Improved method for differential expression proteomics using trypsin-catalyzed 18O labeling with a correction for labeling efficiency.

Mol Cell Proteomics

Protein Chemistry and Proteomics Laboratory, Centro de Biología Molecular Severo Ochoa-Consejo Superior de Investigaciones Científicas, 28049 Cantoblanco, Madrid, Spain.

Published: July 2007

Quantitative strategies relying on stable isotope labeling and isotope dilution mass spectrometry have proven to be a very robust alternative to the well established gel-based techniques for the study of the dynamic proteome. Postdigestion 18O labeling is becoming very popular mainly due to the simplicity of the enzyme-catalyzed exchange reaction, the peptide handling and storage procedures, and the flexibility and versatility introduced by decoupling protein digestion from peptide labeling. Despite recent progresses, peptide quantification by postdigestion 18O labeling still involves several computational problems. In this work we analyzed the behavior of large collections of peptides when they were subjected to postdigestion labeling and concluded that this process can be explained by a universal kinetic model. On the basis of this observation, we developed an advanced quantification algorithm for this kind of labeling. Our method fits the entire isotopic envelope to parameters related with the kinetic exchange model, allowing at the same time an accurate calculation of the relative proportion of peptides in the original samples and of the specific labeling efficiency of each one of the peptides. We demonstrated that the new method eliminates artifacts produced by incomplete oxygen exchange in subsets of peptides that have a relatively low labeling efficiency and that may be considered indicative of false protein ratio deviations. Finally using a rigorous statistical analysis based on the calculation of error rates associated with false expression changes, we showed the validity of the method in the practice by detecting significant expression changes, produced by the activation of a model preparation of T cells, with only 5 microg of protein in three proteins among a pool of more than 100. By allowing a full control over potential artifacts, our method may improve automation of the procedures for relative protein quantification using this labeling strategy.

Download full-text PDF

Source
http://dx.doi.org/10.1074/mcp.T600029-MCP200DOI Listing

Publication Analysis

Top Keywords

18o labeling
12
labeling efficiency
12
labeling
11
postdigestion 18o
8
expression changes
8
improved method
4
method differential
4
differential expression
4
expression proteomics
4
proteomics trypsin-catalyzed
4

Similar Publications

Protein-based stable isotope probing (protein-SIP) can link microbial taxa to substrate assimilation. Traditionally, protein-SIP requires a sample-specific metagenome-derived database for samples with unknown composition. Here, we describe GroEL-prototyping-based stable isotope probing (GroEL-SIP), that uses GroEL as a taxonomic marker protein to identify bacterial taxa (GroEL-proteotyping) coupled to SIP directly linking identified taxa to substrate consumption.

View Article and Find Full Text PDF

The algal macrolide goniodomin A (GDA) undergoes ring-cleavage under unusually mild, alkaline conditions to form mixtures of stereoisomers of seco acids GDA-sa and iso-GDA-sa. In the primary fragmentation pathway, opening of the macrolide ring occurs by displacement of the carboxyl group by a base-catalyzed attack of the C32 hemiketal hydroxy group on C31, yielding an oxirane-carboxylic acid, named goniodomic acid. The oxirane ring is unstable, undergoing solvolytic opening to form mainly GDA-sa.

View Article and Find Full Text PDF

Wastewater ozonation is commonly employed to enhance the subsequent biodegradation of effluent organic matter (EfOM) and contaminants of concern. However, there is evidence suggesting the formation of recalcitrant ozonation products (OPs) from EfOM. To investigate the biodegradability of OPs we conducted batch biodegradation experiments using wastewater effluent ozonated with mass-labeled (O) ozone.

View Article and Find Full Text PDF
Article Synopsis
  • Protein crosslinks caused by oxidative stress are linked to diseases like atherosclerosis, Alzheimer's, and Parkinson's, but their specific nature and locations in proteins remain unclear.
  • A new method utilizing "light" and "heavy" isotope-labeled reagents for efficient amine labeling of crosslinked peptides has shown improved identification and quantification over previous techniques.
  • This approach has led to the successful identification of novel crosslinks in proteins like β-casein and α-synuclein, as well as effective mapping of disulfide bonds in serum albumin, highlighting its versatility for studying protein modifications.
View Article and Find Full Text PDF

Activating the lattice oxygen can significantly improve the kinetics of oxygen evolution reaction (OER), however, it often results in reduced stability due to the bulk structure degradation. Here, we develop a spinel FeCoCrO with active lattice oxygen by high-throughput methods, achieving high OER activity and stability, superior to the benchmark IrO. The oxide exhibits an ultralow overpotential (190 mV at 10 mA cm) with outstanding stability for over 170 h at 100 mA cm.

View Article and Find Full Text PDF

Want AI Summaries of new PubMed Abstracts delivered to your In-box?

Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!