Frequently Asked Questions

RIANA enables protein turnover data analysis from mass spectrometry-based proteomics experiments. Supports multiple labeling strategies, including labeled amino acid and heavy water.

Abstract

This page provides the documentation for the RIANA package, which is a tool for the analysis of stable isotope labeling experiments for protein turnover measurements.

FAQs

Integrate

Q: What does the -D --mass_defect argument do for the integrate function?

A: The -D flag specifies the accurate mass difference between each unit isotopomer and can be set to D, C13, or SILAC, which will dictate the mass difference across each successive peak to be 1.0063 (mass difference of deuterium and protium), 1.003 (mass difference of ¹³C and ¹²C), and 1.001 (custom SILAC mass), respectively. This is set so that RIANA can find the correct accurate mass for integration when multiple atom centers are added. E.g., if deuterium labeled amino acids are used, e.g., D³-leucine, the argument should be set to -D D and the integrated isotopomer argument set to -i 0, 3. Alternatively if ¹³C₆-lysines, the arguments should be -D C13 and i 0,6.

Fit

Q: What are the kinetic models being specified by the different fit options?

A: The simple, guan, and fornasiero options for the model argument refer to a simple exponential (one-pool) model, a two-compartment model, and a two-compartment model that includes label reutilization. See the Documentation page for details.

Q: What does the -l --label argument do?

A: The --label argument determines how the fraction of new proteins at a particular labeling time point is calculated from the relative abundance of isotopomers. The option aa assumes that the first peaks of light or heavy amino acids are used for calculations (e.g., m0, m6 for ¹³C₆-lysine) and hence the initial pre-label fraction of the light peak (m0) is assumed to be 1. The option hw assumes a heavy water type labeling experiment where the entire isotopomer cluster of the peptide is integrated (e.g., m0, m1, m2, m3, m4, m5), and hence the pre-labeling fraction of the m0 peak is calculated from peptide relative isotopomer abundance based on naturally occurring isotopes.

Q: How are the plotted curves separated to the curves_fast_fit, curves_mid_fit, and curve_slow_fit directories?

A: The directories are intended to facilitate navigation only rather than a rigorous indication of data quality. Plots for fitted curves with k < 0.01 are in the “slow” directory, 0.01 <= k <= 1 in the “mid” directory, and 1 < k in the “fast” directory. Plots for fitted curves with R² >= 0.5 are in the “fit” directory, and those with R² < 0.5 are in the “poor” directory.

Q: Does RIANA fit perform protein level curve fitting?

A: RIANA currently performs curve fitting at the peptide level only. Users can perform protein-level curve fitting using the {sample}_riana.txt integration result files. Alternatively, protein-level rate constants can be derived by taking a median or harmonic mean of the peptide-level rate constants with similar results, as we showed in our recent publication.

Q: How do I retrieve half-life information on the peptide and protein?

A: The half-life of a peptide or protein can be calculated from the output k parameter using the equation:

\[ t_{1/2} = ln(2)/k \]

General

Q: How can I contribute to RIANA?

A: Contact us at [edward.lau@cuanschutz.edu]