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Background  The MALDI (matrix-assisted laser desorption/ionization) Biotyper system for bacterial identification has already been utilized in clinical microbiology laboratories as a successful clinical application of protoemics. However, in cases of  Nocardia , mass spectra suitable for MALDI Biotyper identification are often not obtained if such specimens are processed like general bacteria. This problem is related to the insufficiencies in bacterial spectrum databases that preclude accurate specimen identification. Here, we developed a bacterial processing method to improve mass spectra from specimens of the genus  Nocardia . In addition, with the new processing method, we constructed a novel in-house bacterial database that combines a commercial database and mass spectra of  Nocardia  strains from the Department of Clinical Laboratory at Chiba University Hospital (DCLC) and the Medical Mycology Research Center at Chiba University (MMRC).    Results  The newly developed method ( Nocardia  Extraction Method at DCLC [NECLC]) based on ethanol-formic acid extraction (EFAE) improved mass spectra obtained from  Nocardia  specimens. The  Nocardia  in-house database at Chiba University Hospital (NDCUH) was then successfully validated. In brief, prior to introduction of the NECLC and NDCUH, 10 of 64 (15.6%) clinical isolates were identified at the species level and 16 isolates (25.0%) could only be identified at the genus level. In contrast, after the introduction, 58 isolates (90.6%) were identified at the species level and 6 isolates (9.4%) were identified at the genus level.    Conclusions  The results of this study suggest that MALDI-TOF (time-of-flight) Biotyper system can identify  Nocardia  accurately in a short time in combination with a simple processing method and an in-house database.    Electronic supplementary material  The online version of this article (doi:10.1186/s12014-015-9078-5) contains supplementary material, which is available to authorized users.

Identification of Nocardia species using matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry