Benefits of FAIMS to Improve the Proteome Coverage of Deteriorated and/or Cross-Linked TMT 10-Plex FFPE Tissue and Plasma-Derived Exosomes Samples

The proteome characterization of complex, deteriorated, or cross-linked protein mixtures as paired clinical FFPE or exosome samples isolated from low plasma volumes (250 µL) might be a challenge. In this work, we aimed at investigating the benefits of FAIMS technology coupled to the Orbitrap Exploris 480 mass spectrometer for the TMT quantitative proteomics analyses of these complex samples in comparison to the analysis of protein extracts from cells, frozen tissue, and exosomes isolated from large volume plasma samples (3 mL). TMT experiments were performed using a two-hour gradient LC-MS/MS with or without FAIMS and two compensation voltages (CV = -45 and CV = -60). In the TMT experiments of cells, frozen tissue, or exosomes isolated from large plasma volumes (3 mL) with FAIMS, a limited increase in the number of identified and quantified proteins accompanied by a decrease in the number of peptides identified and quantified was observed. However, we demonstrated here a noticeable improvement (>100%) in the number of peptide and protein identifications and quantifications for the plasma exosomes isolated from low plasma volumes (250 µL) and FFPE tissue samples in TMT experiments with FAIMS in comparison to the LC-MS/MS analysis without FAIMS. Our results highlight the potential of mass spectrometry analyses with FAIMS to increase the depth into the proteome of complex samples derived from deteriorated, cross-linked samples and/or those where the material was scarce, such as FFPE and plasma-derived exosomes from low plasma volumes (250 µL), which might aid in the characterization of their proteome and proteoforms and in the identification of dysregulated proteins that could be used as biomarkers.This research was funded by an AES-ISCIII grant from the Instituto de Salud Carlos III (PI20CIII/00019), co-financed by the European Development Regional Fund “A Way to Achieve Europe” (FEDER). A.M.-C. is supported by an FPU predoctoral contract with the Spanish Ministerio de Educación, Cultura y Deporte. R.R.-G. is supported by a predoctoral training contract (FI22CIII/00016) of the Instituto de Salud Carlos III.S

Anti-double stranded DNA antibodies: Electrochemical isotyping in autoimmune and neurological diseases

This work reports the first amperometric biosensor for the simultaneous determination of the single or total content of the most relevant human immunoglobulin isotypes (hIgs) of anti-dsDNA antibodies, dsDNA-hIgG, dsDNA-hIgM, dsDNA-hIgA and dsDNA-three hIgs, which are considered relevant biomarkers in prevalent autoimmune diseases such as systemic lupus erythematosus (SLE) as well as of interest in neurodegenerative diseases such as Alzheimer's disease (AD). The bioplatform involves the use of neutravidin-functionalized magnetic microparticles (NA-MBs) modified with a laboratory-prepared biotinylated human double-stranded DNA (b-dsDNA) for the efficient capture of specific autoantibodies that are enzymatically labeled with horseradish peroxidase (HRP) enzyme using specific secondary antibodies for each isotype or a mixture of secondary antibodies for the total content of the three isotypes. Transduction was performed by amperometry (-0.20 V vs. the Ag pseudo-reference electrode) using the H2O2/hydroquinone (HQ) system after trapping the resulting magnetic bioconjugates on each of the four working electrodes of a disposable quadruple transduction platform (SP4CEs). The bioplatform demonstrated attractive operational characteristics for clinical application and was employed to determine the individual or total hIgs classes in serum from healthy individuals and from patients diagnosed with SLE and AD. The target concentrations in AD patients are provided for the first time in this work. In addition, the results for SLE patients and control individuals agree with those obtained by applying ELISA tests as well as with the clinical ranges reported by other authors, using individual detection methodologies restricted to centralized settings or clinical laboratories.The financial support of PID2019-103899RB-I00 and PID2021-122457OB-I00 (Spanish Ministerio de Ciencia e Innovación) Research Projects, PI17CIII/00045 and PI20CIII/00019 Grants from the AESISCIII Program co-founded by FEDER funds and the TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (Grant S2018/NMT-4349) are gratefully acknowledged. B.A. acknowledges predoctoral contracts from the Spanish Ministerio de Ciencia, Innovación y Universidades (PRE2019-087596). M.G-A. acknowledges the postdoctoral contract Margarita Salas for the requalification of the Spanish University System. A.M-C. was supported by a FPU predoctoral contract supported by the Spanish Ministerio de Educación, Cultura y Deporte.S

