Quantitative Proteomics Analysis of Enzyme

The LC-MS system consisted of a NanoLC Ultra System (Eksigent Technologies, Dublin, CA, USA), a TripleTOF 5600 System (AB SCIEX, Framingham, MA, USA) equipped with a trap column (150 μm I.D. × 10 mm L.; C18, 3 μm, 100 Å; Proteomics Front, Beijing, China) and a separation column (75 μm I.D. × 150 mm L., C18, 3 μm, 100 Å; Proteomics Front). The dried peptides were dissolved in 20 μl of solvent A [5% ACN, 0.1% Formic Acid (FA)], and a 5-μl aliquot was injected into the trap column at a flow rate of 2.0 μl/min. The analytical separation was conducted at a flow rate of 300 nl/min. After a 4-min wash with 5% solvent B (95% ACN, 0.1% FA), a 45-min linear gradient was run with 5–80% solvent B followed by a 5-min linear gradient with 80–95% solvent B. The eluate was directly evaporated at 150 °C with a nitrogen stream of 3 l/min, and the ion spray voltage was set at 2.3 kV. MS was operated in the positive-ion mode with a mass range of 400–4000 m/z. MS/MS was acquired in an automated data-dependent acquisition mode with charge numbers of 2–4 and a mass range of 100–2000 m/z. The data were analyzed with PeakView 1.2 software (AB SCIEX). The sequence coverages were analyzed with ProteinPilot^TM software using Database uniprot_sprot_20121010 specifically towards Accession # sp|P21163|PNGF_ELIMR^{54 (link)}.