Nanoelute lc system

In-gel digestion of protein samples was carried out according to the method of Fuchs et al. [163 (link)]. However, instead of 10% Criterion™ Tris–HCl Protein Gel, a Short 12% SDS polyacrylamide gel was used. LC–MS/MS analysis, using a timsTOF Pro mass spectrometer equipped with a NanoElute LC System (Bruker Daltonik GmbH, Bremen, Germany) on an Aurora column (250 × 0.075 mm, 1.6 µm; IonOpticks, Hanover St., Australia), was carried out according to Engelhart-Straub et al. [164 (link)]. For measurement normalization, quality control samples were prepared by mixing 1 µL of each sample and analyzed at equal intervals between samples.

A total of 2 µL of the reconstituted phosphor-peptides were separated on a nanoElute LC system (Bruker Corporation, Billerica, MA, USA) at 400 nL/min using a reversed phase C18 column (Aurora UHPLC emitter column, 25 cm × 75 µm 1.6 µm, IonOpticks) which was heated to 50 °C. Peptides were loaded onto the column in direct injection mode at 600 bar. Mobile phase A was 0.1% FA (v/v) in water and mobile phase B 0.1% FA (v/v) in can. Peptides were separated, running a linear gradient from 2% to 37% mobile phase B over 45 min. Afterwards, the column was rinsed for 5 min at 95% B followed by equilibration. Eluting peptides were analyzed in positive mode ESI-MS using parallel accumulation serial fragmentation (PASEF) enhanced data-independent acquisition mode (DIA) on a timsTOF Pro 2 mass spectrometer (Bruker Corporation). The dual TIMS (trapped ion mobility spectrometer) was operated at a fixed duty cycle close to 100% using equal accumulation and ramp times of 100 ms each, spanning a mobility range from 1/K₀ = 0.6 Vs cm⁻² to 1.6 Vs cm⁻². We defined 36 × 25 Th isolation windows from m/z 300 to 1165, resulting in fifteen diaPASEF scans per acquisition cycle. The collision energy was ramped linearly as a function of the mobility from 59 eV at 1/K₀ = 1.3 Vs cm⁻² to 20 eV at 1/K₀ = 0.85 Vs cm⁻².

We identified salivary proteins using mass spectrometry-based proteomics. Examining 500 ng of peptides following tryptic digestion and peptide separation mass spectrometry data were obtained using a nanoElute LC system coupled to a timsTOF Pro mass spectrometer (Bruker Daltonics). In accordance with previous procedures [21 (link)], all MS/MS samples were examined using PEAKS Studio X+ (v.10.5, Bioinformatics Solutions). A protein sequence database of reviewed human proteins with 74,823 entries from UniProt was utilized for all searches, assuming the presence of the digestive enzyme trypsin. The InteractiVenn tool [22 (link)] was used to create a Venn diagram that displays the unique and shared proteins from each comparison. The Human Salivary Proteome Wiki (https://www.salivaryproteome.org/), a saliva proteome-focused, open-access database, was used to identify the origin of distinctive proteins [23 (link)]. Protein biological function was determined in FunRich software (v. 3.1.4) (accessed 21 June 2020) [24 (link)] using hypergeometric analysis where significance level was 0.01. In Funrich we add the list of all identified proteins with the “add dataset” function. In the “gene enrichment” option, we selected “compare” to identify the biological processes that presented the greatest differences between the groups. We chose the top 3 of those comparisons.