g., matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). In a second step, relative quantification is performed using trypsin-catalyzed 18O incorporation into all tryptic peptides. This two-stage procedure introduces significant experimental flexibility because it enables postponement of the decision about which pairs of samples from a given set of experiments are to be compared until after the protein ID stage. In-gel digested protein quantities between 50 fmol and 15 pmol are amenable to this new method, with a dynamic range of 1:10 within one sample. Accuracy for measured relative abundances is similar to those reported for other SIL strategies (errors typically <20%), and the method is applicable to protein samples from all kinds of tissue or cell culture. This paper presents quantification data for a set of standard proteins, as well as a study of differential complex formation around the NFκB transcription factor p65 following stimulation with TNF-α.

Original languageEnglish
Pages (from-to)869-876
Number of pages8
JournalRapid Communications in Mass Spectrometry
Volume18
Issue number8
DOIs
StatePublished - 2004
Externally publishedYes

Fingerprint

Dive into the research topics of 'Femtomol sensitivity post-digest 18O labeling for relative quantification of differential protein complex composition'. Together they form a unique fingerprint.

Cite this