Computational protocol: A new dimethyl labeling-based SID-MRM-MS method and its application to three proteases involved in insulin maturation

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Protocol publication

[…] Samples were analyzed on a Thermo Scientific Q Exactive Orbitrap MS with an ESI source. The peptides were separated on an in-house making C18 capillary column with mobile phases 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) at a 78-min gradient from 0 to 8 min, 4%–8% B; 8–58 min, 8%–22% B; 58–70 min, 22%–32% B; 70–71 min, 32%–90% B; 71–78 min, and 90% B at a flow rate of 280 nL/min. MS analysis was performed in data-dependent MS/MS scan mode, spray voltage was 2.0 kV, MS full scan range was 300–1600 m/z, AGC was set as 3e6, resolution at 400 m/z was 70,000, the maximum ion injection time was 60 ms. Top 20 precursor ions were selected into the HCD chamber for MS2 fragmentation analysis, MS/MS scan resolution was 17,500, AGC was set as 5e4, the maximum injection time was 80 ms. Dynamic exclusion time was 50 s. Normalized collision energy was 27%.MS data were searched against Uniprot Rattus norvegicus proteomes datasets (ftp://ftp.uniprot.org/pub/databases/uniprot/current_release/knowledgebase/proteomes/) and 245 common protein contaminants using Proteome Discoverer 1.3 software. Parameters were set as follows: precursor mass tolerance 10 ppm, fragment ion mass tolerance 0.02 Da, two maximum missed cleavages, iodoacetamide on cysteine as fixed modification, oxidation of methionine as variable modifications, peptide false discovery rate of 1% were estimated by Percolator (Kall et al. ), which was implemented as a searching node in SEQUEST search engine with Proteome Discovery software.MALDI-TOF-MS analysis of peptides was performed using an AXIMA-CFR plus MALDI-TOF mass spectrometer (Shimadzu/Kratos, Manchester, UK) equipped with a pulsed nitrogen laser operated at 337 nm. Peptide solution was mixed with 10mg/mL CHCA in 60% acetonitrile containing 0.1% TFA by 1:1 (v/v) in an Eppendorf tube, and 1 μL of the peptide/matrix solution was spotted onto the MALDI sample plate and then crystallized in the air. Positive ion MALDI mass spectra were acquired in the reflection mode under the following parameters: ion source, 20 kV; lens, 6.3 kV; pulsed extraction, −2.5 kV; reflection, 25 kV. Mass spectrometry data were processed with Launchpad 2.7.1 software (Shimadzu/Kratos, Manchester, UK). […]

Pipeline specifications

Software tools Proteome Discoverer, Percolator, Comet
Application MS-based untargeted proteomics
Diseases Diabetes Mellitus