Abstract. Mass spectrometry (MS)-based shotgun proteomics allows protein identifications even in complex biological samples. Protein abundances can then be estimated from the counts of MS/MS spectra attributable to each protein, provided one accounts for differential MS-detectability of contributing peptides. We developed a method, APEX, which calculates Absolute Protein EXpression levels based upon learned correction factors, MS/MS spectral counts, and each protein's probability of correct identification.

The APEX protocol describes APEX-based calculations in three parts:

1. Using training data, peptide sequences and their sequence properties, a model is built to estimate MS-detectability (Oi) for any given protein.
2. Absolute protein abundances are calculated from spectral counts, identification probabilities and the learned Oi-values.
3. Simple statistics allow calculation of differential expression in two distinct biological samples, i.e. measuring relative protein abundances.

APEX-based protein abundances span 3-4 orders of magnitude and are applicable to mixtures of 100s to 1000s of proteins.

Protocol paper (Nature Protocols, 2008). Link to journal website.
Supplementary Notes (to Protocol)

Original citation: Lu, Vogel, Wang, Yao, Marcotte. Absolute protein expression profiling estimates the relative contributions of transcriptional and translational regulation. Nat Biotechnol. 2007 Jan;25(1):117-24

This website provides scripts and data files to conduct analyses described in the APEX Protocol.

Notes/Disclaimers:

• Use of several files on this website requires basic UNIX/LINUX skills as well as some understanding of how to operate a Perl script.
• Perl scripts are written to be easily understood by a non-expert user - they are not optimized for speed.
• Files provided in the Data and Prediction directories are ready to use, even without the use of the Perl scripts. The data is mostly tab-delimited and can be cut-and-pasted into an Excel or other spreadsheet.
• Many files are gzipped. Contact us if you have problems opening the files.

Perl Scripts

• Scripts for download
• Brief description of procedure in terms of script usage

Training

• ThermoFinnigan Surveyor/DecaXP+ iontrap (LCQ) - data from OPD (opd00038_YEAST...opd00042_YEAST)
• ThermoFinnigan LTQ-OrbiTrap (ORBI) - data from OPD (opd00107_YEAST...opd00114_YEAST)

Data

• E.coli - prediction files (.arff, .names, .wekaout, .oi)
• Yeast (S. saccharomyces) - prediction files (.arff, .names, .wekaout, .oi)
• Human (H. sapiens) - prediction files (.arff, .names, .wekaout, .oi)

Predictions

Z-score calculation

Note (August 29, 2008): I am actively working on different ways to calculate significance of differential protein expression. There will be an updated perl script and a more detailed explanaition soon. C.V.

Other links

• PeptideProphet, ProteinProphet - as part of the Transproteomic Pipeline software for mass spectrometry data analysis
• Proteogest - in silico protein digest and description of biophysical peptide properties
• Peptide Detectability Predictor - for LC/MS Ion Traps
• Publication by Mallick et al. on sequence features determining peptide MS detectability
• AAindex - database of amino acid properties
• APEX Quantitative Proteomics Tool - developed by John Braisted and colleagues at the J. Craig Venter Insitute, Rockville, MD.
  Link to pre-publication website.

Enjoy.