Depleted Prefractionated Human Plasma for Data Independent Acquisition Spectral Library

Description We established a spectral library for data-independent analysis of human plasma samples, using a pooled plasma sample from Danish adults. The samples were prepared for proteome analysis using an in-house developed protocol consuming one microliter of plasma per sample, and the tryptic digest and subsequent C18 purification was performed in 96-well plates. The spectral library consists of plasma which was analyzed neat (i.e. non-treated), and top 14 depleted. The samples were subjected to high pH reversed-phase peptide fractionated using C18 tips (8 fraction) and a HPLC system (20 fraction). The resulting peptides were analyzed on a Q Exactive with a capflow reversed-phase C18 LC-MS/MS setup in data-dependent mode. The final spectral library contains 21788 precursors and 1000 proteins (both at FDR<0.01). Sample Processing Protocol EDTA plasma samples from Danish adults were obtained for the study (age: 48.9±18.5 years; sex: 51.6%). The participants had given an oral and written informed consent and the biobank has been approved by Danish Data Protection Agency and the regional Ethical Committee. The highly abundant proteins were depleted from some of the aliquots of the pooled sample using Seppro IgY14 spin columns (Sigma-Aldrich), according to the manufacturer’s instructions. The samples were prepared for proteome analysis using a 96-well PVDF strategy. All solvent transfer and addition steps were performed with a multichannel pipette. Initially, all samples were randomized, and resuspended in 8 M urea, 50 mM ammonium bicarbonate (ABC) at pH 8.5. Cysteine residues were reduced by addition of dithiothreitol and alkylated by addition of iodoacetamide. After incubation for 20 min in the dark, the samples were transferred to a 96-well plate with a polyvinylidene fluoride (PVDF) membrane at the bottom. A vacuum manifold was used to facilitate the liquid transfer through the 96-well PVDF membrane plate. Protein digestion was performed with sequencing grade trypsin (V5111, Promega, nominal enzyme to protein ratio 1:25). After incubation for 2 h at 37 °C in a humidified incubator, the remaining digestion buffer was evacuated from the 96-well PVDF membrane plate. The eluents were pooled in a 96-well plate and centrifuged to dryness in a vacuum centrifuge. The peptides were resuspended and transferred to a 96-well MACROSPIN C18 plate (TARGA, Nest Group). The samples were washed with 0.1% FA, and peptides were eluted with 40/70% ACN, 0.1% FA. The samples were subjected to high pH reversed-phase peptide fractionated using C18 tips (8 fraction) and a HPLC system (20 fraction). iRT peptides (Biognosys, Schlieren, Switzerland) were spiked into the samples according to manufacturer instructions. 0.5 µg peptide material was loaded onto a capflow PicoChip column (150 µm x 10 cm Acquity BEH C18 1.7 µm 130 Å, New Objective, Woburn, MA) with 2 µL min solvent A (0.1% FA). The proteolytic peptides were eluted from the column using 2% solvent B (0.1% FA in ACN) in solvent A, which was increased from 2-30% on a 40 min ramp gradient and to 35% on a 5 min ramp gradient, at a flowrate of 1000 nL/min. The PicoChip containing an emitter for nanospray ionization was kept at 50 °C, and mounted directly at the inlet to a Orbitrap Q Exactive mass spectrometer (Thermo Scientific, Bremen, Germany). The mass spectrometer was operated in positive DDA top 12 mode with the following MS1 scan settings: mass-to-charge (m/z) range 375-1400, resolution 70000 @ m/z 200, AGC target 3e6, max IT 60 ms. MS2 scan settings: resolution 17500 @ m/z 200, AGC target 1e5, max IT 100 ms, isolation window m/z 1.6, NCE 27, underfill ratio 1% (intensity threshold 1e4), charge state exclusion unassigned, 1, >6, peptide match preferred, exclude isotopes on, dynamic exclusion 40 s. Data Processing Protocol The .RAW-files were directly processed with MaxQuant v1.5.4.1. using the UniProt Human reference proteome (downloaded April 7th 2016, 70634 protein entries) with the iRT peptide sequences added. Standard settings were employed in MaxQuant, including a first search with 20 ppm mass tolerance followed by data calibration, and a main search with 4.5 ppm mass tolerance. A maximum of three tryptic missed cleavages were allowed. Additionally, the following modifications were found to be abundant with the applied MStern blotting protocol, and were included in the search: carbamidomethylated cysteine residues (fixed), acetylation of the N-terminal of proteins (variable), oxidation of methionine (variable), deamidation of asparginine and glutamine residues (variable). Identified proteins and peptide spectral matches were filtered to <1% false discovery rate (FDR) using a forward/reverse database search strategy in MaxQuant. For the DDA analysis of the pooled plasma sample, the match between runs feature was enabled in MaxQuant using standard parameters. This allow the transfer of MS2 identifications across LC-MS samples based on accurate m/z and retention time. LFQ was performed with a minimum ratio count of one. Contaminants were filtered from the final protein-list. Contact Tue Bjerg Bennike, Aalborg University Hanno Steen, Departments of Pathology, Boston Children’s Hospital and Harvard Medical School, Boston, MA, USA (lab head) Submission Date 08/11/2017 Publication Date 20/04/2018 Project PXD008163