Immobilized pH gradient-driven paper-based IEF: a new method for fractionating complex peptide mixtures before MS analysis

Clinical Proteomics

KH Chandramouli³  Beerelli Seshi¹  Kumaraguru Raja² 

[1] Department of Pathology and Laboratory Medicine, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, USADepartment of Pathology and Laboratory Medicine, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, USADepartment of Pathology and Laboratory Medicine, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, USA;Department of Pathology and Laboratory Medicine, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, USAGetzville, USADepartment of Pathology and Laboratory Medicine, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, USADepartment of Pathology and Laboratory Medicine, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, USAGetzville, USAGetzville, USADepartment of Pathology and Laboratory Medicine, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, USAGetzville, USA;Department of Pathology and Laboratory Medicine, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, USASection of Marine Ecology and Biotechnology, Division of Life Science, The Hong Kong University of Science and Technology, Kowloon, Hong Kong SARDepartment of Pathology and Laboratory Medicine, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, USADepartment of Pathology and Laboratory Medicine, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, USASection of Marine Ecology and Biotechnology, Division of Life Science, The Hong Kong University of Science and Technology, Kowloon, Hong Kong SARSection of Marine Ecology and Biotechnology, Division of Life Science, The Hong Kong University of Science and Technology, Kowloon, Hong Kong SARDepartment of Pathology and Laboratory Medicine, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, USASection of Marine Ecology and Biotechnology, Division of Life Science, The Hong Kong University of Science and Technology, Kowloon, Hong Kong SAR

关键词: Mass spectrometry;    iTRAQ;    Offgel electrophoresis;    Paper IEF;    Progenitor cells;    Clinical proteomics;   

DOI  :  10.1186/1559-0275-8-10

来源: Humana Press Inc

【 摘 要 】

Abstract

Introduction

The vast difference in the abundance of different proteins in biological samples limits the determination of the complete proteome of a cell type, requiring fractionation of proteins and peptides before MS analysis.

Methods

We present a method consisting of electrophoresis of complex mixtures of peptides using a strip of filter paper cut into 20 sections laid end to end over a 24-cm-long IPG strip, the pH gradient of which would drive the electrophoresis. Peptides absorbed onto individual paper pads after electrophoresis are subsequently recovered into a buffer solution, thus dividing a complex peptide mixture according to pI into 20 liquid fractions. This paper-based IEF method (PIEF) was compared side-by-side with a similar but liquid-based Offgel electrophoresis (OGE) by analyzing iTRAQ-labeled peptide mixtures of membrane proteins from four different cell types.

Results

PIEF outperformed OGE in resolving acidic peptides, whereas OGE did a better job in recovering relatively basic peptides. OGE and PIEF were quite comparable in their coverage, identifying almost equal number of distinct proteins (PIEF =1174; OGE = 1080). Interestingly, however, only 675 were identified by both of them, each method identifying many unique proteins (PIEF = 499; OGE = 415). Thus, the two methods uncovered almost 40% more proteins compared to what is obtained by only one method. Conclusion: This initial investigation demonstrates the technical feasibility of PIEF for complementing OGE. PIEF uses standard IPG IEF equipment, requires no specialized apparatus (e.g., OGE fractionator) and may be integrated into peptide mapping strategies for clinical samples.

【 授权许可】

Unknown