Computational protocol: Assay of Protein and PeptideAdducts of CholesterolOzonolysis Products by Hydrophobic and Click Enrichment Methods

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Protocol publication

[…] The peptide solution (secosterol-treated, trypsinized protein) was separated using an Eksigent NanoLC Ultra HPLC and autosampler. The analytical column was packed with C18 resin (Jupiter, 3 μm, 300 Å, Phenomenex) in a capillary column (20 cm length, 360 μm O.D. × 100 μm I.D.). Peptides were eluted using a gradient as follows: 0–14.5 min, 2% B; 14.5–15 min, 2 to 5% B; 15–35 min, 5 to 35% B; 35–55 min, 35 to 99% B; 55–75 min, 99% B; and 75–90 min, 99 to 2% B at a flow rate of 500 nL/min (solvent A: 0.1% formic acid in H2O, solvent B: 0.1% formic acid in CH3CN). Eluting peptides were mass analyzed on an LTQ Orbitrap Velos mass spectrometer (Thermo Scientific) followed by a data dependent neutral loss (DDNL) MS3 method with dynamic exclusion enabled. Full-scan (m/z 350–2000) spectra were acquired with the Orbitrap mass analyzer (resolution 30,000), and the six most abundant ions in each MS scan were selected for fragmentation in the LTQ. An isolation width of 2 m/z units, activation time of 10 ms, and 35% normalized collision energy were used to generate MS2 spectra followed by MS3 neutral loss from MS2 with an isolation width of 2.2 m/z units. Neutral loss mass scans are enabled with the five most abundant in each MS2 with loss of m/z 390.349 (singly charged species), 195.175 (doubly charged species), and 130.120 (triply charged species). Dynamic exclusion settings allowed for a repeat count of 1 within a repeat duration of 15 s, and the exclusion duration time was set to 30 s. For identification of HSA peptides, tandem mass spectra were processed in BumberDash Tools, version 1.4.110, and searched by Myrimatch algorithm with a custom-built subset database originated from Homo_sapience_GRCh37-59_Cntm_r (2010_11-18). The sequence database was reversed so that each protein sequence appeared in both normal and reversed orientations, enabling false discovery rate estimation. MyriMatch was configured to have dynamic modifications of +57.021 for carbamidomethyl to Cys and 400.3342, 402.3498, 404.3654, 384.3342, and 386.3498 Da for [Cys,Lys,His]. MS3 spectra generated from DDNL were separately processed using ProteoWizard msConvert tool to convert to mzXML format, followed by Myrimatch search. In this case the configuration was consistent with dynamic modifications of +57.021 for carbamidomethyl to Cys and +12.00 for Lys residue. Protein assembly was handled by IDPicker algorithm, version 2.1.6710. […]

Pipeline specifications

Software tools BumberDash, MyriMatch, ProteoWizard, IDPicker
Application MS-based untargeted proteomics
Diseases Alzheimer Disease, Asthma, Parkinson Disease, Atherosclerosis
Chemicals Cholesterol, Ozone