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Abstract Background Antibodies constitute a powerful tool to study protein function, protein localization and protein-protein interactions, as well as for diagnostic and therapeutic purposes. High-throughput antibody development requires faster methodologies with lower antigen consumption. Results Here, we describe a novel methodology to select human monoclonal recombinant antibodies by combining in vitro protein expression, phage display antibody libraries and antibody microarrays. The application of this combination of methodologies permitted us to generate human single-chain variable fragments (scFvs) against two proteins: green fluorescent protein (GFP) and thioredoxin (Trx) in a short time, using as low as 5 μg of purified protein. These scFvs showed specific reactivity against their respective targets and worked well by ELISA and western blot. The scFvs were able to recognise as low as 31 ng of protein of their respective targets by western blot. Conclusion This work describes a novel and miniaturized methodology to obtain human monoclonal recombinant antibodies against any target in a shorter time than other methodologies using only 5 μg of protein. The protocol could be easily adapted to a high-throughput procedure for antibody production. ; RB was supported by a JAE-Doc contract (CSIC). AP is a recipient of a predoctoral FPI grant of the Spanish Ministry of Science and Innovation. This research was supported by grants from the Spanish Ministry of Science and Innovation BIO2009-08818 and Colomics Programme of the regional government of Madrid.We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI). ; Peer Reviewed

Metastasis is the primary cause of colorectal cancer (CRC) death. The liver and lung, besides adjacent lymph nodes, are the most common sites of metastasis. Here, we aimed to study the lymph nodes, liver, and lung CRC metastasis by quantitative spatial proteomics analysis using CRC cell-based models that recapitulate these metastases. The isogenic KM12 cell system composed of the non-metastatic KM12C cells, liver metastatic KM12SM cells, and liver and lung metastatic KM12L4a cells, and the isogenic non-metastatic SW480 and lymph nodes metastatic SW620 cells, were used. Cells were fractionated to study by proteomics five subcellular fractions corresponding to cytoplasm, membrane, nucleus, chromatin-bound proteins, and cytoskeletal proteins, and the secretome. Trypsin digested extracts were labeled with TMT 11-plex and fractionated prior to proteomics analysis on a Q Exactive. We provide data on protein abundance and localization of 4710 proteins in their different subcellular fractions, depicting dysregulation of proteins in abundance and/or localization in the most common sites of CRC metastasis. After bioinformatics, alterations in abundance and localization for selected proteins from diverse subcellular localizations were validated via WB, IF, IHC, and ELISA using CRC cells, patient tissues, and plasma samples. Results supported the relevance of the proteomics results in an actual CRC scenario. It was particularly relevant that the measurement of GLG1 in plasma showed diagnostic ability of advanced stages of the disease, and that the mislocalization of MUC5AC and BAIAP2 in the nucleus and membrane, respectively, was significantly associated with poor prognosis of CRC patients. Our results demonstrate that the analysis of cell extracts dilutes protein alterations in abundance in specific localizations that might only be observed studying specific subcellular fractions, as here observed for BAIAP2, GLG1, PHYHIPL, TNFRSF10A, or CDKN2AIP, which are interesting proteins that should be further analyzed in CRC metastasis. ; This research was funded by the Instituto de Salud Carlos III (ISCIII) through the PI20CIII/00019 grants from the AES-ISCIII program to R.B., co-financed by the European Development Regional Fund "A way to achieve Europe" (FEDER). J. Hofkens. acknowledges financial support from the Research Foundation–Flanders (FWO, grant No. ZW15_09-G0H6316N), the Flemish government through long-term structural funding Methusalem (CASAS2, Meth/15/04), and the MPI as MPI fellow. S.R. acknowledges the financial support of the KU Leuven through the internal C1 funding (KU Leuven (C14/16/053)). G.S.-F. is the recipient of a predoctoral contract (grant number 1193818N) supported by The Research Foundation–Flanders (FWO). The FPU predoctoral contract to A.M.-C. is supported by the Spanish Ministerio de Educación, Cultura y Deporte. ; Sí