Software

Software in use by the Protein Profiling Group:

ProteinLynx Global SERVER (PLGS, Waters Corp.)Waters

The basic software provided by Waters for analysis of raw data from our instruments. PLGS will convert raw data files into processed files which are then searched against a selected database of protein sequences. Identification and quantification of peptides is carried out using the “Top 3” approach with spiked standards as benchmarks (Silva et al, 2006). Data is exported in .csv format (Excel compatible) or as zipped files for uploading into Scaffold software for visualisation.

Scaffold (Proteome Software Inc.)

This sofScaffoldtware is used for the visualisation and validation of complex proteomics experiments. Identification of peptides is performed against a selected protein sequence database. Quantification is calculated differently to PLGS (variation on “Top 3”), so results do not match PLGS output. The software has a very good user interface and graphical display of results.

 

There is a free viewer available at www.proteomesoftware.com:

 

ProgenesisQI for Proteomics (Nonlinear Dynamics, A Waters Company)

This is suitable for largerProgenesis datasets. Will currently only calculate relative quantitation (compare samples in one experimental file) and does not report absolute quantification values. The software will use all available peptides, not “Top 3”. Progenesis provides excellent visual aids including PCA analysis, dendrograms, 3D montages of peptide peaks and filtering options within datasets. There is no free viewer available and results are exported as .csv files only.

Ion Intensity Map

Principal Components Analysis

3D Montage of retention time vs m/z

ISOQuant (Jörg Kuharev and Stefan Tenzer, University of Mainz, Germany)

This sofISOQuanttware is used for absolute quantitation of proteins. Processed / searched PLGS data files are loaded into ISOQuant for further quantitative analysis. ISOQuant only returns an absolute protein amount if three reliable and strong peptide hits are available for quantification (“Top 3”) (Distler et al, 2014). Spiked ADH1 (yeast) tryptic digests are used to calculate the absolute amounts of identified proteins. For analysis of protein complexes of interest, one component of each complex can be used to normalize the data and calculate stoichiometric ratios. Output is in Excel format only.

References

Absolute Quantification of Proteins by LCMSE: a virtue of parallel MS acquisition.

Silva, J. C., Gorenstein, M. V., Li, G.-Z., Vissers, J. P. C., Geromanos, S. J.

Mol Cell Proteomics. 2006 Jan;5(1):144-56.

Drift time-specific collision energies enable deep-coverage data-independent acquisition proteomics.

Distler, U., Kuharev, J., Navarro, P., Levin, Y., Schild, H., & Tenzer, S.

Nat Methods. 2014 Feb;11(2):167-70. doi: 10.1038/nmeth.2767.

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