Medical College of Wisconsin
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A comparison of 2-D chromatography separations using UV and (18)O quantification of proteins in similar proteomes. J Sep Sci 2008 Feb;31(2):314-20 PMID: 18246581

Abstract

Proteins present in two mitochondria preparations were separated by 2-D chromatography using the ProteomeLab PF-2D Protein Fractional System, protein fractionation in two dimensions (PF-2D). The proteins in each first-dimension fraction were determined by trypsinization and LC-MS/MS. Chromatography peaks were quantified by UV detection using the "Mapping Tools" software (Beckman). The proteins present in UV detected peaks were trypsinized and identified by automated MS/MS sequencing. Relative amounts of the proteins present in the equivalent peak for each sample were assessed by comparison of the intensities of the constituent peptides and a predicted PF-2D value was calculated from the total ion count (TIC) for each peptide. Relative quantification for (18)O labeled peptides was performed using the ZoomQuant (v1.43b) software [1, 2]. We found that the chromatography peaks detected by UV generally contained several proteins. Using (18)O labeling we determined that in each peak the ratios of the constituent proteins were different. When these ratios were normalized using the TIC to account for abundance, the resulting ratio corresponded to that determined by UV. The predicted value for the PF-2D score corresponded to the observed value for each peak irrespective of the number or proteins detected.

Author List

Smith JR, Matus I, Greene AS

Author

Andrew S. Greene PhD Interim Vice Chair, Chief, Professor in the Biomedical Engineering department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Chromatography, Liquid
Mass Spectrometry
Oxygen Isotopes
Proteins
Proteome
Spectrophotometry, Ultraviolet



View this publication's entry at the Pubmed website PMID: 18246581
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