A systematic approach for peptide characterization of B-cell receptor in chronic lymphocytic leukemia cells

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.We gratefully acknowledge financial support from the Spanish Health Institute Carlos III (ISCIII) for the grants: FIS PI11/02114 and FIS PI114/01538. We also acknowledge Fondos FEDER (EU) and Junta Castilla León (grant BIO/SA07/15). This work has been also sponsored by Fundación Solórzano (FS/23-2015). The Proteomics Unit belongs to ProteoRed, PRB2-ISCIII, supported by grant PT13/0001, of the PE I+D+I 2013-2016, funded by ISCIII and FEDER. The authors would like to thank all the clinicians and technicians in the Cytometry and Cell Purification Services of the University of Salamanca, the Spanish National DNA Bank (Banco Nacional de DNA Carlos III, University of Salamanca) and the Genomic Unit of Cancer Research Centre (IBMCC, USAL-CSIC) for their support in the data collection for the preparation of this manuscript. P.D. is supported by a JCYL-EDU/346/2013 Ph.D. scholarship.Peer Reviewe

Dynamic Intracellular Metabolic Cell Signaling Profiles During Ag-Dependent B-Cell Differentiation

© 2021 Díez, Pérez-Andrés, Bøgsted, Azkargorta, García-Valiente, Dégano, Blanco, Mateos-Gomez, Bárcena, Santa Cruz, Góngora, Elortza, Landeira-Viñuela, Juanes-Velasco, Segura, Manzano-Román, Almeida, Dybkaer, Orfao and Fuentes.Human B-cell differentiation has been extensively investigated on genomic and transcriptomic grounds; however, no studies have accomplished so far detailed analysis of antigen-dependent maturation-associated human B-cell populations from a proteomic perspective. Here, we investigate for the first time the quantitative proteomic profiles of B-cells undergoing antigen-dependent maturation using a label-free LC-MS/MS approach applied on 5 purified B-cell subpopulations (naive, centroblasts, centrocytes, memory and plasma B-cells) from human tonsils (data are available via ProteomeXchange with identifier PXD006191). Our results revealed that the actual differences among these B-cell subpopulations are a combination of expression of a few maturation stage-specific proteins within each B-cell subset and maturation-associated changes in relative protein expression levels, which are related with metabolic regulation. The considerable overlap of the proteome of the 5 studied B-cell subsets strengthens the key role of the regulation of the stoichiometry of molecules associated with metabolic regulation and programming, among other signaling cascades (such as antigen recognition and presentation and cell survival) crucial for the transition between each B-cell maturation stage.We gratefully acknowledge financial support from the Spanish Health Institute Carlos III (ISCIII) for the grants: FIS PI14/01538, FIS PI17/01930 and CB16/12/00400. We also acknowledge Fondos FEDER (EU) and Junta Castilla-León (COVID19 grant COV20EDU/00187). Fundación Solórzano FS/38-2017.The Proteomics Unit belongs to ProteoRed, PRB3-ISCIII, supported by grant PT17/0019/0023, of the PE I + D + I 2017-2020, funded by ISCIII and FEDER. AL-V is supported by VIII Centenario-USAL PhD Program. PJ-V is supported by JCYL PhD Program and scholarship JCYL-EDU/601/2020. PD and EB are supported by a JCYL-EDU/346/2013 Ph.D. scholarship

Determining the Plasmodium vivax VCG-1 strain blood stage proteome

This is an open access article under the CC BY-NC-ND license.Plasmodium vivax is the second most prevalent parasite species causing malaria in humans living in tropical and subtropical areas throughout the world. There have been few P. vivax proteomic studies to date and they have focused on using clinical isolates, given the technical difficulties concerning how to maintain an in vitro culture of this species. This study was thus focused on identifying the P. vivax VCG-1 strain proteome during its blood lifecycle through LC-MS/MS; this led to identifying 734 proteins, thus increasing the overall number reported for P. vivax to date. Some of them have previously been related to reticulocyte invasion, parasite virulence and growth and others are new molecules possibly playing a functional role during metabolic processes, as predicted by Database for Annotation, Visualization and Integrated Discovery (DAVID) functional analysis. This is the first large-scale proteomic analysis of a P. vivax strain adapted to a non-human primate model showing the parasite protein repertoire during the blood lifecycle. Database searches facilitated the in silico prediction of proteins proposed for evaluation in further experimental assays regarding their potential as pharmacologic targets or as component of a totally efficient vaccine against malaria caused by P. vivax. Biological significance: P. vivax malaria continues being a public health problem around world. Although considerable progress has been made in understanding genome- and transcriptome-related P. vivax biology, there are few proteome studies, currently representing only 8.5% of the predicted in silico proteome reported in public databases. A high-throughput proteomic assay was used for discovering new P. vivax intra-reticulocyte asexual stage molecules taken from parasites maintained in vivo in a primate model. The methodology avoided the main problem related to standardising an in vitro culture system to obtain enough samples for protein identification and annotation. This study provides a source of potential information contributing towards a basic understanding of P. vivax biology related to parasite proteins which are of significant importance for the malaria research community.We would also like to thank the ProteoRed, PRB2-ISCIII proteomics facility at the Instituto de Biología Molecular y Celular del Cáncer USAL-CSIC, financed by grant PT13/0001. This research was supported by the “Instituto Colombiano para el Desarrollo de la Ciencia ‘Francisco José de Caldas’” (COLCIENCIAS) through contracts RC#309-2013 and 709-2013.Peer Reviewe

