title: Comparison of fractionation strategies for offline two-dimensional liquid chromatography tandem mass spectrometry analysis of proteins from mouse adipose tissue.
abstract: In the frame of protein identification from mouse adipose tissue, two strategies were compared for the offline elution of peptides from a strong cation exchange (SCX) column in two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS) analyses. First, the salt gradient (using K(+) as displacing agent) was evaluated from 25 to 500mM KCl. Then, a less investigated elution mode using a pH gradient (using citric acid and ammonium hydroxide) was carried out from pH 2.5 to 9.0. Equal amounts of peptide digest derived from mouse adipose tissue were loaded onto the SCX column and fractionated according to the two approaches. A total of 15 fractions were collected in two independent experiments for each SCX elution strategy. Then, each fraction was analyzed on a nanoLC-MS/MS platform equipped with a column-switching unit for desalting and enrichment. No substantial differences in peptide quality characteristics (molecular weight, isoelectric point, or GRAVY [grand average of hydropathicity] index distributions) were observed between the two datasets. The pH gradient approach was found to be superior, with 27.5% more unique peptide identifications and 10% more distinct protein identifications compared with the salt-based elution method. In conclusion, our data imply that the pH gradient SCX fractionation is more desirable for proteomics analysis of entire adipose tissue.
authors.affiliation: Life Sciences Mass Spectrometry, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, 1211 Geneva 4, Switzerland; Department of Cellular Physiology and Metabolism, University of Geneva, 1211 Geneva 4, Switzerland.
authors.forename: Tatjana
authors.initials: T
authors.lastname: Sajic
authors.affiliation: Life Sciences Mass Spectrometry, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, 1211 Geneva 4, Switzerland. Electronic address: emmanuel.varesio@unige.ch.
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Varesio
authors.affiliation: Department of Cellular Physiology and Metabolism, University of Geneva, 1211 Geneva 4, Switzerland; Department of Internal Medicine Specialties, Geneva University Hospitals, 1205 Geneva, Switzerland.
authors.forename: Ildiko
authors.initials: I
authors.lastname: Szanto
authors.affiliation: Life Sciences Mass Spectrometry, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, 1211 Geneva 4, Switzerland.
authors.forename: Gérard
authors.initials: G
authors.lastname: Hopfgartner
mesh_terms: D000273:Adipose Tissue / Q000378:metabolism*; D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D008297:Male; D051379:Mice; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
keywords: Adipose tissue; Offline 2D–LC–MS/MS; Peptides; SCX fractionation; Salt gradient SCX; pH gradient SCX