Functional Genomics Center Zurich - p2242

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Quantitative proteomics analysis of Angiostrongylus vasorum-induced alterations in dog serum

  • Organism: Canis lupus familiaris
  • Instrument: Q Exactive HF-X
  • SpikeIn: Yes
  • Keywords: Angiostrongylus vasorum
Blood contains hundreds of proteins, reflecting ongoing cellular processes and immune reactions. Angiostrongylus vasorum infection is associated with a perturbed blood protein profile in dogs. However, the literature currently available lacks the necessary depth of analysis in order to resolve the observed pathologies in A. vasorum infections, including bleeding disorders. Using sera from 8 experimentally-infected dogs (i) before infection with A. vasorum, (ii) 34 days post-infection (p.i.; immature infection), and (iii) 75 days p.i. (mature patent infection), serum proteins were measured using liquid chromatography, tandem mass spectrometry (LC-MS/MS). For 2 dogs, serum was analyzed at days 104 and 230 p.i. additionally. A data-independent acquisition workflow was employed in order to generate quantitative data. Following computational analysis, we identified 139 up- and down-regulated proteins following infection (log2 ratio cutoff ≥ 1.0; q-value ≤ 0.05). Differences in serum profiles were most pronounced at day 75 p.i. compared to before infection. Among up-regulated proteins, chitinase 3, several saposin-like proteins, and heat shock proteins were found greatly increased (log2 fold-changes ≥ 5). Levels of pulmonary surfactant protein B were elevated on day 34 p.i. already, in the prepatent phase. Pathway enrichment revealed that complement (especially the lectin pathway) and coagulation cascades as significantly affected upon analysis of down-regulated proteins. Among them were mannan-binding lectin serine peptidases, ficolin, and coagulation factors. These results reflect the ongoing immune response and stress imposed to the lungs by the parasite. In addition, they bring new elements towards understanding the coagulopathies observed in some A. vasorum-infected dogs.
Experiment Description
Briefly, eight beagles (5 males and 3 females, 11 months of age at the start of the experiment) were inoculated with 200 A. vasorum L3 as described in previous papers based on the same animals [20,21]. For proteomics analysis and for each dog, time points were selected in order to reflect serum profiles (i) before infection (one week prior to infection; day -7), (ii) during prepatency (day 34 post-infection (p.i.)), and (iii) after patency (day 75 p.i.) (Fig. 1). Hence, each dog is represented by an uninfected (baseline) profile and at least two time points during ongoing infection. Serum from two further time points were chosen for two dogs additionally (days 104 and 230 p.i.).
Sample Description
Blood was collected from the jugular vein into serum tubes (Greiner Bio-One Vacuette Z serum clot activator (silica particles)) and spun down for 10 minutes at XXX x g, prior to storage at -20°C for 9 years. Protein enrichment was performed using the ProteoMiner small capacity kit (Bio-Rad) following the manufacturer’s instructions. Proteins were precipitated using trichloroacetic acid (TCA) according to a modified protocol by Thermo Fisher/Pierce. Briefly, 50 µl protein eluates were mixed with 450 µl dH2O, 100 µl sodium deoxycholate 0.15 % (w/v), and 100 µl TCA 72 % (w/v). After a 30 min incubation at 4 °C samples were vortexed, spun 30 min at 16’900 x g, 4 °C, and pellets were washed 3 times with pure ice-cold acetone. Pellets were air-dried and resuspended in 50 µl 4 % SDS, Tris-HCl 0.1 M, pH = 7.6, 0.1 M DTT. Protein in samples was quantified by Qubit protein assay (ThermoFisher Scientific). Samples were further alkylated and digested using a modified filter-aided sample preparation [22]. Thirty µg protein were mixed with 200 µl 8 M urea in 100 mM Tris-HCl pH 8.2, prior to be loaded onto Microcon 30 filters (Millipore, Cat. MRCF0R030) and spun at 14,000 x g for 25 min at 35 °C. Samples were washed again with 200 µl 8 M urea buffer and 100 µl iodoacetamide 0.05 M were added to the filter units. Samples were shaken for 1 min in a thermo-mixer (600 rpm) and 5 min at RT °C and spun as before. Filters were washed 2 x with 100 µl 8 M urea buffer and with 100 µl 0.5 M NaCl. Finally, 120 µl 0.05 M Triethylammoniumbicarbonate and 1.5 µl sequencing grade modified trypsin 0.4 µg/µl (Promega, V5113) were added, mixed 1 min (600 rpm) and incubated overnight in a wet cell at RT °C. Filter units were spun at 14,000 x g for 25 min and the flow-through acidified with trifluoroacetic acid (TFA) to reach a 0.5 % final concentration. Peptides were then desalted using C18 stage tips [23]. Briefly, equilibrated columns were loaded with 150 µl sample (in 3 % acetonitrile (ACN), 0.1 % TFA) and spun at 2,000 x g for 1 min. Samples were washed 2 x with 150 µl 3 % ACN, 0.1 % TFA and eluted in 150 µl 60 % ACN, 0.1 % TFA. Samples were placed into a speed-vac and dried to completeness. Pellets were resusp ended in 3 % ACN, 0.1 % formic acid (FA) and frozen at -20 °C pending use.
Created on 12/20/19, 8:44 AM

