Chip cube interface

Equivalent amounts of soluble Cashewgurt protein and cashew nut extract were trypsinized and analyzed by LC–MS/MS to characterize allergen content. To ensure complete characterization, the results of at least 13 independent sample runs for each of the cashew nut and Cashewgurt protein extracts were merged to show the overall percent of the allergens that were identified (percent coverage). Samples treated with trypsin as previously described [13 (link)] were run on an Agilent 6520 Q-TOF LC/MS using a Chip Cube interface with a Large Capacity Chip (II) (Agilent, Santa Clara, CA, USA) and analyzed using Mascot software (Matrix Science, Boston, MA, USA) to determine protein identification and percent of protein coverage.

As previously published by our group and others7 (link)9 (link)49 (link), we used SRM-LC-MS/MS to validate differential proteins identified in the discovery phase. Urine samples from different individuals’ cohorts were used. In particular, 16 patients and 7 healthy subjects were recruited. Urine samples were concentrated and desalted. Total protein content was quantified by Bradford assay and protein samples were reduced, alkylated and digested with sequencing grade trypsin (Roche). Tryptic peptides solutions were cleaned with C18 spin columns (Protea Biosciences) according to manufacturer’s instructions and mixed 1:1 with mobile phase A (0.1% formic acid in MilliQ water). A 6460 Triple Quadrupole mass spectrometer was used on-line connected to nano-chromatography in a Chip-format configuration (ChipCube interface, ProtID Zorbax 300B-C18-5 μm chip, 43 × 0.075-mm analytical column and 40 nL enrichment column, Agilent Technologies). The system was controlled by Mass Hunter Software (v4.0 Agilent Technologies). Theoretical SRM transitions were designed using Skyline (v.1.1.0.2905) and peptide specificity was confirmed by protein blast. Only those proteotypic (specific) peptides were selected50 (link). In this case, one specific proteotypic peptide was measured per protein, by monitoring two transitions for RBP4 and three transitions for KNG1 (see Supplementary material).

Semi-automated in-gel tryptic digestion was performed on a liquid handler as previously described [53 ]. Peptides were analyzed using a 1200 Series nano high performance liquid chromatography coupled with Quadrupole-Time of Flight 6520 with a Chip-Cube interface (Agilent Technologies) as described [53 ]. Spectrum Mill (Agilent, B.04.00.127) was used for data analysis. Data were extracted with carbamidomethylated cysteine as a fixed modification and SILAC amino acids N-Lys, ¹³C₆¹⁵N₂-Lys, N-Arg and ¹³C₆¹⁵N₄-Arg as a mix modification. Extracted data were searched against the SwissProt (release-2011_01) human database with the same modifications as above, and oxidized methionine as a variable modification. Precursor and product mass tolerance was set to ± 20 ppm and ± 50 ppm respectively. Reverse database scores were calculated with Spectrum Mill search engine by using the percentage of false positive identifications using the following cut-off: protein score > 11, peptide score >10 and scored peak intensity > 60%. All peptides identified had a global false discovery rate of less than 0.9%. Mean SILAC ratio (H/L) and standard deviations were calculated using all the peptide ratios matched to a protein, and p-values were calculated using peptide SILAC ratios as previously described [25 -27 (link)].