Targeting Peptide Termini, a Novel Immunoaffinity Approach to Reduce Complexity in Mass Spectrometric Protein Identification

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, ␤-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.

Epitope‐specificity of recombinant antibodies reveals promiscuous peptide‐binding properties

“…In order to make sure that the phenomenon of degenerate peptide-binding specificity was biologically relevant and not a bias introduced by the library design, we examined the reactivity pattern of two conventional antibodies raised in rabbits against similar tryptic peptide motifs. 33 Of note, these antibodies are used within a set-up similar to our GPS technology platform, [21][22][23] denoted Triple X proteomics (TXP). 33 Most interestingly, we found three semi-conserved resides (P4-x-P2-P1 or P4-P3-x-P1) to be essential for establishing the peptide-binding specificity also for these antibodies, while a higher degree of wobbling was allowed in the other positions (Supporting Information Fig.…”

What Can Proteomics Tell Us About Platelets?

“…Accordingly, current global proteomics procedures might result in underestimation of heavily modified and membrane proteins with high hydrophobicity; splice variants, isoforms of related proteins, and proteins expressed at low copy numbers (<0.5k). More dedicated approaches using enrichment procedures 170,171 or targeted MS analyses described above need to be used in these cases.…”

Identification and Quantification of a New Family of Peptide Endocannabinoids (Pepcans) Showing Negative Allosteric Modulation at CB1 Receptors

“…To determine the minimal sequence required for antibody binding, a bead-based peptide array was prepared as previously described (22)(23)(24). The array consisted of the RVD-Hp␣ peptide (RVDPVNFKLLSH), a carboxamide version of the RVDHp␣ peptide, and 22 truncated peptides.…”

“…Paramagnetic protein G-coated beads (Dynabeads) were washed four times with PBS, 0.3% N-octyl glucoside, and 5 l of the bead suspension were added to each sample. After a 60-min incubation at room temperature, the beads were washed four times and eluted in 1% formic acid as described previously (24,25). 1 l of each sample was spotted in triplicates onto a Prespotted HCCA AnchorChip (Bruker Daltonics).…”