Multiplexed peptide analysis using data-independent acquisition and Skyline

• Published in: Nature protocols Vol. 10; no. 6; pp. 887 - 903
• Main Authors: Egertson, Jarrett D, MacLean, Brendan, Johnson, Richard, Xuan, Yue, MacCoss, Michael J
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.06.2015; Nature Publishing Group
• Subjects: 631/1647/2196; 631/1647/2230/1452; 631/45/607/275; 631/80/458/1733; Analytical Chemistry; Biological Techniques; Computational Biology/Bioinformatics; Data acquisition; Data entry; Database searching; Identification and classification; Libraries; Life Sciences; Mass Spectrometry; Mass spectroscopy; Methods; Microarrays; Organic Chemistry; Peptide Library; Peptides; Peptides - analysis; Proteomics; protocol; Quality Control; Quantitation; Software; Spectra; Target acquisition; United States
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/nprot.2015.055

Skyline is a targeted proteomics environment that can be used in the detection and quantitation of peptides. In this protocol the MS/MS peptide data is generated by data-independent acquisition on a Q-Exactive mass spectrometer. Here we describe the use of data-independent acquisition (DIA) on a Q-Exactive mass spectrometer for the detection and quantification of peptides in complex mixtures using the Skyline Targeted Proteomics Environment (freely available online at http://skyline.maccosslab.org ). The systematic acquisition of mass spectrometry (MS) or tandem MS (MS/MS) spectra by DIA is in contrast to DDA, in which the acquired MS/MS spectra are only suitable for the identification of a stochastically sampled set of peptides. Similarly to selected reaction monitoring (SRM), peptides can be quantified from DIA data using targeted chromatogram extraction. Unlike SRM, data acquisition is not constrained to a predetermined set of target peptides. In this protocol, a spectral library is generated using data-dependent acquisition (DDA), and chromatograms are extracted from the DIA data for all peptides in the library. As in SRM, quantification using DIA data is based on the area under the curve of extracted MS/MS chromatograms. In addition, a quality control (QC) method suitable for DIA based on targeted MS/MS acquisition is detailed. Not including time spent acquiring data, and time for database searching, the procedure takes ∼1–2 h to complete. Typically, data acquisition requires roughly 1–4 h per sample, and a database search will take 0.5–2 h to complete.