Angiogenesis inhibitor or aggressiveness marker? The function of endostatin in cancer through electrochemical biosensing

This work reports the first electrochemical bioplatform developed for the determination of human endostatin (HE), a biomarker with recognized antiangiogenic potential whose elevated circulating levels have also been associated with the development of aggressive cancers. The developed electroanalytical biotool combines the benefits of using magnetic microparticles for the implementation of sandwich immunoassays and amperometric transduction on disposable carbon electrodes. A limit of detection (LOD) of 34.1 pg mL-1 for HE standards and a selectivity suitable for its foray into the clinical oncology area, are demonstrated. The determination of HE in clinical samples such as lysates and secretomes of colorectal cancer (CRC) cells, plasma, and tissue samples from patients with CRC in different stages, has been faced with satisfactory results showing the ability for discriminating the metastatic capabilities of cells and for identifying and staging CRC patients. The developed bioplatform allows precise quantitative determinations, requiring minimal pre-treatments and sample amounts in only 75 min. In addition, due to the instrumentation and the type of substrates used in the detection step, the biotool is compatible with implementation in multiplexed and/or point-of-need devices, features in which this bioplatform is advantageous with respect to the enzyme linked immunosorbent assay (ELISA) or immunoblotting technologies.The financial support of PID2019-103899RB-I00 (Spanish Ministerio de Ciencia e Innovación) Research Project and PI20CIII/00019 Grant from the AES-ISCIII Program co-founded by FEDER funds and the TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (Grant S2018/NMT-4349) are gratefully acknowledged. M.G-A. acknowledges the postdoctoral contract Margarita Salas for the requalification of the Spanish University System. A.M-C. was supported by a FPU predoctoral contract supported by the Spanish Ministerio de Educación, Cultura y Deporte. S.T.M. acknowledges a predoctoral contract from the Spanish Ministerio de Ciencia e Innovación (PRE2020-092859).S

The specific seroreactivity to ∆Np73 isoforms shows higher diagnostic ability in colorectal cancer patients than the canonical p73 protein

The p53-family is tightly regulated at transcriptional level. Due to alternative splicing, up to 40 different theoretical proteoforms have been described for p73 and at least 20 and 10 for p53 and p63, respectively. However, only the canonical proteins have been evaluated as autoantibody targets in cancer patients for diagnosis. In this study, we have cloned and expressed in vitro the most upregulated proteoforms of p73, ΔNp73α and ΔNp73β, for the analysis of their seroreactivity by a developed luminescence based immunoassay test using 145 individual plasma from colorectal cancer, premalignant individuals and healthy controls. ∆Np73α seroreactivity showed the highest diagnostic ability to discriminate between groups. The combination of ∆Np73α, ∆Np73β and p73 proteoforms seroreactivity were able to improve their individual diagnostic ability. Competitive inhibition experiments further demonstrated the presence of unique specific epitopes in ΔNp73 isoforms not present in p73, with several colorectal patients showing unique and specific seroreactivity to the ΔNp73 proteoforms. Overall, we have increased the complexity of the humoral immune response to the p53-family in cancer patients, showing that the proteoforms derived from the alternative splicing of p73 possess a higher diagnostic ability than the canonical protein, which might be extensive for p53 and p63 proteins.This work was supported by the Ramon y Cajal programme of the MINECO and the financial support of the PI17CIII/00045 grant from the AES-ISCIII program to R.B., cofounded by FEDER funds. G.D. acknowledges the financial support of PI15/00246 grant of the FIS and Cátedra UAM-Roche en Medicina de Innovación. M.G-A. was supported by a contract of the Programa Operativo de Empleo Juvenil y la Iniciativa de Empleo Juvenil (YEI) with the participation of the Consejería de Educación, Juventud y Deporte de la Comunidad de Madrid y del Fondo Social Europeo. We thank the excellent technical support of Maricruz Sánchez. A.M-C. is a recipient of a FPU fellowship from the Ministerio de Educación, Cultura y Deporte.S

Tackling CD147 exosome-based cell-cell signaling by electrochemical biosensing for early colorectal cancer detection

