TY - CHAP
T1 - Three-dimensional peptide fractionation for highly sensitive nanoscale LC-based shotgun proteomic analysis of complex protein mixtures
AU - Bandhakavi, Sricharan
AU - Markowski, Todd W.
AU - Xie, Hongwei
AU - Griffin, Timothy J.
PY - 2011/11/10
Y1 - 2011/11/10
N2 - 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.
AB - 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.
KW - 2D-peptide fractionation
KW - 3D-peptide fractionation
KW - Nanoscale reversed-phase HPLC
KW - Preparative isoelectric focusing
KW - Shotgun proteomics
KW - Strong cation exchange HPLC
UR - http://www.scopus.com/inward/record.url?scp=80555139572&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=80555139572&partnerID=8YFLogxK
U2 - 10.1007/978-1-61779-319-6_4
DO - 10.1007/978-1-61779-319-6_4
M3 - Chapter
C2 - 21948405
AN - SCOPUS:80555139572
SN - 9781617793189
T3 - Methods in Molecular Biology
SP - 47
EP - 56
BT - Nanoproteomics
A2 - Toms, Steven
A2 - Weil, Robert
ER -