Liquid chromatography‐tandem mass spectrometry analysis of peptides separated from the insoluble matrix by in‐sample tryptic protein digestion for rapid discrimination of various induced pathological states of human bone models in oral surgery

For the understanding of pathological states of bone tissues in oral surgery, it would be desirable to have the possibility to simulate these processes on bone cell models in vitro. These cultures, similarly to bone tissues, contain numerous proteins entrapped in the insoluble matrix. The major goal of this study was to verify whether a method based on direct in‐matrix protein digestion could be suitable for the discrimination between different induced pathological states of bone cell models cultivated in vitro. Using in‐sample specific protein digestion with trypsin followed by liquid chromatography‐tandem mass spectrometry analysis of released peptides, 446 proteins (in average per sample) were identified in a bone cell in vitro model with induced cancer, 440 proteins were found in a model with induced inflammation, 451 proteins were detected in control in vitro culture, and 491 proteins were distinguished in samples of vestibular laminas of maxillary bone tissues originating from six different patients. Subsequent partial least squares – discrimination analysis of obtained liquid chromatography‐tandem mass spectrometry data was able to discriminate among in vitro cultures with induced cancer, with induced inflammation, and control cultivation. Thus, the direct in‐sample protein digestion by trypsin followed by liquid chromatography‐tandem mass spectrometry analysis of released specific peptide fragments from the insoluble matrix and mathematical analysis of the mass spectrometry data seems to be a promising tool for the routine proteomic characterization of in vitro human bone models with induced different pathological states.