The great opportunities represented by exosomes in liquid biopsy diagnostics and the relevance of CD147 protein as diagnostic and prognostic cancer biomarker led us to develop the first bio-electroanalytical platform for the determination of exosomal CD147 (exoCD147) by exploiting micro-sized magnetic beads coated with specific anti-CD147 antibodies. The captured exosomal target protein was sandwiched by specific biotin functionalized detector antibodies followed by attaching streptavidin-HRP conjugate to perform the amperometric reading using screen-printed carbon electrodes (SPCEs) as electrode transducers in the presence of hydroquinone (HQ) and H2O2. The analytical and operational characteristics achieved by implementing this simple methodology allowed the sensitive (LOD 29 pg mL-1) and selective determination of CD147 and the analysis of exoCD147 in different but inter-related real clinical scenarios including lysed and entire exosomes previously isolated from CRC cell lines with different metastatic potential. The obtained results, in agreement with those provided by ELISA and WB, proved the reliability of the developed immunosensor and its potential to isolate or identify specific subpopulations of exosomes based on the differential expression of characteristic surface biomarkers.The financial support of PID2019-103899RB-I00 (Spanish Ministerio de Ciencia e Innovación) Research Project and the TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (Grant S2018/NMT-4349) are gratefully acknowledged. R.B. acknowledges the financial support of PI20CIII/00019 grant from the AES-ISCIII program. A.M-C. acknowledges a FPU predoctoral contract supported by the Spanish Ministerio de Educación, Cultura y Deporte.S

First bioelectronic immunoplatform for quantitative secretomic analysis of total and metastasis-driven glycosylated haptoglobin

The glycosylation status of proteins is increasingly used as biomarker to improve the reliability in the diagnosis and prognosis of diseases as relevant as cancer. This feeds the need for tools that allow its simple and reliable analysis and are compatible with applicability in the clinic. With this objective in mind, this work reports the first bioelectronic immunoplatforms described to date for the determination of glycosylated haptoglobin (Hp) and the simultaneous determination of total and glycosylated Hp. The bioelectronic immunoplatform is based on the implementation of non-competitive bioassays using two different antibodies or an antibody and a lectin on the surface of commercial magnetic microcarriers. The resulting bioconjugates are labeled with the horseradish peroxidase (HRP) enzyme, and after their magnetic capture on disposable electroplatforms, the amperometric transduction using the H2O2/hydroquinone (HQ) system allows the single or multiple detection. The developed immunoplatform achieves limits of detection (LODs) of 0.07 and 0.46 ng mL-1 for total and glycosylated Hp in buffer solution, respectively. The immunoplatform allows accurate determination using simple and relatively short protocols (approx. 75 min) of total and glycosylated Hp in the secretomes of in vitro-cultured colorectal cancer (CRC) cells with different metastatic potentials, which is not feasible, due to lack of sensitivity, by means of some commercial ELISA kits and Western blot methodology.Funding Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. Spanish Ministerio de Ciencia e Innovación (PID2019-103899RB-I00 and RTI2018-095756-B-I00), AES-ISCIII Program co-founded by FEDER funds (PI17CIII/00045 and PI20CIII/00019 grants), TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (Grant S2018/NMT-4349).S

Metabolic Reprogramming Helps to Define Different Metastatic Tropisms in Colorectal Cancer

