Three-Dimensional Peptide Fractionation for Highly Sensitive Nanoscale LC-Based Shotgun Proteomic Analysis of Complex Protein Mixtures

Sricharan Bandhakavi, Todd W. Markowski, Hongwei Xie, Timothy J. Griffin

Research output: Chapter in Book/Report/Conference proceedingChapter

4 Scopus citations


To sensitively analyze complex protein mixtures by mass spectrometry-based shotgun proteomics, researchers have employed platforms that couple orthogonal peptide fractionation methods using nanoscale HPLC. Commonly used platforms have coupled either strong cation exchange (SCX) HPLC or preparative isoelectric focusing (IEF) with nanoscale reversed-phase (nanoRP) HPLC fractionation of peptides. Coupling two dimensions of peptide fractionation, prior to mass spectrometric analysis, increases the sensitivity for identifying low abundance proteins. However, the large dynamic range of protein abundance and high level of complexity of protein mixtures derived from many biological sources, such as bodily fluids, require additional steps of peptide fractionation. To address this shortcoming, we have developed a platform combining three dimensions of peptide fractionation as follows: (1) preparative IEF; (2) SCX HPLC; and (3) nanoRP HPLC. This platform significantly increases the sensitivity of shotgun proteomic analysis in complex protein mixtures. Here, we describe the implementation of this three-dimensional peptide fractionation platform for proteomic studies of complex mixtures.

Original languageEnglish (US)
Title of host publicationNanoproteomics
Subtitle of host publicationMethods and Protocols
PublisherHumana Press Inc.
Number of pages10
ISBN (Print)9781617793189
StatePublished - 2011

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Bibliographical note

Publisher Copyright:
© 2011, Springer Science+Business Media, LLC.


  • 2D-peptide fractionation
  • 3D-peptide fractionation
  • Nanoscale reversed-phase HPLC
  • Preparative isoelectric focusing
  • Shotgun proteomics
  • Strong cation exchange HPLC


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