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 data sets. Although significant technical advances in peptide and protein identification have been made, accurate quantitation of high-throughput data sets 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 noncommercial users at http://www.proteomeumb.org/MZw.html .

Figure 1. Framework of IsoQuant

Figure 2. User interface of IsoQuant

A) IsoQuant consists of three major components: Raw file conversion, Peptide/ Protein Quantitation and Visual Validation. B) Interface of the peptide and protein quantitation module. Currently, IsoQuant supports the following quantitation features: 1. double and triple label SILAC experiments; 2. Single or multiple .raw files; and 3. SEQUEST and Mascot peptide identification results (only SEQUEST search result is shown here).

Figure 3. IsoQuant overcomes the limitation of under-sampling in data-dependent instrument methods, thereby increasing the accuracy of SILAC-based quantitation

A) Extracted ion chromatogram (XIC) of 572.77 m/z (SILAC heavy-labeled peptide). 572.77 m/z was selected for MS2 fragmentation and was identified by SEQUEST in the first two scans (indicated by *; retention time (RT) 32.70 min and 32.75 min). B) MS1 scan at RT 32.70 min (568.0 – 575.0 m/z) demonstrating an incomplete isotope envelope of 569.76 m/z (SILAC light-labeled peptide). C) MS1 spectrum from the most intense peak in A) at RT 32.84 min. Although 572.77 m/z was not selected for MS2 fragmentation due to dynamic exclusion, its complete isotopic envelope permitted quantitation by IsoQuant.

Figure 4. Representative extracted ion chromatograms (XIC) of model peptide from a SILAC H/L = 2 mixture

(A) Nuclear migration protein nudC (NUDC) peptide, LVSSDPEINTK, was identified at scan # 7605, as indicated by the long arrow. The peak intensities of isotopic envelopes corresponding to the heavy and light peptides were then extracted by an IsoQuant MS1 peaks extraction module. Two XICs corresponding to individual heavy (square) and light (diamond) peptides were then reconstructed. The H/L ratio calculated by IsoQuant was 2.05 and close to the expected H/L ratio of 2.0. The beginning (scan # 7601) and ending (scan # 7634) points of the XICs are indicated by the two short arrows. (B) MS1 spectra of peptide LVSSDPEINTK used for ratio calculation.

Figure 5. Quantitation accuracy of IsoQuant using standard SILAC mixtures

SILAC standard mixtures at H/L = 1:1, 1:2, 1:5, 1:10, 1:25, 1:50 and 1:100 were prepared in triplicate by mixing ¹³C₆-lysine and ¹³C₆-arginine labeled SKBR3 cells with ¹²C₆-lysine and ¹²C₆-arginine labeled SKBR3 cells. The same raw data were analyzed by IsoQuant (white) and Proteome Discoverer (gray). The percentage of quantified peptides (A) and proteins (B) from the quantitation results of IsoQuant (white) and Proteome Discoverer (gray) are shown. Error bars represent standard deviations from three technical replicates. Expected ratios and experimental ratios of peptides (C) and proteins (D) are shown. Error bars represent standard deviations from three technical replicates.

Figure 5. Quantitation accuracy of IsoQuant using standard SILAC mixtures

SILAC standard mixtures at H/L = 1:1, 1:2, 1:5, 1:10, 1:25, 1:50 and 1:100 were prepared in triplicate by mixing ¹³C₆-lysine and ¹³C₆-arginine labeled SKBR3 cells with ¹²C₆-lysine and ¹²C₆-arginine labeled SKBR3 cells. The same raw data were analyzed by IsoQuant (white) and Proteome Discoverer (gray). The percentage of quantified peptides (A) and proteins (B) from the quantitation results of IsoQuant (white) and Proteome Discoverer (gray) are shown. Error bars represent standard deviations from three technical replicates. Expected ratios and experimental ratios of peptides (C) and proteins (D) are shown. Error bars represent standard deviations from three technical replicates.

Figure 6. Interface of IsoQuant visualization module permits users to validate quantitation ratios

A) Screenshot of quantitative SILAC peptide summary report and overall peptide quantitation ratio distribution from a representative double (Lys4, Arg6) SILAC label experiment. B) Screenshot of a representative spectrum from which the peptide ratios were calculated. Users can move a slider at the top of the “MS1 spectrum” view in the “Validate” window to allow the visualization of neighboring MS1 scans used for peptide quantitation. C) Screenshot of protein ratio report