C18 ziptip

Major protein gel bands of major peanut allergens were excised, and in-gel trypsin digested as previously described [15 (link)]. Briefly, protein bands were resolved on 12% PAA gels and excised at positions corresponding to the four major peanut allergens (Figure 1a). Proteins in excised bands had their disulphide bridges reduced by 10 mM dithiothreitol, and cysteines alkylated with 55 mM iodoacetamide. Both samples were digested with proteomics-grade trypsin (Sigma-Aldrich, Taufkirchen, Germany) in a 1:50 enzyme-to-substrate ratio overnight at 37 °C. After trypsin digestion, sample cleanup with C18 ZipTips (Sigma-Aldrich, Taufkirchen, Germany) was done, and samples were ready for HRMS analysis.

Each of 4 peptides-fractions (for human milk glycolproteomics) was dissolved in 40 μL enrichment buffer solution (80% acetonitrile/1% trifluoroacetic acid), and transferred to a hydrophilic microcolumn (HILIC column, Dalian Institute of Chemistry Physics, Chinese Academy of Sciences, China) (13 (link)), and the enrichment was completed by centrifugation at 4000 g for 15 min. The microcolumns were then washed 3 times with enrichment buffer. The glycopeptides were then eluted with 10% acetonitrile and lyophilized. Then, the lyophilized glycopeptides were reconstituted in 50 μL of 50 mM ammonium bicarbonate buffer dissolved in heavy oxygen water (H₂¹⁸O), 2 μL PNGase F glycosidase (New England Biolabs, United Kingdom) was added, and the peptides were digested overnight at 37°C. Finally, the salt was removed according to the instructions of C18 ZipTips (Sigma, United States), and the samples were freeze-dried for LC/MS analysis.

20 μg of protein from each sample was reduced with 25 mM DTT for 30 min at 37°C and alkylated with 55 mM iodoacetamide for 20 min in the dark at room temperature. The reaction was diluted with three volumes of 50 mM Tris-HCl (pH 8). Trypsin (mass spectrometry grade, Promega, USA) which was reconstituted in 50 mM ammonium bicarbonate in a 1 : 20 ratio was added and incubated at 37°C for 16 h. Subsequently, the reaction was quenched by 10% TFA in ammonium bicarbonate. Peptides were desalted using C18 zip tips (Sigma-Aldrich, USA) of 0.6 μl bed volume following the manufacturer's protocol, lyophilized by vacuum centrifugation, reconstituted in 0.1% (v/v) FA in LC-MS/MS grade water (Sigma-Aldrich, USA), and quantified as per the Scopes method. Peptide concentration was normalized as 0.1 μg/μl.

The protein digestion was performed following a standard enhanced, filter-aided sample preparation protocol (FASP) [32 (link)]. Briefly, after washing, proteins were reduced and alkylated. On-filter digestion was performed with 1.2 µg Trypsin/Lys-C mix (Promega, Madison, USA) for 14 h at 37 °C. Digested peptides were recovered, dried, and redissolved in 0.1% aqueous trifluoroacetic acid. After desalting and sample cleanup by C18 ZipTips (Sigma-Aldrich), all samples were spiked with standardized indexed retention time reference peptides for facilitation of retention time alignment (iRT kit, Biognosys, Switzerland). For each liquid chromatography (LC)–mass spectrometer (MS) analysis, 3 µg of digested protein was loaded onto the LC column.

LC-MS/MS analysis following an in-gel digestion was performed by International Research Resource Center in Biomolecular Mass Spectrometry and Proteomics at University of California San Francisco (UCSF). The in-gel digestion was carried out using their standard sample preparation procedure, available
here. The proteins in each gel band were reduced, alkylated, and finally digested overnight with 100 ng of Trypsin (
Trypsin/sequencing-grade-modified-Trypsin/?catNum=V5111">Promega, v511c). The resultant peptide mixture was desalted with µC18-ZipTips (
Millipore, ZTC18M960), speed vacuum dried, suspended in 0.1% formic acid, and analyzed on a Velos Pro Elite Orbitrap Mass Spectrometer. The mass spectrometric data was obtained in a data-dependent acquisition mode. The data was then converted into peak lists with PAVA
^{29 (link)}
, a software developed by International Research Resource Center in Biomolecular Mass Spectrometry and Proteomics. The raw data can be also processed by software package
MaxQuant. Using
ProteinProspector search engine (v5.20.1), the peak lists were searched against the
SwissProt
C. elegans database and the proteins detected by the LC-MS/MS were subsequently identified.

Five and three fragments immunoprecipitated with anti-µNS (4 and 5 wpc) and anti-µ1C (5 wpc), respectively, that were not observed at 0 wpc, were excised and in-gel digested with 0.1 µg of trypsin in 20 µL of 50 mM ammonium bicarbonate, pH 7.8 for 16 h at 37 °C (Promega, Madison, WI, USA). The peptides were purified with µ-C18 ZipTips (Millipore, Billerica, MA, USA), and analyzed using an Ultimate 3000 nano-UHPLC system (Dionex, Sunnyvale, CA, USA) connected to a Q Exactive mass spectrometer (ThermoElectron, Bremen, Germany). Liquid chromatography and mass spectrometry was performed as previously described [37 (link)]. Data were acquired using Xcalibur v2.5.5 and raw files were processed to generate peak list in Mascot generic format (*.mgf) using ProteoWizard release (Version 3.0.331). Database searches were performed using Mascot (Version 2.4.0) against the protein sequences of λ1, λ2, λ3, µNS, µ1, µ2, σNS, σ1, σ2 and σ3 assuming the digestion enzyme trypsin and semi-trypsin, at a maximum of one missed cleavage site, fragment ion mass tolerance of 0.05 Da, parent ion tolerance of 10 ppm and oxidation of methionines, propionamidylation of cysteines, acetylation of the protein N-terminus as variable modifications. Scaffold 4.4.8 (Proteome Software Inc., Portland, OR, USA) was used to validate MS/MS based peptide and protein identifications.