NAPPA as a real new method for protein microarray generation

This is an open access article distributed under the Creative Commons Attribution License.-- This article belongs to the Special Issue "New and Old Technologies for Generation of Microarrays".Nucleic Acid Programmable Protein Arrays (NAPPA) have emerged as a powerful and innovative technology for the screening of biomarkers and the study of protein-protein interactions, among others possible applications. The principal advantages are the high specificity and sensitivity that this platform offers. Moreover, compared to conventional protein microarrays, NAPPA technology avoids the necessity of protein purification, which is expensive and time-consuming, by substituting expression in situ with an in vitro transcription/translation kit. In summary, NAPPA arrays have been broadly employed in different studies improving knowledge about diseases and responses to treatments. Here, we review the principal advances and applications performed using this platform during the last years.We gratefully acknowledge financial support from the Carlos III Health Institute of Spain (ISCIII, FIS PI14/01538)-Fondos FEDER (EU). The Proteomics Unit belongs to ProteoRed, PRB2-ISCIII, supported by grant PT13/0001. P.D. is supported by a JCYL-EDU/346/2013 PhD scholarship.We acknowledge the support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).Peer reviewe

CSF analysis for protein biomarker identification in patients with leptomeningeal metastases from CNS lymphoma

[Introduction]: Leptomeningeal metastases (LM) from lymphoma remain a difficult complication for oncologist due to the high incidence in morbidity and mortality. Early diagnostic and initiation of treatment are essential to prevent neurological deterioration. [Areas covered]: In this review, several proteomic approaches are described in order to help and provide the basis for the identification of biomarkers useful in early diagnosis, also in discovery novel targets for therapeutic agents. In fact, the identification of biomarkers will have a high potential to detect leptomeningeal lymphoma, as well as to predict its progression and treatment response. [Expert commentary]: In the case of LM by Central nervous system (CNS) lymphoma, these studies generated the first insights into the utility of proteomic analysis for biomarker identification and will be demonstrated that identifying specific proteins in cerebrospinal fluid (CSF) had much greater sensitivity for detecting LM in comparison to standard cytological protocols.We gratefully acknowledge financial support from the Spanish Health Institute Carlos III (ISCIII) [FIS PI114/01538]. We also acknowledge Fondos FEDER (EU), Junta Castilla-León [grant BIO/SA07/15] and Fundación Solórzano [FS23/2015]. The Proteomics Unit belongs to ProteoRed, PRB2-ISCIII [PT13/0001], of the PE I+D+I 2013-2016, funded by ISCIII and FEDER. N.G. is supported by Consejo Nacional de Ciencia y Tecnología (CONACYT) and P.D. is supported by a JCYL Ph.D. scholarship [EDU/346/2013].Peer Reviewe

Methods for selecting phage display antibody libraries

The selection process aims sequential enrichment of phage antibody display library in clones that recognize the target of interest or antigen as the library undergoes successive rounds of selection. In this review, selection methods most commonly used for phage display antibody libraries have been comprehensively described.We gratefully acknowledge financial support from the Carlos III Health Institute of Spain (FIS PI14/01538), Fondos FEDER (EU) and Junta Castilla-Leon (BIO/SA07/15) and Fundación Solórzano FS/23-2015. The proteomics Unit belongs to ProteoRedISCIII, PRB2-ISCII, supported by grant PT13/001. P.D. is supported by a JCYL-EDU/346/2013 PhD. scholarship.Peer Reviewe

Quest for missing proteins: Update 2015 on chromosome-centric human proteome project

49 p.-3 fig.-1 tab.-- Horvatovich, Péter et al.This paper summarizes the recent activities of the Chromosome-Centric Human Proteome Project (C-HPP) consortium, which develops new technologies to identify yet-to-be annotated proteins (termed “missing proteins”) in biological samples that lack sufficient experimental evidence at the protein level for confident protein identification. The C-HPP also aims to identify new protein forms that may be caused by genetic variability, post-translational modifications, and alternative splicing. Proteogenomic data integration forms the basis of the C-HPP’s activities; therefore, we have summarized some of key approaches and their roles in the project. We present new analytical technologies that improve the chemical space and lower detection limits coupled with bioinformatics tools and some publicly available resources that can be used to improve data analysis or support the development of analytical assays. Most of this paper’s contents have been compiled from posters, slides, and discussions presented in the series of C-HPP workshops held during 2014. All data (posters, presentations) used are available at the C-HPP Wiki (http://c-hpp.webhosting.rug.nl/) and in the supporting information.J.A.V. acknowledges the EU FP7 grants ‘ProteomeXchange’ [grant number 260558] and PRIME-XS [grant number 262067]. G.S.O. acknowledges grant U54ES017885 from the NIH. Carol L. Nilsson acknowledges the Cancer Prevention and Research Institute of Texas (CPRIT, RML 1122) and the University of Texas Medical Branch. Y.K.P acknowledges the C-HPP grant from the Korean Ministry of Health and Welfare (to Y.K.P., HI13C2098).Peer reviewe

