%0 Journal Article
%A An, Yanming
%A Fu, Zongming
%A Gutierrez, Peter
%A Fenselau, Catherine
%D 2005
%T Solution Isoelectric Focusing for Peptide Analysis: Comparative
Investigation of an Insoluble Nuclear Protein Fraction
%U https://acs.figshare.com/articles/journal_contribution/Solution_Isoelectric_Focusing_for_Peptide_Analysis_Comparative_Investigation_of_an_Insoluble_Nuclear_Protein_Fraction/3372991
%R 10.1021/pr050221+.s001
%2 https://acs.figshare.com/ndownloader/files/5216455
%K Insoluble Nuclear Protein Fraction
%K separation method
%K dimension
%K ribonucleoprotein
%K protein
%K parent cell line
%K MCF
%K sIEF
%K peptide
%K chromatography
%K solution isoelectric
%K abundance
%K 18 O labels
%K HPLC
%K LC
%K analysis
%K spectrometry
%X In this study, a solution isoelectric focusing apparatus was modified and built into a two-dimensional
separation method for peptides. Newly commercialized isoelectric membranes, which carry immobilized
ampholytes, were integrated to establish the pH boundaries in this apparatus. High-performance liquid
chromatography was employed as the second dimension, interfaced with mass spectrometry. An
insoluble nuclear protein fraction was used to evaluate and optimize this method. This two-dimensional
separation method dramatically improves peptide detection and identification compared with a single
dimension LC−MS analysis. Off-line reversed-phase HPLC was used to ascertain reproducibility. The
two-dimensional separation method was combined with 18O labeling for comparative analysis of protein
expression in two cell lines. Separation of peptides by solution isoelectric focusing (sIEF) offers the
advantage that it can be accomplished after the 18O labels are introduced. The labeled peptides can be
mixed with unlabeled ones before fractionation by sIEF. The relative abundances of nuclear proteins
from a drug resistant MCF-7 cancer cell line were compared to those from the drug susceptible parent
cell line using this combined strategy. The abundances of several heterogeneous nuclear ribonucleoproteins were found to be increased in the mitoxantrone-resistant line.
Keywords: solution isoelectric focusing • shotgun proteomics • 18O labeling • mass spectrometry • drug resistance
• nuclear proteins • reversed-phase liquid chromatography • MCF-7 cancer cells • heterogeneous nuclear
ribonucleoproteins
%I ACS Publications