IsoQuant: A Software Tool for Stable Isotope Labeling by Amino Acids in Cell Culture-Based Mass Spectrometry Quantitation

Abstract

Accurate protein identification and quantitation are critical when interpreting the biological relevance of large-scale shotgun proteomics datasets. Although significant technical advances in peptide and protein identification have been made, accurate quantitation of high throughput datasets remains a key challenge in mass spectrometry data analysis and is a labor intensive process for many proteomics laboratories. Here, we report a new SILAC-based proteomics quantitation software tool, named IsoQuant, which is used to process high mass accuracy mass spectrometry data. IsoQuant offers a convenient quantitation framework to calculate peptide/protein relative abundance ratios. At the same time, it also includes a visualization platform that permits users to validate the quality of SILAC peptide and protein ratios. The program is written in the C# programming language under the Microsoft .NET framework version 4.0 and has been tested to be compatible with both 32-bit and 64-bit Windows 7. It is freely available to non-commercial users at http://www.proteomeumb.org/MZw.html.

An Internal Standard-Assisted Synthesis and Degradation Proteomic Approach Reveals the Potential Linkage between VPS4B Depletion and Activation of Fatty Acidβ-Oxidation in Breast Cancer Cells

“…At the end of each chase period, SKBR3_shVPS4B and SKBR3 cells were collected and analyzed by a FASP-based SAX fractionation and LC-MS/MS analysis using a hybrid Orbitrap mass spectrometer. MS/MS raw files were then subjected to database search and quantification analysis using our in-house software IsoQuant [38]. However, in order to compare the degradation rate constants and synthesis rates of each peptide in SKBR3_shVPS4B and SKBR3 cells at the proteome level, we required that the “same” peptides should be identified and quantified at each time point in both cell lines throughout the entire course of the experiment in all five time points.…”

“…Similarly, the relative abundance of labeled peptide ( A m ) was defined as the ratio of the peak intensities of labeled peptide ( I m ) to I h , (2). An in-house SILAC-based mass spectrometry quantitation software, IsoQuant (http://www.proteomeumb.org/MZw.html) [38], was used to automatically integrate the isotopic peak intensities of each peptide and calculate A l and A m . The relative abundance of total peptide ( A tot ) was calculated by summing the relative abundance of light unlabeled peptide ( A l ) and labeled peptide ( A m ), (3) $\begin{array}{c}{A}_l=\frac{I_l}{I_h},\end{array}$ $\begin{array}{c}{A}_m=\mathrm{\hspace{0.17em}\hspace{0.17em}}\frac{I_m}{I_h},\end{array}$ $\begin{array}{c}{A}_{\text{tot}}=A_l+A_m.\end{array}$ …”

Human DNA Ligase I Interacts with and Is Targeted for Degradation by the DCAF7 Specificity Factor of the Cul4-DDB1 Ubiquitin Ligase Complex

“…A database search was carried out as described previously to identify the proteins from which the tryptic peptides were derived (52). The ratios of peptides from labeled and non-labeled cells to parental cells were calculated using the IsoQuant software (58).…”

Mass Spectrometry Analysis of Lysine Posttranslational Modifications of Tau Protein from Alzheimer’s Disease Brain

“…If the sample was isotopically labeled (e.g., chemical labeling with iTRAQ or TMT tags or metabolically labeled using SILAC), use a software package such as MaxQuant [62, 63] or QuantiMORE [66] to calculate the peptide and protein ratios ( see Notes 15 and 16). …”