Multidimensional liquid phase protein separations in conjunction with stable isotope labelling for quantitative proteomics

Abstract

A method for quantitative protein profiling has been developed utilising multidimensional liquid phase protein separations in conjunction with stable isotope labelling. This approach combines the advantages of high throughput, automated, reproducible protein separations with accurate protein quantitation performed in the mass spectrometer. Escherichia coli cells were grown in the presence and absence of the DNA methylation inhibitor 5-Azacytidine on 14 N and 15 N enriched media. Protein separations were performed using ion exchange chromatography in the first dimension and RP capillary chromatography in the second dimension. UV absorbance measurements were used for the initial semiquantitative identification of differentially expressed proteins. Selected peaks from the mixed 15 N/ 14 N lysates were used for the accurate quantitation performed in the mass spectrometer using the ratios of the stable isotopes. Using this approach, a number of differentially expressed proteins have been identified. Moreover, this approach overcomes a number of caveats associated with multidimensional liquid phase protein separations, including the presence of multiple proteins present in a single chromatographic peak.

Identification and Characterization of Sulfolobus solfataricus P2 Proteome Using Multidimensional Liquid Phase Protein Separations

“…The overall resolution of protein separations using 2DLC from S. solfataricus protein lysates was lower than that observed previously for a soluble protein lysate generated from Escherichia coli . A number of broad peaks were observed in the chromatogram possibly due to the nature of the thermophilic proteins from S.…”

“…The presence of a number of proteins present in a single peak will generate problems using such approaches for quantitative work. However, the use of stable isotope labeling in conjunction with 2DLC protein separations can overcome such problems . In addition, the identification of low-abundance proteins may be hampered if co-eluting with high-abundance proteins during the chromatography.…”

“…We have previously demonstrated the high resolution separation of complex mixtures of proteins with short analysis times (20 min gradients) under such conditions. 31 In addition, the capillary chromatography enables sensitive UV absorbance detection of the proteins in the second dimension. Proteins in the low nanogram range can be detected using this approach.…”

Multi-dimensional liquid chromatography in proteomics—A review

“…The ratio of peak intensities for such peptide pairs accurately reflects the abundance ratio for the proteins from which these peptides are derived. 18 O- or 15 N-based metabolic labeling techniques have been used in many quantitative proteomics projects [128-131]. It should be recognized however, that stable isotope incorporation rates in metabolic labeling experiments do not reach 100% [129], thereby confounding analyses.…”

Proteomic Technologies for the Discovery of Type 1 Diabetes Biomarkers

“…The first strategy involves processing the protein samples on a traditional 2D gel system or higher-dimensional intact protein separation systems that use multiple in-solution protein separation techniques, such as isoelectric focusing (IEF) coupled with liquid chromatography and followed by SDS-PAGE. [24][25][26] Mass spectrometry is then used in offline mode to analyze the much simplified peptide mixture digested from protein fractions containing a single or limited number of proteins after separation. This approach of multidimensional separation has a better resolution but is more labor intensive compared to 2D electrophoresis.…”