Orbitrap fusion lumos tribrid spectrometer

The dried peptides were reconstituted in a solution containing 15 μL of 0.1% formic acid (FA) and 2% acetonitrile (ACN). For proteomic analysis, liquid chromatography–tandem mass spectrometry (LC-MS/MS) was performed using a Dionex UltiMate 3000 RSL nano system (Thermo Scientific, Waltham, MA, USA) in conjunction with an Orbitrap Fusion Lumos Tribrid spectrometer (Thermo Scientific, USA). The samples were separated on a custom-packed 15 cm column (internal diameter of 150 μm) filled with C18 AQ beads (1.9 μm; Dr. Maisch, GmbH, Ammerbuch, Germany). The mobile phase composition was designed as follows: mobile phase A consisted of 98% water and 2% ACN, while mobile phase B comprised 80% ACN and 20% water; both phases contained 0.1% FA. During the proteomic analysis, a constant flow rate of 600 nL/min was maintained, employing a linear gradient spanning 100 min. Specifically, the elution gradient was initially set to 1% mobile phase B for 10 min, followed by a linear increase from 1% to 34% mobile phase B over 60 min. Subsequently, the gradient rose linearly from 34% to 90% mobile phase B within 15 min. After maintaining 90% mobile phase B for 12 min, the content of mobile phase B was gradually returned to 1% and held steady for 3 min. The mass spectrometer operated in a data-dependent mode, enabling precise proteomic analysis.

Protein extracts were subjected
to filter-aided sample preparation as described elsewhere.^{40 (link)} The resulting peptides from recent teeth were
analyzed by liquid chromatography–tandem mass spectrometry
(LC–MS/MS) performed using an UltiMate 3000 RSLCnano system
connected to an Orbitrap Fusion Lumos Tribrid spectrometer (Thermo
Fisher Scientific). Peptides from the ancient tooth and dental calculus
were analyzed on a nanoElute system online coupled with a timsTOF
Pro spectrometer (Bruker). See the Supporting Information file for full details regarding the analyses and
data evaluation, including the custom database described in Table S3.