Pepswift monolithic nano column

Samples digested with endolysosomal proteases were desalted using C18 ZipTips (EMD Millipore, Billerica, MA, USA). Resulting peptides were separated by reverse-phase nano-HPLC (Dionex Ultimate 3000, Thermo Fisher Scientific, Bremen, Germany, column: PepSwift Monolithic Nano Column, 100 μm × 25 cm, Dionex), where the column was eluted with an acetonitrile gradient (Solvent A: 0.1% (v/v) FA/0.01% (v/v) TFA/5% (v/v) ACN; solvent B: 0.1% (v/v) FA/0.01% (v/v) TFA/90% (v/v) ACN; 5–45% B in 60 min) at a flow rate of 1 μL/min at 55 °C. The peptides were analyzed by a Q Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific) directly coupled to the HPLC. Capillary voltage at the nano-electrospray head was 2 kV. The instrument was tuned for maximum sensitivity. For peptide assignments, a top 12 method was used with the normalized fragmentation energy at 27%. Survey and fragment spectra were analyzed with Proteome Discoverer version 1.4 with SequestHT as search engine (Thermo Fisher Scientific) or PEAKS Studio 8 (Bioinformatics Solutions, Waterloo, ON, Canada), respectively. Searches were conducted with single allergen sequences. Only peptides with high confidence scores (XCorr ≥ 2.3 for SequestHT, −10lgP ≥ 35 for PEAKS) were considered.

5 μL of A. lumbricoides extract (1000 μg/mL) were digested with the ProteoExtract All-in-One Trypsin Digestion Kit (EMD Millipore, Billerica, MA, USA) and desalted using C18ZipTips (EMD Millipore, Billerica, MA, USA). Resulting peptides were separated by reverse-phase nano-high performance liquid chromatography (HPLC, Dionex Ultimate 3000, Thermo Fisher Scientific, Bremen, Germany, column: PepSwift Monolithic Nano Column, 100 μM × 25 cm, Dionex). The column was developed with an acetonitrile gradient (Solvent A: 0.1% (v/v) FA/0.01% (v/v) TFA/5% (v/v) ACN; solvent B: 0.1% (v/v) FA/0.01% (v/v) TFA/90% (v/v) ACN; 5–45% B in 60 min) at a flow rate of 1 μL/min at 55 °C). The HPLC was directly coupled via nano electrospray to a Q Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific). Capillary voltage was 2 kV. For peptide assignment, a top 12 MS/MS method was used with the normalized fragmentation energy set to 27%. Proteins were identified with PEAKS Studio 8 (Bioinformatics Solutions, Waterloo, Canada), using the UniProt (SwissProt/TrEMBL) sequence database. For the identification of post-translational modifications and amino acid exchanges, the PTM and Spider modules of PEAKS Studio were used. Only peptides with high confidence scores (−10lgp ≥ 35, corresponding to false discovery rate (FDR) < 0.5%) were considered in the database searches.

A. lumbricoides extract (1000 µg/mL) was digested using the ProteoExtract All-in-One Trypsin Digestion Kit (EMD Millipore, Billerica, MA, USA) and desalted using C18ZipTips (EMD Millipore, Billerica, MA, USA). Resulting peptides were separated by reverse-phase nano-high performance liquid chromatography (HPLC, Dionex Ultimate 3000, Thermo Fisher Scientific, Bremen, Germany, column: PepSwift Monolithic Nano Column, 100 µM × 25 cm, Dionex). The column was developed with an acetonitrile gradient (Solvent A: 0.1% (v/v) FA/0.01% (v/v) TFA/5% (v/v) ACN; solvent B: 0.1% (v/v) FA/0.01% (v/v) TFA/90% (v/v) ACN; 5–45% B in 60 min) at a flow rate of 1 µL/min at 55 °C. The HPLC was directly coupled via nano electrospray to a Q Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific). Capillary voltage was 2 kV. For peptide assignment, a top 12 MS/MS method was used with the normalized fragmentation energy set to 27%. The protein was identified with PEAKS Studio 8 (Bioinformatics Solutions, Waterloo, ON, Canada) using the UniProt (SwissProt/TrEMBL) sequence database. Only peptides with high confidence scores (−10lgp ≥ 35, corresponding to false discovery rate (FDR) < 0.5%) were considered in the database searches [18 (link)]. Abundance of Al-CPI in the extract was calculated based on the “Area” parameter from the summarized proteomic search results [23 (link)].