Easy nlc 2

Aliquots of the digested peptides (250 ng), spiked with heavy labeled peptides and index retention time (iRT) peptides, were analyzed with Easy-nLC-II coupled to a TSQ Vantage mass spectrometer (Thermo Fisher Scientific)^{32 (link)}. The peptide mixture was separated with a 150 mm × 75 µm ID column packed with ReproSil-Pur C18-AQ 5 µm resin (Dr. Maisch GmbH). An unscheduled analysis of the sample was carried out to generate iRT values of target peptides and their heavy counterparts. Skyline software was used to build up the scheduled method for the selected targets using the unscheduled run. The scheduled method was then edited by removing interfering signals^{32 (link)} to monitor 99 transitions from 33 peptides, representing 10 proteins and the iRT peptides.
The 86 serum samples (N = 43 vs. 43) were prepared without depletion and analyzed as randomized batches. To monitor the variation of peak areas and retention time across and within the batches, a pooled digest of the undepleted serum was included in each batch.

The fraction U₁-SCRTX-Lg1a was analyzed in positive ion mode on an LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) coupled to an Easy-nLC II (Thermo Fisher Scientific, Bremen, Germany), according to Abreu et al. [12 (link)] with some modifications. The mass spectrometer was programmed for a full scan, recorded between m/z 300 and 2000 with a resolution of 60,000 (at m/z 400). The 10 most abundant peaks were fragmented using collision-induced dissociation (CID) and analyzed in an ion trap. The isolation window for precursor ions was set to 2 m/z, the minimum count of ions to trigger events (MS²) was 10,000, and the dynamic exclusion time was set to 90 s. Normalized collision energy was set to 35%. In order to identify the U₁-SCRTX-Lg1a fraction, mass spectrometry (MS) raw data were processed and searched in the PEAKS Studio software (v8; Bioinformatics Solutions, Waterloo, ON, Canada) [73 (link)]. To determine its amino acid sequence, we performed de novo sequencing from MS/MS data with the following parameters: a precursor mass tolerance of 10 ppm and a fragment ion mass tolerance of 0.5. De novo peptides, whose ALC scores ≥80% were searched against the UniProt-SwissProt database using the PEAKS DB tool. The peptide false discovery rate (FDR) was predictable by the decoy fusion method and was selected at a maximum of 1%.

The concentration of various biological markers for inflammation and stress in blood and saliva [33 (link), 34 (link)] will be measured using multiplex immunoassay technology (Meso Scale Discovery, MSD). This multi-array technology enables the detection of up to 72 substances in multiplex format. Untargeted biomarker analysis will be performed using proteomics. This will be done by using mass spectrometry in combination with various separation methods such as two-dimensional gel electrophoresis (2-DE) and liquid chromatography (LC). A venous blood sample of about 10 ml is taken from the arm. The saliva sample is taken 15 min after washing the mouth with water by placing a cotton swab (Salivette) in the mouth for 3 min. All samples will be unidentified (marked with a code number). 2-DE instruments in combination with digitizing camera and special software (PDQuest, Bio Rad) for protein separation and quantification are available at the PAINOMICS® laboratory (Linköping University). The laboratory is also equipped with a MESO QUICKPLEX SQ 120 instrument (Meso Scale Discovery, Maryland, USA). EASY-nLC II (Thermo Scientific) combined with LTQ Orbitrap Velos Pro hybrid mass spectrometer (Thermo Scientific) with a nano-electrospray source available at the Core Facility at the Medical Faculty, Linköping University will be used.

Peptides were injected onto a reverse phase nanobore HPLC column (AcuTech Scientific, C18, 1.8um particle size, 360 um x 20 cm, 150 um ID), equilibrated in solvent A (water/acetonitrile/FA, 98/2/0.1, v/v/v) and eluted (300 nL/min) with an increasing concentration of solvent B (acetonitrile/water/FA, 98/2/0.1, v/v/v: min/% F; 0/0, 5/3, 18/7, 74/12, 144/24, 153/27, 162/40, 164/80, 174/80, 176/0, 180/0) using an EASY-nLC II (Thermo Fisher Scientific). The effluent from the column was directed to a nanospray ionization source connected to a hybrid quadrupole-Orbitrap mass spectrometer (Q Exactive Plus, Thermo Fisher Scientific) acquiring mass spectra in a data-dependent mode alternating between a full scan (m/z 350-1700, Automated Gain Control (AGC) target 3 x 106, 50 ms maximum injection time, FWHM resolution 70,000 at m/z 200) and up to 15 MS/MS scans (quadrupole isolation of charge states 2-7, isolation window 0.7 m/z) with previously optimized fragmentation conditions (normalized collision energy of 32, dynamic exclusion of 30 s, AGC target 1 x 105, 100 ms maximum injection time, FWHM resolution 35,000 at m/z 200).