Mass spectrometry and peptide-centric approaches are powerful techniques for the identification of differentially expressed proteins. Despite enormous improvements in MS technologies, sample preparation and efficient fractionation of target analytes are still major bottlenecks in MS-based protein analysis. The complexity of tryptically digested whole proteomes needs to be considerably reduced before low abundance proteins can be effectively analyzed using MS/MS. Sample preparation strategies that use peptide-specific antibodies are able to reduce the complexity of tryptic digests and lead to a substantial increase in throughput and sensitivity; however, the number of peptide-specific capture reagents is low, and consequently immunoaffinity-based approaches are only capable of detecting small sets of protein-derived peptides. In this proof-of-principle study, special anti-peptide antibodies were used to enrich peptides from a complex mixture. These antibodies recognize short amino acid sequences that are found directly at the termini of the peptides. The recognized epitopes consist of three or four amino acids only and include the terminally charged group of the peptide. Because of its limited length, antibodies recognizing the epitope will enrich not only one peptide but a whole class of peptides that share this terminal epitope. In this study, β-catenin-derived peptides were used to demonstrate that it is possible (i) to effectively generate antibodies that recognize short C-terminal peptide epitopes and (ii) to enrich and identify peptide classes from a complex mixture using these antibodies in an immunoaffinity MS approach. The expected β-catenin peptides and a set of 38 epitope-containing peptides were identified from trypsin-digested cell lysates. This might be a first step in the development of proteomics applications that are based on the use of peptide class-specific antibodies.
@article{Hoeppe2011,
author = {Hoeppe, Sibylle and Schreiber, Thomas D. and Planatscher, Hannes
and Zell, Andreas and Templin, Markus F. and Stoll, Dieter and Joos,
Thomas O. and Poetz, Oliver},
title = {Targeting peptide termini, a novel immunoaffinity approach to reduce
complexity in mass spectrometric protein identification},
journal = {Mol Cell Proteomics},
year = {2011},
volume = {10},
pages = {M110.002857},
number = {2},
month = feb,
abstract = {Mass spectrometry and peptide-centric approaches are powerful techniques
for the identification of differentially expressed proteins. Despite
enormous improvements in MS technologies, sample preparation and
efficient fractionation of target analytes are still major bottlenecks
in MS-based protein analysis. The complexity of tryptically digested
whole proteomes needs to be considerably reduced before low abundance
proteins can be effectively analyzed using MS/MS. Sample preparation
strategies that use peptide-specific antibodies are able to reduce
the complexity of tryptic digests and lead to a substantial increase
in throughput and sensitivity; however, the number of peptide-specific
capture reagents is low, and consequently immunoaffinity-based approaches
are only capable of detecting small sets of protein-derived peptides.
In this proof-of-principle study, special anti-peptide antibodies
were used to enrich peptides from a complex mixture. These antibodies
recognize short amino acid sequences that are found directly at the
termini of the peptides. The recognized epitopes consist of three
or four amino acids only and include the terminally charged group
of the peptide. Because of its limited length, antibodies recognizing
the epitope will enrich not only one peptide but a whole class of
peptides that share this terminal epitope. In this study, $\beta$-catenin-derived
peptides were used to demonstrate that it is possible (i) to effectively
generate antibodies that recognize short C-terminal peptide epitopes
and (ii) to enrich and identify peptide classes from a complex mixture
using these antibodies in an immunoaffinity MS approach. The expected
$\beta$-catenin peptides and a set of 38 epitope-containing peptides
were identified from trypsin-digested cell lysates. This might be
a first step in the development of proteomics applications that are
based on the use of peptide class-specific antibodies.},
doi = {10.1074/mcp.M110.002857},
institution = {NMI Natural and Medical Sciences Institute at the University of Tuebingen,
Reutlingen, Germany},
keywords = {Antibodies, chemistry; Cell Line; Epitopes, chemistry; Gene Library;
HEK293 Cells; Humans; Mass Spectrometry, methods; Peptides, chemistry;
Protein Binding; Protein Structure, Tertiary; Proteins, chemistry;
Proteome; Proteomics, methods; Spectrometry, Mass, Matrix-Assisted
Laser Desorption-Ionization; Trypsin, chemistry},
language = {eng},
medline-pst = {ppublish},
pdf = {http://www.mcponline.org/content/10/2/M110.002857.pdf},
pii = {M110.002857},
pmid = {20962300},
url = {http://dx.doi.org/10.1074/mcp.M110.002857}
}