ProtQuanter SAQ (Semi Absolute Quantification)

Bioinformatic pipeline implemented to determine semi-absolute quantification (fmol) from label-free proteomics data.

# clone with ssh 
git@forgemia.inra.fr:aaron.millan-oropeza/protquanter-saq.git

# clone with https
https://forgemia.inra.fr/aaron.millan-oropeza/protquanter-saq.git

The available methods of quantifications are:

• NSAF: defined by Zibailov et al. (2006) as:

NSAF_k = (SC / L)_k / SUM (SC / L) _N

k = a given protein

N = total quantified proteins

SC = Spectral counts

L = protein length in aa

• SAF: The spectral counts associated to a given protein over the length of the protein in aminoacids.

• PAI: the number of observed peptides counts per protein over the number of observable peptides per protein (Rappsilber et al., 2002)

• emPAI: exponentially modified protein abundance index (emPAI) was proposed by Ishihama et al., (2005) as:

  emPAI = (10 ^PAI ) – 1

• IBAQ: the sum of the MS1 intensities of all the peptides associated to a protein divided by the number of theoretically observable peptides (Schwanhäusser et al., 2011).

• TOP3: The sum of the top three most intense peptides of a protein (Silva et al., 2006).

• SUM of eXtracted Ion Chromatograms (XICs) normalized by protein length (aa).

The two strategies to convert relative quantification into absolute quantification are:

• By using the external standard UPS2 (SIgma)

• By using the Total Protein Approach (TPA, Wiśniewski et al., 2014)

Generated LC-MS/MS data

Files present in inputs directory contain the detected proteins in an Orbitrap Fusion Lumos Tribrid Mass Spectrometer (Thermo Fischer Scientific) and identified by the search engine X!Tandempipeline (Langella et al. 2017)

Data inputs

• compar_spectra.csv : protein abundances expressed as spectral counts per sample, including the groups, subgroups and proteins nomenclature from X!Tandempipeline.

• G1_proteins_modif.tsv: peptide list with their associated proteins, this file was generated from the eXtracted Ion Chromatogram (XIC) by using MassChroQ (Valot et al.. 2011)

• match_table.csv: file containing the protein IDs from UNIPROT and column indicating the type of protein (ex. UPS, Yeast)

• metadata.csv: file containing the sample names with the different conditions and replicates

• peptides_q1_G1.tsv: quantification of peptides XICs using MassChroQ (Valot et al.. 2011)

• theo_peptides.csv: file containing proteins length and molecular weight, this file corresponds to the sheet "proteins" present in the export of results (.ods) of X!Tandempipeline (Langella et al. 2017).