Approximately 25% of colorectal cancer (CRC) patients experience systemic metastases, with the most frequent target organs being the liver and lung. Metabolic reprogramming has been recognized as one of the hallmarks of cancer. Here, metabolic and functional differences between two CRC cells with different metastatic organotropisms (metastatic KM12SM CRC cells to the liver and KM12L4a to the lung when injected in the spleen and in the tail vein of mice) were analysed in comparison to their parental non-metastatic isogenic KM12C cells, for a subsequent investigation of identified metabolic targets in CRC patients. Meta-analysis from proteomic and transcriptomic data deposited in databases, qPCR, WB, in vitro cell-based assays, and in vivo experiments were used to survey for metabolic alterations contributing to their different organotropism and for the subsequent analysis of identified metabolic markers in CRC patients. Although no changes in cell proliferation were observed between metastatic cells, KM12SM cells were highly dependent on oxidative phosphorylation at mitochondria, whereas KM12L4a cells were characterized by being more energetically efficient with lower basal respiration levels and a better redox management. Lipid metabolism-related targets were found altered in both cell lines, including LDLR, CD36, FABP4, SCD, AGPAT1, and FASN, which were also associated with the prognosis of CRC patients. Moreover, CD36 association with lung metastatic tropism of CRC cells was validated in vivo. Altogether, our results suggest that LDLR, CD36, FABP4, SCD, FASN, LPL, and APOA1 metabolic targets are associated with CRC metastatic tropism to the liver or lung. These features exemplify specific metabolic adaptations for invasive cancer cells which stem at the primary tumour.This work was supported by grants cofounded by Fondo Europeo de Desarrollo Regional -FEDER- PI17CIII/00045 and PI20CIII/00019 from the AES-ISCIII program to RB from the Instituto de Salud Carlos III (ISCIII) and grants from Spanish Ministry of Science (Plan Nacional I+D+i PID2019-110183RBC21), Regional Government of Community of Madrid (P2018/BAA-4343-ALIBIRD2020-CM, and Y2020/BIO-6350), and Ramón Areces Foundation (CIVP19A5937) to AR. AM-C FPU predoctoral contract is supported by the Spanish Ministerio de Educació n, Cultura y Deporte. AQ-F acknowledges Comunidad de Madrid for the Garantıa Juvenil PEJD-2017-RE/BMD-3394 contract. GS-F is a recipient of a predoctoral contract (grant number 1193818N) supported by the Flanders Research Foundation (FWO).S

In-depth proteomics characterization of ∆Np73 effectors identifies key proteins with diagnostic potential implicated in lymphangiogenesis, vasculogenesis and metastasis in colorectal cancer

Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related death worldwide. Alterations in proteins of the p53-family are a common event in CRC. ΔNp73, a p53-family member, shows oncogenic properties and its effectors are largely unknown. We performed an in-depth proteomics characterization of transcriptional control by ∆Np73 of the secretome of human colon cancer cells and validated its clinical potential. The secretome was analyzed using high-density antibody microarrays and stable isotopic metabolic labeling. Validation was performed by semiquantitative PCR, ELISA, dot-blot and western blot analysis. Evaluation of selected effectors was carried out using 60 plasma samples from CRC patients, individuals carrying premalignant colorectal lesions and colonoscopy-negative controls. In total, 51 dysregulated proteins were observed showing at least 1.5-foldchange in expression. We found an important association between the overexpression of ∆Np73 and effectors related to lymphangiogenesis, vasculogenesis and metastasis, such as brain-derived neurotrophic factor (BDNF) and the putative aminoacyl tRNA synthase complex-interacting multifunctional protein 1 (EMAP-II)-vascular endothelial growth factor C-vascular endothelial growth factor receptor 3 axis. We further demonstrated the usefulness of BDNF as a potential CRC biomarker able to discriminate between CRC patients and premalignant individuals from controls with high sensitivity and specificity.This study has been funded by Instituto de Salud Carlos III (ISCIII) through the project “PI18/00473” and co-funded by the European Union (FEDER funds) and Cátedra UAM-Roche en Medicina de Innovación to GD, and the Ramón y Cajal Programme of the MINECO, PI17CIII/00045 and PI20CIII/00019 research projects from AES-ISCIII to RB. MG-A and JR-C were supported by contracts of the Programa Operativo de Empleo Juvenil y la Iniciativa de Empleo Juvenil (YEI) with the participation of the Consejería de Educación, Juventud y Deporte de la Comunidad de Madrid y del Fondo Social Europeo. AM-C FPU predoctoral contract is supported by the MECD. GS-F is a recipient of a predoctoral contract (grant num-ber 1193818N) supported by The Flanders Research Foundation (FWO).S

Disposable electrochemical immunoplatform to shed light on the role of the multifunctional glycoprotein TIM-1 in cancer cells invasion

