U of Masaryk RECETOX - Vidova et al. Sci Rep 2021 Supplementary

Simultaneous quantitative profiling of clinically relevant immune markers in neonatal stool swabs to reveal inflammation
Data License: CC BY 4.0 | ProteomeXchange: PXD027988 | doi: https://doi.org/10.6069/jwjg-sb14
  • Organism: Homo sapiens
  • Instrument: 6495B Triple Quadrupole LC/MS
  • SpikeIn: Yes
  • Keywords: fecal inflammatory markers, microbial colonization, immune response, targeted quantitative proteomics, mass spectrometry
  • Lab head: Zdenek Spacil Submitter: Zdenek Spacil
Abstract
An aberrant immune response developed early in life may trigger inflammatory bowel disease (IBD) and food allergies (e.g., celiac disease). Fecal levels of immune markers categorize an inflammatory response (e.g., food allergy, autoimmune) paralleled with the initial microbial colonization. The immunoaffinity assays are routinely applied to quantify circulating immune protein markers in blood/serum. However, a reliable, multiplex assay to quantify fecal levels of immune proteins is unavailable. We developed mass spectrometry assays to simultaneously quantify fecal calprotectin, myeloperoxidase, eosinophil-derived neurotoxin, eosinophil cationic protein, alpha 1 antitrypsin 1, and adaptive immunity effectors in 134 neonatal stool swabs. We optimized extraction and proteolytic protocol and validated the multiplex assay in terms of linearity of response (>100; typically 0.04 to 14.77 µg/mg of total protein), coefficient of determination (R2; >0.99), the limit of detection (LOD; 0.003 to 0.04 µg/mg of total protein), the limit of quantification (LOQ; 0.009 to 0.122 µg/mg of total protein) and robustness. The median CV of intra- and interday precision was 9.8% and 14.1%, respectively. We quantified breast milk-derived IGHA2 to differentiate meconium from feces samples and to detect the first food intake. An early life profiling of immune markers reflects disrupted intestinal homeostasis, and it is perhaps suitable for pre-symptomatic interception of IBD and food allergies.
Experiment Description
We precipitated proteins by adding 1 ml of 80% IPA to the sample and orbital shaking (5 min, 1600 rpm). We centrifuged samples (2 min, 12 000x g) and removed 50 µL of supernatant. The residual sample volume (950 µL), including the swab, was dried in speed vac overnight (minimum 6 hours). Dried samples were reconstituted in 1500 µL of buffer (50 mM ABB with 5 g/l SDC) and homogenized (Benchmark Scientifics, Bead Blaster 24 homogenizer, 4 pulses x 30 s; 4 m/s; inter-time 10 s; ambient temperature). Next, we centrifuged samples (3000x g; 10 min), transferred a supernatant (500 µL) into a clean vial, and centrifuged the vial again (12 000x g; 5 min). After the second centrifugation, the supernatant (400 µL) was transferred into another clean vial for the trypsin digestion of extracted proteins. Total protein concentration in 134 neonatal meconium or feces swabs extracts were assessed in 10 µL of protein extract using the BCA assay (cat. #23225). We subjected the protein extract (50 µL) to the trypsin digestion protocol. We reduced and alkylated protein extracts (50 µL) by adding 5 µL of 200 mM DTT (10 min at 95˚C) and consequently adding 5 µL of 400 mM IAA (30 min at ambient temperature in the dark). We added a working solution (10 µL; 500-600 nM) of the SIL-TCT peptide internal standards into a sample. Next, we added trypsin (3 µL; 1 µg/µL) approximately in the ratio of 1:70 to the total protein content and incubated samples at 37 ˚C (orbital shaking 200 rpm). We quenched the trypsin digestion after 5 hours by adding 200 µL of 2% FA and peptides were purified using solid-phase extraction (SPE, Oasis HLB prime; 96-well plate format, 30 mg; Waters, Milford, MA). Samples were loaded on SPE, washed with 300 µL of 2% FA, and eluted with 200 µL 50% ACN with 2% FA. SPE eluates were dried in speed vac, reconstituted in 50 µL 5% ACN with 0.1% FA, resulting in 100-120 nM SIL-TCT internal standard concentrations in the sample, and analyzed by UHPLC/SRM-MS. Samples were injected (2 µL) on the UHPLC system (1260 series Agilent, CA) equipped with an analytical column (C18 Peptide CSH; 1.7 µm, 2.1 mm i.d. x 100 mm; cat. #186006937; Waters, Milford, MA) thermostated at 40 °C. The mobile phase consisted of solution A (0.1% FA in water) and solution B (0.1% FA in ACN). The flow rate was 300 µL/min, and the gradient elution program consisted of analytical (0-30.9 min) and re-equilibration part (31-35 min): 0.0 min 5% B; 25 min 30% B; 25.5 min 95% B; 30.9 min 95% B; 31 min 5% B; 35 min 5% B. A standard-flow electrospray was used to couple the UHPLC system with a triple quadrupole mass spectrometer (AJS 6495A, Agilent, CA). Electrospray source operated in positive ion mode (capillary voltage 3.5 kV; gas flow rate 11 L/min at 130 °C; sheath gas pressure 25 PSI at 400 °C; nozzle voltage 500 V). We monitored 98 transitions per the dynamic SRM mode analysis, with 2 min window scheduled around peptide experimental RT.
Sample Description
Trained hospital personnel collected feces swabs from neonates (n=134) during the first, second, or third day following the birth using FLOQSwabs (cat. #520CS01, Copan, Italy). Meconium or feces were scooped up from a conventional diaper using a swab, then the swab was placed inside a 2 ml vial, and the handle was broken off. The vial was sealed, immediately placed in a -80˚C freezer, and stored until pre-analytical sample processing and analysis. The study subjects were female (n=56) and male (n=78) neonates at gestation age [weeks + days] ranging from 38 + 2 to 41 + 3. An average birth weight [g] 4394.1 ± 427.0 SD ranged from 2470 to 4900g. Samples were from neonates delivered vaginally (n=114) or via cesarean section (n=20).
Created on 8/17/21, 8:18 PM
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Calibration peptide TPLTATLSK.sky.zip2021-08-17 20:18:281121033
Calibration, peptide SAVQGPPER.sky.zip2021-08-17 20:18:281121254
Calibration peptide VVLEGGIDPILR.sky.zip2021-08-17 20:18:28112630
Calibration peptide LGHPDTLNQGEFK.sky.zip2021-08-17 20:18:28112824
Calibration peptide NQNTFLR.sky.zip2021-08-17 20:18:28112830
Calibration, peptide DPPQYPVVPVHLDR.sky.zip2021-08-17 20:18:281121026
Calibration peptide DLQNFLK.sky.zip2021-08-17 20:18:281121030
Calibration peptide DASGATFTWTPSSGK.sky.zip2021-08-17 20:18:281121027
Calibration peptide AVLTIDEK.sky.zip2021-08-17 20:18:281121430
Calibration peptide ALNSIIDVYHK.sky.zip2021-08-17 20:18:281121024
Proteomic analysis of 134 newborn samples.sky.zip2021-08-17 20:18:289102093134
Trypsin digestion time lapse.sky.zip2021-08-17 20:18:28910209321
Matrix effect on protein quantification.sky.zip2021-08-17 20:18:28910209821