Multipronged functional proteomics approaches for global identification of altered cell signalling pathways in B-cell chronic lymphocytic leukaemia

Chronic lymphocytic leukaemia (CLL) is a malignant B cell disorder characterized by its high heterogeneity. Although genomic alterations have been broadly reported, protein studies are still in their early stages. Herein, a 224-antibody microarray has been employed to study the intracellular signalling pathways in a cohort of 14 newly diagnosed B-CLL patients as a preliminary study for further investigations. Several protein profiles were differentially identified across the cytogenetic and molecular alterations presented in the samples (deletion 13q14 and 17p13.1, trisomy 12, and NOTCH1 mutations) by a combination of affinity and MS/MS proteomics approaches. Among others altered cell signalling pathways, PKC family members were identified as down-regulated in nearly 75% of the samples tested with the antibody arrays. This might explain the rapid progression of the disease when showing p53, Rb1, or NOTCH1 mutations due to PKC-proteins family plays a critical role favouring the slowly progressive indolent behaviour of CLL. Additionally, the antibody microarray results were validated by a LC-MS/MS quantification strategy and compared to a transcriptomic CLL database. In summary, this research displays the usefulness of proteomic strategies to globally evaluate the protein alterations in CLL cells and select the possible biomarkers to be further studied with larger sample sizes.We gratefully acknowledge financial support from the Carlos III Health Institute of Spain (ISCIII, FIS PI11/02114 and FIS PI14/01538), Fondos FEDER (EU), Junta Castilla-Leon SA198A12-2 and BIO/SA03/15. The proteomics analysis was performed in the Proteomics facility of Centro de Investigacion del Cancer (Salamanca, Spain) that belongs to ProteoRed, PRB2- ISCIII, supported by grant PT13/0001. P.D. is supported by a JCYL-EDU/346/2013 PhD scholarship.Peer Reviewe

In-depth proteomic characterization of classical and non-classical monocyte subsets

Monocytes are bone marrow-derived leukocytes that are part of the innate immune system. Monocytes are divided into three subsets: classical, intermediate and non-classical, which can be differentiated by their expression of some surface antigens, mainly CD14 and CD16. These cells are key players in the inflammation process underlying the mechanism of many diseases. Thus, the molecular characterization of these cells may provide very useful information for understanding their biology in health and disease. We performed a multicentric proteomic study with pure classical and non-classical populations derived from 12 healthy donors. The robust workflow used provided reproducible results among the five participating laboratories. Over 5000 proteins were identified, and about half of them were quantified using a spectral counting approach. The results represent the protein abundance catalogue of pure classical and enriched non-classical blood peripheral monocytes, and could serve as a reference dataset of the healthy population. The functional analysis of the differences between cell subsets supports the consensus roles assigned to human monocytes.All participating laboratories are members of the PRBB-ISCIII platform. This work was supported by the Carlos III Health Institute of Spain (ISCIII, FIS PI11/02114 and FIS PI14/01538)-Fondos FEDEREU); grants SAF2014-5478-R from Ministerio de Economía y Competitividad to FJC; grant DPI2015-68982-R from Ministerio de Ciencia e Innovación

Functional insights into the cellular response triggered by a bile-acid platinum compound conjugated to biocompatible ferric nanoparticles using quantitative proteomic approaches

At present, bioferrofluids are employed as powerful multifunctional tools for biomedical applications such as drug delivery, among others. The present study explores the cellular response evoked when bile-acid platinum derivatives are conjugated with bioferrofluids by testing the biological activity in osteosarcoma (MG-63) and T-cell leukemia (Jurkat) cells. The aim of this work is to evaluate the biocompatibility of a bile-acid platinum derivative conjugated with multi-functional polymer coated bioferrofluids by observing the effects on the protein expression profiles and in intracellular pathways of nanoparticle-stimulated cells. To this end, a mass spectrometry-based approach termed SILAC has been applied to determine in a high-throughput manner the key proteins involved in the cellular response process (including specific quantitatively identified proteins related to the vesicular transport, cellular structure, cell cycle, biosynthetic process, apoptosis and regulation of the cell cycle). Finally, biocompatibility was evaluated and validated by conventional strategies also (such as flow cytometry, MTT, etc.).We gratefully acknowledge financial support from the Spanish Health Institute Carlos III (ISCIII) for the grants: FIS PI11/02114 and FIS PI14/01538. We also acknowledge Fondos FEDER (EU) and Junta Castilla-León (grant BIO/SA07/15). The Proteomics Unit belongs to ProteoRed, PRB2-ISCIII, supported by grant PT13/0001, of the PE I + D + I 2013–2016, funded by ISCIII and FEDER. P. D. is supported by a JCYL-EDU/346/2013 Ph.D. scholarship. This work was partially supported by the Spanish Ministry of Science and Innovation (MAT2014-54975-R).Peer Reviewe