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, ␤-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 ␤-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.

MS-based strategies are key technologies for identifying proteins in proteomic research. Despite significant improvements in recent years efficient fractionation processes of target analytes remain major bottlenecks in MS-based protein analysis. Immunoaffinity-based sample fractionation strategies have shown their potential for the enrichment of analyte peptides of interest, but only small numbers of analytes can be quantified in one experiment. The lack of appropriate capture reagents limits the application of immunoaffinity-based approaches and only biased biomarker discovery approaches are possible. This perspective discusses the current status of immunoaffinity MS-based approaches and introduces a novel concept that uses group specific anti-peptide antibodies -- Triple X Proteomics Antibodies -- for the enrichment of signature peptides. Classes of peptides with identical termini can be fractionated based on TXP immunoaffinity enrichment steps and can subsequently be identified using established tandem MS procedures. Based on bioinformatic algorithms minimal sets of TXP epitopes can be specified, that cover a wide range of given proteome landscapes of one or even several different species. This opens the possibility to use a minimal number of TXP antibodies as a universal toolbox for general immunoaffinity-based approaches in proteome analysis.