Project PXD011695

Summary

Title

Interactive Peptide Spectral Annotator Experimental Data

Description

Abstract Here we present IPSA, an innovative web-based spectrum annotator that visualizes and characterizes peptide tandem mass spectra. A tool for the scientific community, IPSA can visualize peptides collected using a wide variety of experimental and instrumental configurations. Annotated spectra are customizable via a selection of interactive features and can be exported as editable scalable vector graphics to aid in the production of publication-quality figures. Single spectra can be analyzed through provided forms, while multiple peptide spectral matches can be uploaded directly to the server as CSV, MGF, or mzML files. IPSA facilitates the characterization of experimental MS/MS performance through the optional export of fragment ion statistics from one to many peptide spectral matches. This resource is made freely accessible at http://interactivepeptidespectralannotator.com, and the source code is available for inspection at https://github.com/dbrademan/IPSA-dev for custom implementations. This repository contains the raw data, sequence databases, and peptide identifications utilized in the manuscript.

Sample Processing Protocol

Cell pellets of Saccharomyces cerevisiae (strain BY4742) containing approximately 1 x 108 cells were harvested from liquid culture by centrifugation (3,000 g, 3 min, 4°C). The supernatant was removed, and the cell pellet was resuspended in 8 M urea, 100 mM tris (pH 8.0). Methanol was added to 90% by volume and vortexed to lyse the cells and induce protein precipitation. The resulting solution was centrifuged (14,000 g, 3 min) to form a protein pellet. The supernatant was removed, and the pellet was resuspended in 8 M urea, 100 mM tris (pH 8.0), 10 mM tris(2-carboxyethyl)phosphine, and 40 mM chloroacetamide. The solution was then diluted to 1.5 M urea with 50 mM tris. Trypsin was added (1:50 enzyme:protein) and was allowed to digest overnight (22°C). The resultant peptides were acidified (pH < 2.0) using 0.1% trifluoroacetic acid (TFA) and were desalted using polymeric reverse phase Strata-X columns. Columns were equilibrated using one bed volume of 100% acetonitrile (ACN), then one bed volume of 0.1% TFA. Peptides were loaded onto the column and washed with two bed volumes of 0.1% TFA. Peptides were eluted by an addition of 500 µL 40% ACN, 0.1% TFA followed by an addition of 650 µL 70% ACN, 0.1% TFA and were then dried and resuspended in 0.2% formic acid. Peptide concentration was determined using a Pierce quantitative colorimetric peptide assay (Thermo Scientific). Low pH reverse-phase liquid chromatography was conducted using a Dionex UltiMate 3000 UPLC as described previously.(1, 2) Eluting peptides were analyzed using a Q Exactive HF hybrid quadrupole Orbitrap mass spectrometer (Thermo Scientific) and were fragmented at HCD at 25% normalized collisional energy. Survey scans were taken at a resolution of 60,000 at 200 m/z, while tandem mass spectra were collected using a resolution of 15,000 at 200 m/z.

Data Processing Protocol

The resulting tandem mass spectra were searched using the Coon OMSSA Proteomic Analysis Software Suite.(27, 28) A precursor mass tolerance of ±150 ppm was used, while fragment ions were searched using a mass tolerance of ±0.01 Da. A maximum of 3 missed tryptic cleavages were permitted. Carbamidomethylation of cysteine was set as a fixed modification, while oxidation of methionine was set as a variable modification. Data was searched against a canonical and isoform Saccharomyces cerevisiae database (UniProt, June 10, 2016) concatenated with the reverse protein sequence for decoy generation. A 1% FDR threshold was utilized at the peptide level, using both e-value and precursor mass accuracy to filter results.

Contact

Dain Brademan, UW-Madison Joshua J. Coon Research Group Chemistry Department
Joshua J. Coon, Department of Chemistry, University of Wisconsin–Madison, Madison, WI 53706, USA Morgridge Institute for Research, Madison, WI 53715, USA Genome Center of Wisconsin, Madison, WI 53706, USA Department of Biomolecular Chemistry, University of Wisconsin–Madison, Madison, WI 53706, USA ( lab head )

Submission Date

14/11/2018

Publication Date

15/05/2019

Software

Not available

Quantification

Not available

Experiment Type

Shotgun proteomics

Publication

    Brademan DR, Riley NM, Kwiecien NW, Coon JJ. Interactive Peptide Spectral Annotator: A Versatile Web-Based Tool for Proteomic Applications. Mol Cell Proteomics. 2019 PubMed: 31088857