%\VignetteIndexEntry{UPSprot25 and UPSpep25 datasets description}
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\begin{document}
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\title{Description of the UPS\{pep/prot\}2 datasets}
\author{
Samuel Wieczorek\footnote{firstname.lastname@cea.fr} ,
Florence Combes$^\ast$,
Claire Ramus,
Quentin Giai Gianetto,\\
Yohann Coute,
Myriam Ferro,
Christophe Bruley
and Thomas Burger$^\ast$
}
\maketitle





This dataset results from a controlled relative quantification proteomics 
experiment where Sigma UPS1 human proteins were spiked in a similar yeast 
lysate in 2 different concentrations (with a ratio of 2).
As a consequence, it can be used to benchmark the quality of a statistical 
analysis: in the ideal case, after the differential analysis, only and all the 
human proteins should be selected.

\section{Preparation and digestion of proteins and peptides}
UPS1 was spiked in yeast extract in 8M urea, with a concentration of 10fmol 
for one condition, and of 5fmol for the other. The solution was diluted twice 
before 1h incubation with 10mM DTT at 37\celsius, 45min incubation with 55mM 
iodoacetamide in the dark and at room temperature. Proteins were then 
submitted to LysC digestion for 4 hours at 37\celsius. After dilution, 
proteins were submitted to overnight digestion with modified trypsin (Promega, 
sequencing grade) at 37\celsius. Digestion was stopped through acidification 
with trifluoroacetic acid. Resulting peptides were desalted using C18 
MacroSpin columns (Harvard Apparatus).

\section{Nano-LC-MS/MS analyses}
Dried peptides were resuspended in 5\% acetonitrile and 0.1\% trifluoroacetic 
acid and analyzed by online nanoLC-MS/MS (Ultimate 3000, Dionex and Q-Exactive 
Plus, Thermo Scientific). Peptides were sampled on a 300 µm x 5 mm PepMap 
C18 precolumn and separated on a 75 µm x 250 mm C18 column (PepMap, Dionex). 
The nanoLC method consisted of a 120-minutes gradient at a flow rate of 
300 nl/min, ranging from 5\% to 37\% acetronitrile in 0.1\% formic acid for 
114 min, before reaching 72\% acetronitrile in 0.1\% formic acid for the last 
6 min. MS and MS/MS data were acquired using Xcalibur (Thermo Scientific). 
Spray voltage was set at 1.6 kV; heated capillary was adjusted to 250\celsius. 
Survey full-scan MS spectra (m/z = 400–1600) were acquired in the Orbitrap 
with a resolution of 70,000 after accumulation of 106 ions (maximum filling 
time 200 ms). MS/MS spectra were acquired for up to the ten most intense ions 
from the MS scan after higher energy collisional dissociation (accumulation 
of 105 ions, maximum filling time of 50 ms, resolution of 17,500).

\section{Computational analyses}
RAW files were processed using MaxQuant version 1.5.1.2. Spectra were searched 
against the Uniprot database (Saccharomyces cerevisiae 
(strain ATCC 204508 / S288c) taxonomy, June 2015 version), the UPS database 
and the frequently observed contaminants database embedded in MaxQuant. 
Trypsin was chosen as the enzyme and 2 missed cleavages were allowed. 
Precursor mass error tolerances were set respectively at 20 ppm and 4.5 ppm 
for first and main searches. Fragment mass error tolerance was set to 0.5 Da. 
Peptide modifications allowed during the search were: carbamidomethylation 
(C, fixed), acetyl (Protein N-ter, variable) and oxidation (M, variable). 
Minimum peptide length was set to 7 amino acids. Minimum number of peptides, 
razor + unique peptides and unique peptides were all set to 1. Maximum false 
discovery rates -calculated by employing a reverse database strategy - were 
set to 0.01 at peptide and protein levels. Raw intensity IBAQ and LFQ values 
were calculated using MaxQuant as previously described (2), from MS intensity 
of unique peptides. 
%The resulting file was exported into the CSV format.

\section{Datasets}
The package DAPARdata contains two datasets:
\begin{itemize}
\item USPpep2 which contains the quantitative data at the peptide level,
\item USPprot2 which contains the quantitative data at the protein level.
\end{itemize}
Both datasets are available in CSV and MSnSet formats.




\end{document}