Solution Isoelectric Focusing for Peptide Analysis: Comparative
Investigation of an Insoluble Nuclear Protein Fraction
Yanming An
Zongming Fu
Peter Gutierrez
Catherine Fenselau
10.1021/pr050221+.s001
https://acs.figshare.com/articles/journal_contribution/Solution_Isoelectric_Focusing_for_Peptide_Analysis_Comparative_Investigation_of_an_Insoluble_Nuclear_Protein_Fraction/3372991
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 <sup>18</sup>O 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 <sup>18</sup>O 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 • <sup>18</sup>O labeling • mass spectrometry • drug resistance
• nuclear proteins • reversed-phase liquid chromatography • MCF-7 cancer cells • heterogeneous nuclear
ribonucleoproteins
2005-12-12 00:00:00
Insoluble Nuclear Protein Fraction
separation method
dimension
ribonucleoprotein
protein
parent cell line
MCF
sIEF
peptide
chromatography
solution isoelectric
abundance
18 O labels
HPLC
LC
analysis
spectrometry