Detecting overexpression of cancer biomarkers is an excellent tool for diagnostic/prognostic and follow-up of patients with cancer or their response to treatment. This work illustrates the relevance of interrogating the levels of T-cell immunoglobulin and mucin domain 1 (TIM-1) protein as a diagnostic/prognostic biomarker of high-prevalence breast and lung cancers by using an amperometric disposable magnetic microparticles-assisted immunoplatform. The developed method integrates the inherent advantages of carboxylic acid-functionalized magnetic beads (HOOC-MBs) as pre-concentrator support and the amperometric transduction at screen-printed carbon electrodes (SPCEs). The immunoplatform involves a sandwich-type immunoassay assembled on HOOC-MBs through the specific capture/labeling of TIM-1 using capture antibodies and horseradish peroxidase (HRP)-conjugated biotinylated detection antibodies as biorecognition elements. The magnetic immunoconjugates were confined onto the working electrode (WE) surface of the SPCEs for amperometric detection using the hydroquinone/hydrogen peroxide/HRP (HQ/H2O2/HRP) redox system. The method allows the selective detection of TIM-1 protein over the 87-7500 pg mL-1 concentration range in only 45 min, with a limit of detection of 26 pg mL-1. The developed bioplatform was successfully applied to the analysis of breast and lung cancer cell extracts, providing the first quantitative results of the target glycoprotein in these types of samples.The financial support of PID2019-103899RB-I00 (Spanish Ministerio de Ciencia e Innovación) Research Projects and PI20CIII/00019 Grant from the AES-ISCIII Program co-founded by FEDER funds and the TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (Grant S2018/NMT-4349) are gratefully acknowledged. A.M-C. was supported by a FPU predoctoral contract supported by the Spanish Ministerio de Educación, Cultura y Deporte. J.Q. was founded by Minciencias, Mineducacion, MINCIT, and ICETEX through the Program Ecosistema Cientifico Cod. FP44842-211–2018, project number 58536. J.O. thanks support from the University of Antioquia and the Max Planck Society through the cooperation agreement 566–1, 2014.S

Electrochemical immunosensing of Growth arrest‐specific 6 in human plasma and tumor cell secretomes

Growth arrest-specific 6 (GAS6) protein plays a key role in processes related toproliferation,inflammation,angiogenesis,andatheroscleroticplaqueformation.In addition, it has been reported that plasma levels of GAS6 are related to cancerprognosis and other relevant pathologies, such as heart failure or sepsis. Wereport here the first electrochemical immunoplatform for the determination ofGAS6, which has demonstrated to be competitive with other available method-ologies in terms of cost, simplicity, and decentralized application. The developedimmunoplatform involves a sandwich immunoassay using magnetic microparti-cles (MBs) and uses amperometric detection at disposable screen-printed carbonelectrodes (SPCEs). The MBs were modified with an antibody specific to GAS6for its selective capture, which is further recognized by a biotinylated secondaryantibody subsequently labeled with a streptavidin-horseradish peroxidase(Strep-HRP) conjugate. The electrochemical detection was carried out using thehydroquinone (HQ)/H2O2system. The developed bioplatform exhibits a greatselectivity and low limit of detection (27 pg/mL) that allowed the determinationof the GAS6 circulating level in plasma samples from patients suffering heartfailure (HF) and diagnosed with pancreatic ductal adenocarcinoma (PDAC),as well as the determination of the target protein in raw secretomes of humancolorectal cancer cell lines.This work is part of the POSITION-II project funded by the ECSEL Joint Undertaking under grant number Ecsel-783132-Position-II-2017-IA; www.position-2.eu, and PCI2018-093067 (Spanish Ministerio de Ciencia e Innovación) to M.P. The financial support of PID2019-103899RB-I00 (Spanish Ministerio de Ciencia e Innovación) Research Project to S.C., PI17CIII/00045 and PI20CIII/00019 grants from the AES-ISCIII program to R.B. and the TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (Grant S2018/NMT-4349) to S.C., RTI2018-095672-B-I00 (Spanish Ministerio de Ciencia e Innovación) to P.G.F.; Fundació la Marató de TV3 project 081010 to M.B.; research project PI20/00625, from the AES-ISCIII/FEDER program, to P.N, are gratefully acknowledged. A. Montero-Calle acknowledges the support of the FPU predoctoral contracts by the Spanish Ministerio de Educación, Cultura y Deporte. G.S-F. is recipient of a predoctoral contract (grant number 1193818N) supported by The Flanders Research Foundation (FWO). C. Muñoz-San Martín acknowledges a predoctoral contract from Complutense University of Madrid. R.M. Torrente-Rodríguez acknowledges a Talento-Contract from Comunidad de Madrid (2019-T2/IND-15965).S