Use of two-dimensional liquid fractionation for separation of proteins from cell lysates without the presence of methionine oxidation

Use of two-dimensional liquid fractionation for separation of proteins from cell lysates without the presence of methionine oxidation

A novel two-dimensional (2D) chromatographic method is developed to separate proteins from malignant breast cancer whole cell lysates. Protein mixtures are first separated according to their pIby chromatofocusing followed by an orthogonal non-porous reversed-phase separation. An important advantage...

Full description

Saved in:
Bibliographic Details
Published in:Journal of Chromatography A Vol. 1053; no. 1-2; pp. 133 - 142
Main Authors: Zhua, Kan, Kachman, Maureen T, Miller, Fred R, Lubman, David M, Zand, Robert
Format: Journal Article
Language:English
Published: Netherlands 22-10-2004
Subjects:
Acetylation
Amino Acid Sequence
Chromatography, Liquid - methods
Methionine - chemistry
Molecular Sequence Data
Oxidation-Reduction
Proteins - isolation & purification
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:A novel two-dimensional (2D) chromatographic method is developed to separate proteins from malignant breast cancer whole cell lysates. Protein mixtures are first separated according to their pIby chromatofocusing followed by an orthogonal non-porous reversed-phase separation. An important advantage of this 2D chromatographic method is that, unlike gel-based methods, it does not result in methionine oxidation. The lack of methionine oxidation during separation is demonstrated by the analysis of protein tryptic digests using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS. Our novel 2D chromatographic method used in combination with on-target light-induced methionine oxidation provides a means for studying methionine-containing peptides. Methionine residues in peptide sequences are partially oxidized with light exposure. Neither the location nor the modification of methionine in the peptide sequence affects the oxidation. As a result, multiple peaks are observed in MALDI-TOF-MS spectra after light exposure. Sequence information derived from light-induced methionine can be applied to enhance the database search results obtained through peptide mass fingerprinting.
Bibliography:ObjectType-Article-1
SourceType-Scholarly Journals-1
ObjectType-Feature-2
content type line 23
ISSN:0021-9673
DOI:10.1016/S0021-9673(04)01435-9