DIA analysis of serum samples

Sample concentration was adjusted to 0.5 µg/ml in 3 % ACN, 0.1 % FA and retention time normalization peptides (iRT, Biognosys) were added at a 1:20 ratio. Peptide samples were analyzed in random order in data independent acquisition (DIA) mode on a hybrid quadrupole-orbitrap mass spectrometer (Q Exactive HF, Thermo Scientific) operated in line with a nano UHPLC system (Easy-nLC 1000, Thermo Scientific). For each sample 3 μl of purified peptide mixture was loaded onto a custom-made fused-silica column (150 mm x 75 um ID) filled with porous reverse-phase chromatography resin (Reprosil-Pur C18 AQ, 1.9 μm spherical silica particles, 120 Å pore diameter, M/N:, Dr. Maisch GmbH, Ammerbuch, Germany). Bound peptides were eluted from the analytical column by running a linear gradient from 5 % to 35 % B in 120 min (Solvent A: 0.1% FA in water, solvent B: 0.1% FA in acetonitrile) at a linear flow rate of 300 nl/min. Eluted peptides were injected into the MS utilizing a nano ESI source (10 μm fused-silica spray emitter and Digital PicoView 565, O/N: DPV-550-565, New Objective, Woburn, MA). Mass spectra were recorded at a resolution of 60,000 (at 200 m/z) in profile mode using positive polarity. MS1 scans covering 350-1800 m/z were acquired using an AGC target of 3e6 and a maximum IT of 200 ms, and a centroid spectrum data type.

The acquired raw files were deposited at PRIDE under ... 

PRM assay development and characterization

A blank sample and 6 dilutions of peptides were included and measured in duplicates. Detection window consisted of five min before and after expected retention time for each peptide; these are provided in Table X. Response curves made of a serial dilution of heavy peptides in a digested serum matrix from a healthy beagle was used to determine the spiked-in concentrations. The analysis was conducted in Skyline (v. 4.2 [24]), using the top 5 predicted y fragment ions were extracted from the MS2 spectra to generate elution profiles, excluding y1 and y2 because of their small size. One peptide was monitored by extracted ion chromatogram (XIC) of 4 products due to the absence of a 5th.

Spectral libraries


NIST-formated library containing HCD spectra of 14 target peptides predicted by PROSIT for NCE=27, z=2.



BiblioSpec library generated in Skyline by importing Mascot search results generated from 20190604_003_Peptide_Mix_FF61456.raw (Cutoff score=0.95). The raw data was searched against UP000002254 using the following settings: Enzyme=Trypsin; FixedModification=Carbamidomethyl(C); VariableModification=Oxidation(M), 13C(6)15N(2)(K), 13C(6)15N(4)(R); PeptideMassTolerance=10ppm, FragmentMassTolerance=0.05Da; MaxMissedCleavages=1; InstrumentType=Q-Exactive

Validation of DIA data by PRM

Bound peptides were eluted from the analytical column by running a linear gradient from 5 % to 35 % B in 45 min (Solvent A: 0.1% FA in water, solvent B: 0.1% FA in acetonitrile) at a linear flow rate of 300 nl/min. MS1 scans were recorded at a resolution of 120,000 at a scan range of 350-1800 m/z in profile mode using positive polarity. We used AGC target of 3e6 and a maximum IT of 55 ms, and a centroid spectrum data type. Each MS1 scan was followed by 15 multiplexed fragment ion scans. Parallel reaction monitoring (PRM) scans were recorded at a resolution of 120,000 in profile mode using positive polarity. PRM scans were acquired as centroid spectrum data using an AGC target of 1e5; isolated precursors were fragmented using HCD at a NCE of 28. Further settings included maximum IT of 247 ms, isolation windows of 1.4 m/z. Fixed first mass was set at 100 m/z. The maximum injection time per PRM scan was selected by the instrument software to assure acquisition at a maximum scan speed of 3 Hz. Accordingly, the instrument cycle time was around 3.7 s at a chromatographic peak width of 15 s. The inclusion list encompassed 9 endogenous peptides, 9 heavy peptides (C-terminal R or K, carbamidomethyl C, z = .2 and .3; PEPotec, Thermo Scientific) listed in supplemental Table X), as well as the retention time normalization peptides (iRT).