Differential expression analysis of Escherichia coli proteins using a novel software for relative quantitation of LC-MS/MS data.
The study of changes in protein levels between samples derived from cells representing different biological conditions is a key to the understanding of cellular function. There are two main methods available that allow both for global scanning for significantly varying proteins and targeted profiling of proteins of interest. One method is based on 2-D gel electrophoresis and image analysis of labelled proteins. The other method is based on LC-MS/MS analysis of either unlabelled peptides or peptides derived from isotopically labelled proteins or peptides. In this study, the non-labelling approach was used involving a new software, DeCyder MS Differential Analysis Software (DeCyder MS) intended for automated detection and relative quantitation of unlabelled peptides in LC-MS/MS data. Total protein extracts of E. coli strains expressing varying levels of dihydrofolate reductase and integron integrase were digested with trypsin and analyzed using a nanoscale liquid chromatography system, Ettan MDLC, online connected to an LTQTM linear ion-trap mass spectrometer fitted with a nanospray interface. Acquired MS data were subjected to DeCyder MS analysis where 2-D representations of the peptide patterns from individual LC-MS/MS analyses were matched and compared. This approach to unlabelled quantitative analysis of the E. coli proteome resulted in relative protein abundances that were in good agreement with results obtained from traditional methods for measuring protein levels.
Johansson
Carolina
C
Uppsala University, Department of Medical Biochemistry and Microbiology (IMBlm), and GE Healthcare, Biosciences AB, Sweden.
Samskog
Jenny
J
Sundström
Lars
L
Wadensten
Henrik
H
Björkesten
Lennart
L
Flensburg
John
J
D000595:Amino Acid Sequence; D015180:Electrophoresis, Gel, Two-Dimensional; D029968:Escherichia coli Proteins / Q000032:analysis*; D019426:Integrases / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D040901:Proteomics; D012984:Software; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism
D029968:Escherichia coli Proteins; D013762:Tetrahydrofolate Dehydrogenase; D019426:Integrases