Platform-independent and Label-free Quantitation of Proteomic Data Using MS1 Extracted Ion Chromatograms in Skyline

Abstract

Despite advances in metabolic and postmetabolic labeling methods for quantitative proteomics, there remains a need for improved label-free approaches. This need is particularly pressing for workflows that incorporate affinity enrichment at the peptide level, where isobaric chemical labels such as isobaric tags for relative and absolute quantitation and tandem mass tags may prove problematic or where stable isotope labeling with amino acids in cell culture labeling cannot be readily applied. Skyline is a freely available, open source software tool for quantitative data processing and proteomic analysis. We expanded the capabilities of Skyline to process ion intensity chromatograms of peptide analytes from full scan mass spectral data (MS1) acquired during HPLC MS/MS proteomic experiments. Moreover, unlike existing programs, Skyline MS1 filtering can be used with mass spectrometers from four major vendors, which allows results to be compared directly across laboratories. The new quantitative and graphical tools now available in Skyline specifically support interrogation of multiple acquisitions for MS1 filtering, including visual inspection of peak picking and both automated and manual integration, key features often lacking in existing software. In addition, Skyline MS1 filtering displays retention time indicators from underlying MS/MS data contained within the spectral library to ensure proper peak selection. The modular structure of Skyline also provides well defined, customizable data reports and thus allows users to directly connect to existing statistical programs for post hoc data analysis. To demonstrate the utility of the MS1 filtering approach, we have carried out experiments on several MS platforms and have specifically examined the performance of this method to quantify two important post-translational modifications: acetylation and phosphorylation, in peptide-centric affinity workflows of increasing complexity using mouse and human models.

Modulation of Protein Phosphorylation, N-Glycosylation and Lys-Acetylation in Grape (Vitis vinifera) Mesocarp and Exocarp Owing to Lobesia botrana Infection

“…2B). Even though we identified more Lysacetylation sites than previous plant acetylproteome studies (37,38), it is important to mention that iTRAQ can interfere with the Lys-acetylation immunoprecipitation efficiency, as previously reported (39) and this can reduce the number of identified Lys-acetylated peptides. The distribution of the 603 phosphorylation sites identified in the exocarp, and the 878 sites detected in the mesocarp was, respectively 89%/92% ( p S); 10%/7% ( p T); and 1%/1% ( p Y) (Fig.…”

Quantitative Profiling of Post-translational Modifications by Immunoaffinity Enrichment and LC-MS/MS in Cancer Serum without Immunodepletion

“…All quantitative results were generated using Progenesis V4.1 (Waters Cooperation) and Skyline Version 3.1 to extract the integrated peak area of the corresponding peptide assignments according to previously published protocols (21,22). Extracted ion chromatograms for peptide ions that changed in abundance between samples were manually reviewed to ensure accurate quantitation in Skyline.…”

“…Both programs perform retention time alignment across LC-MS/MS runs by either common MS2 identifications (Skyline) or common ion features (Progenesis) so that a narrow and accurate precursor window can be applied to each unique peptide or ion feature (21). This methodology allows determination of peptide peak intensities across all samples even if a particular peptide was not MS/MS identified in every sample run (14,21,25).…”

Lysine desuccinylase SIRT5 binds to cardiolipin and regulates the electron transport chain

“…Peptides were searched against Swiss-Prot entry P49748 and chosen based on a 95% confidence level followed by manual inspection. Raw MS data files were imported into Skyline, and precursor ion chromatograms were extracted for label-free quantification using MS1 filtering (32). Peptide areas were then averaged across all sample acquisitions, and a ratio was generated.…”