Nanolockspray

Peptides were sequenced on a Synapt G2 HDMS (Waters, Milfords, MS, EUA) mass spectrometer coupled to a UPLC NanoAcquity system with 1D technology (Waters, Milfords, MS, EUA) and captured by a C18 Symmetry column (5 μm, 180 μm × 20 mm) (Waters, Milfords, MS, EUA). The peptides were separated by using a 2–90% acetonitrile gradient in 0.1% formic acid and an HSS T3 analytical column (1.8 μm, 75 μm × 100 mm) (Waters, Milfords, MS, EUA) with a flow of 300 μL min^− 1 for 120 min. The data were acquired on a Waters Synapt G2S Q-TOF mass spectrometer equipped with a NanoLockSpray (Waters, Milfords, MS, EUA). The experiments were performed in the HDMSE mode (data-independent analysis). The mass spectra were processed with the ProteinLynxGlobalServer (PLGS) software version 3.1. The proteins were identified by comparison to the Aspergillus UNIPROT database (207,966 proteins) [42 ]. The defined parameters were automatic tolerance for precursors and ion products, minimum of three corresponding ion fragments per peptide, minimum of seven corresponding ion fragments per protein, trypsin missed cleavage, carbamidomethylation as a fixed modification, oxidation of methionine as a variable modification, and 4% FDR peptide.

Samples were processed as described previously by Duarte Escalante et al. (2018) (link) with some modifications. Briefly, total protein extracts from fibroblasts NA2, NA3, AD1, and AD3 were treated with a 0.05 M DTT solution as a reducing agent. After 45 min, 0.03 M iodoacetamide was added and the mixture was incubated for 2 h at room temperature in the dark. The samples were then washed 3 times with 0.1 M ammonium bicarbonate solution and dehydrated with 100% acetonitrile under vacuum. Digestion was performed by adding 30 μl of a modified porcine trypsin solution (20 ng/μl; Promega, Madison, WI, United States) in 0.05 M ammonium bicarbonate solution, followed by incubation for 24 h at 37°C.
The resulting peptides were extracted twice in 50% (v/v) acetonitrile, and 5% (v/v) formic acid for 30 min with sonication. The volume was reduced by evaporation in a vacuum centrifuge and adjusted to 20 μl with 1% (v/v) formic acid. Mass spectrometric analysis was performed with an integrated nano-LC-ESI MS/MS system: quadrupole/time of light, synapt G2 high-definition mass spectrometer (Waters Corporation, Milford, MA, United States) equipped with a NanoLockSpray ion source, coupled online to a nanoacquity UPLC system (Waters Corporation, Milford, MA, United States).

Qualitative and quantitative proteomic analyses were performed in a bidimensional micro UPLC tandem nanoESI-HDMSE platform by multiplexed data-independent acquisition (DIA) experiments82 (link). The Acquity UPLC M-Class System (Waters Corporation, Milford, MA) coupled to a Synapt G2-Si mass spectrometer (Waters Corporation) was used.
Peptide loads were separated in a nanoACQUITY UPLC HSS T3 Column (1.8 μm, 75 μm X 150 mm, Waters Corporation). Peptides were eluted using an acetonitrile gradient ranging from 7% to 40% (v/v) for 95 min at a flow rate of 0.4 μL/min directly into a Synapt G2-Si. For every measurement, the mass spectrometer was operated in resolution mode with an m/z resolving power of approximately 40 000 FWHM and using ion mobility with a cross-section resolving power at least 40 Ω/ΔΩ. The effective resolution obtained with the conjoined ion mobility was 1 800 000 FWHM. MS/MS analyses were performed by nanoelectrospray ionization in positive ion mode nanoESI (+) and with a NanoLock Spray (Waters Corporation, Manchester, UK) ionization source. The lock mass channel was sampled every 30 sec. The mass spectrometer was calibrated with an MS/MS spectrum of [Glu1]-Fibrinopeptide B human (Glu-Fib) solution that was delivered through the reference sprayer of the NanoLock Spray source.