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A common problem encountered when performing large-scale MS proteome analysis is the loss of information due to the high percentage of unassigned spectra. To determine the causes behind this loss we have analyzed the proteome of one of the smallest living bacteria that can be grown axenically, Mycoplasma pneumoniae (729 ORFs). The proteome of M. pneumoniae cells, grown in defined media, was analyzed by MS. An initial search with both Mascot and a species-specific NCBInr database with common contaminants (NCBImpn), resulted in around 79% of the acquired spectra not having an assignment. The percentage of non-assigned spectra was reduced to 27% after re-analysis of the data with the PEAKS software, thereby increasing the proteome coverage of M. pneumoniae from the initial 60% to over 76%. Nonetheless, 33 413 spectra with assigned amino acid sequences could not be mapped to any NCBInr database protein sequence. Approximately, 1% of these unassigned peptides corresponded to PTMs and 4% to M. pneumoniae protein variants (deamidation and translation inaccuracies). The most abundant peptide sequence variants (Phe-Tyr and Ala-Ser) could be explained by alterations in the editing capacity of the corresponding tRNA synthases. About another 1% of the peptides not associated to any protein had repetitions of the same aromatic/hydrophobic amino acid at the N-terminus, or had Arg/Lys at the C-terminus. Thus, in a model system, we have maximized the number of assigned spectra to 73% (51 453 out of the 70 040 initial acquired spectra). ; This work was sup-ported by the European Research Council (ERC), the Fundación Marcelino Botín, the Spanish Ministerio de Economía y Com-petitividad BIO2007-61762 and the ISCIII (PI10/01702). TheCRG/UPF Proteomics Unit is part of the "Plataforma de Re-cursos Biomoleculares y Bioinform´aticos (ProteoRed)" supportedby grant PT13/0001 of Instituto de Salud Carlos III (ISCIII).We acknowledge the support of the Spanish Ministry of Economyand Competitiveness, 'Centro de Excelencia Severo Ochoa 2013–2017', SEV-2012-0208. This project has received funding fromthe European Union's Horizon 2020 research and innovationprogram under grant agreement No 634942

AL amyloidosis is characterized by widespread deposition of immunoglobulin light chains (LCs) as amyloid fibrils. Cardiac involvement is frequent and leads to life-threatening cardiomyopathy. Besides the tissue alteration caused by fibrils, clinical and experimental evidence indicates that cardiac damage is also caused by proteotoxicity of prefibrillar amyloidogenic species. As in other amyloidoses, the damage mechanisms at cellular level are complex and largely undefined. We have characterized the molecular changes in primary human cardiac fibroblasts (hCFs) exposed in vitro to soluble amyloidogenic cardiotoxic LCs from AL cardiomyopathy patients. To evaluate proteome alterations caused by a representative cardiotropic LC, we combined gel-based with label-free shotgun analysis and performed bioinformatics and data validation studies. To assess the generalizability of our results we explored the effects of multiple LCs on hCF viability and on levels of a subset of cellular proteins. Our results indicate that exposure of hCFs to cardiotropic LCs translates into proteome remodeling, associated with apoptosis activation and oxidative stress. The proteome alterations affect proteins involved in cytoskeletal organization, protein synthesis and quality control, mitochondrial activity and metabolism, signal transduction and molecular trafficking. These results support and expand the concept that soluble amyloidogenic cardiotropic LCs exert toxic effects on cardiac cells. ; This work was supported by the PRIME-XS project, grant agreement number 262067, funded by the European Union 7th Framework Programme; the Italian Ministry of Health (GR-2010-2317596); Associazione Italiana per la Ricerca sul Cancro special program "5 per mille" (number 9965); Fondazione Cariplo (2013-0964, 2015-0591 and 2016-0489); the Italian Ministry of Health, research target project "Cardiac amyloidosis: molecular mechanism and innovative therapies for a challenging aging" (RF-2013-02355259); POR Campania FSE 2007-2013, Project DIAINTECH; and by Grant PON03PE_00060_2 and PON03PE_00060_7 (Campania - Bioscience) from the Italian Ministry of University and Research. The CRG/UPF Proteomics Unit is part of the "Plataforma de Recursos Biomoleculares y Bioinformáticos (ProteoRed)" supported by grant PT13/0001 of ISCIII and Spanish Ministry of Economy and Competitiveness. We acknowledge support of the Spanish Ministry of Economy and Competitiveness, "Centro de Excelencia Severo Ochoa 2013-2017", SEV-2012-0208, and from "Secretaria d'Universitats i Recerca del Departament d'Economia i Coneixement de la Generalitat de Catalunya" (2014SGR678).