*2.3. Plasma Manipulation and Digestion*

Plasma samples were sequentially subjected to high abundant plasma protein depletion and trypsin/Lys-C digestion. To remove the 14 most abundant plasma proteins (albumin, IgA, IgG, IgM, α1-antitrypsin, α1-acid glycoprotein, apolipoprotein A1, apolipoprotein A2, complement C3, transferrin, α2-macroglobulin, transthyretin, haptoglobin, and fibrinogen), a 40 μL aliquot of plasma diluted 4-fold with a proprietary "Buffer A" was injected into a MARS14 depletion column (Agilent Technology, Palo Alto, CA, USA) on a binary HPLC system (20A Prominence, Shimadzu, Tokyo, Japan). The unbound fraction was buffer-exchanged into 8 M urea in 50 mM Tris (pH 8), concentrated to approximately 50 μL by ultrafiltration using a Vivaspin 500 3 kDa cutoff filter (Sartorius, Goettingen, Germany), and then transferred to a new filter unit (Nanosep, 30 kDa; Pall Corporation, NY, USA). A 200 μL aliquot of 8 M urea in 50 mM Tris (pH 8.5) was added, and the mixture was centrifuged at 14,000× *g* for 15 min, with the procedure repeated twice. The flow-through from the collection tube was discarded, 100 μL of 0.05 M iodoacetamide solution was added, and the preparation was mixed at 600 rpm in a thermo-mixer for 1 min and incubated without mixing for 20 min. The filter units were centrifuged at 14,000× *g* for 10 min; 100 μL of 8 M urea in 50 mM Tris (pH 8.5) was added, and the filter units were again centrifuged at 14,000× *g* for 15 min, with this step repeated twice. A 100 μL aliquot of 0.05 M ammonium bicarbonate was added to the filter unit, and the unit was centrifuged at 14,000× *g* for 10 min, with this step also repeated twice. A 40 μL aliquot of 0.05 M ammonium bicarbonate containing 2.5 μg Lys-C/trypsin was added, and the preparation was mixed at 600 rpm in a thermo-mixer for 1 min. The units were incubated in a wet chamber at 37 ◦C for 12 h and transferred to new collection tubes. The filter units were centrifuged at 14,000× *g* for 10 min, 40 μL of 0.5 M NaCl was added, and the filter units were again centrifuged at 14,000× *g* for 10 min. The digestion reaction was stopped by the addition of formic acid to a final concentration of 0.3%. The peptide mixture was desalted with a Sep Pak C-18 cartridge (Waters, Milford, MA, USA), lyophilized with a cold trap (CentriVap Cold Traps, Labconco, Kansas City, MO, USA), and stored at −80 ◦C until used.

## *2.4. Nano-LC-ESI-MS*/*MS Analysis*

Peptides were separated using a Dionex UltiMate 3000 RSLCnano system (Thermo Fisher Scientific, Waltham, MA, USA). Tryptic peptides from a bead column were reconstituted in 0.1% formic acid and separated on a 50 cm Easy-Spray column with a 75 μm inner diameter packed with 2 μm C18 resin (Thermo Fisher Scientific) over 200 min (250 nL/min). The column was developed using a 0–45% acetonitrile gradient in 0.1% formic acid and 5% DMSO at 50 ◦C. The LC was coupled to a Q Exactive mass spectrometer with a nano-ESI source. Mass spectra were acquired in a data-dependent mode with an automatic switch between a full scan and 20 data-dependent MS/MS scans. The target value for the full scan MS spectra was 3,000,000, with a maximum injection time of 120 ms and a resolution of 70,000 at m/z 400. Repeated peptides were dynamically excluded for 20 s. All MS data have been deposited in the PRIDE archive (www.ebi.ac.uk/pride/archive/projects/PXD017211) under Project PXD017211 [24].
