Abstract
A wide variety of immunoglobulins (Ig) is produced by the immune system thanks to different mechanisms (V(D)J recombination, somatic hypermutation, and antigen selection). The profiling of Ig sequences (at both DNA and peptide levels) are of great relevance to developing targeted vaccines or treatments for specific diseases or infections. Thus, genomics and proteomics techniques (such as Next-Generation Sequencing (NGS) and mass spectrometry (MS)) have notably increased the knowledge in Ig sequencing and serum Ig peptide profiling in a high-throughput manner. However, the peptide characterization of membrane-bound Ig (e.g., B-cell receptors, BCR) is still a challenge mainly due to the poor recovery of mentioned Ig.Herein, we have evaluated three different sample processing methods for peptide sequencing of BCR belonging to chronic lymphocytic leukemia (CLL) B cells identifying up to 426 different peptide sequences (MS/MS data are available via ProteomeXchange with identifier PXD004466). Moreover, as a consequence of the results here obtained, recommended guidelines have been described for BCR-sequencing of B-CLL samples by MS approaches.For this purpose, an in–house algorithm has been designed and developed to compare the MS/MS results with those obtained by molecular biology in order to integrate both proteomics and genomics results and establish the steps to follow when sequencing membrane-bound Ig by MS/MS.
Highlights
The immune system has the capacity to produce a vast repertoire of immunoglobulins, (Ig) in response to the wide number of existing antigens, by processes such as V(D)J recombination [1, 2], somatic hypermutation [3, 4] and antigen selection [5].The Ig structure comprises four chains: two identical heavy chains and two identical light chains linked by disulfide bonds
We have evaluated three different sample processing methods for peptide sequencing of B-cell receptors (BCR) belonging to chronic lymphocytic leukemia (CLL) B cells identifying up to 426 different peptide sequences (MS/mass spectrometry (MS) data are available via ProteomeXchange with identifier PXD004466)
We have evaluated three approaches for peptide sequencing of BCR as well as B-cell proteins associated to BCR (MHC-I, MHC-II, CD20, CD79b, among others) and related to the immune system using an MS scan and tandem mass spectra (MS/MS) approach
Summary
The immune system has the capacity to produce a vast repertoire of immunoglobulins, (Ig) in response to the wide number of existing antigens, by processes such as V(D)J recombination (it occurs during B-cell maturation) [1, 2], somatic hypermutation (generated during B-cell affinity maturation) [3, 4] and antigen selection (during B-cell activation) [5]. Determining the protein profiles of B-CLL cells could have a great impact on disease knowledge, progression, origin, and identification of new drug targets; referring to immune-system proteins, antibodies and immunoglobulins In this sense, highthroughput DNA sequencing has been the approach of choice for extracting the most Ig biological information [11]. Since little variations in antibody sequences (even one single amino acid) could lead to modified specific antigen bindings; MS/MS strategies seem a good alternative for providing confidence in peptide sequencing [25] To this end, we have evaluated three approaches for peptide sequencing of BCR as well as B-cell proteins associated to BCR (MHC-I, MHC-II, CD20, CD79b, among others) and related to the immune system using an MS/MS approach. An in-house algorithm has been designed for the comparison of sequences obtained by MS/MS and molecular sequencing (Figure 1) to demonstrate the potential of MS/MS approaches in B cells to characterize Ig sequences (426 unique peptide sequences have been identified in this study) and correlate them with DNA sequence libraries
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