A challenge of quantitative analysis of complex protein mixtures using isotope coded affinity tags is to correctly identify and obtain accurate quantitative information for ICAT reagent pairs on a spectrum generated by mass spectrometry. Because MALDI-TOF mass spectrometry can easily generate peptide mass fingerprint profiles and relative quantitation, it can be used to quickly screen the ICAT reagent pairs representing the up- and down-regulated proteins from complex mixtures. The challenge lies in how to distinguish true signal from noise present in the analysis.
Accuracy of the quantitative results and dynamic range depends on peak detection, data processing, and interpretation strategies. We describe here the software implemented for ICAT reagent analysis using MALDI-TOF mass spectrometry (Figure 1). Examples of ICAT reagent data analysis using this software are demonstrated and show the experimental accuracy and dynamic range.
ICAT reagent experiments can be performed on a MALDI-TOF instrument to achieve reliable quantitative results.
We performed ICAT reagent experiments on proteins such as BSA, a complex 10-protein mixture using the ICAT reagent kit for protein labeling. The purified biotinylated peptides were generated from a mixture and further fractionated by reverse-phase separation and analyzed by MALDITOF mass spectrometry. Data was submitted to a software program written in Microsoft Visual Basic for Applications available for use with mass spectrometry software. The software offers intelligent features such as intensity-based peak selection based on a userdefined mass range increment. It contains mass filters that automatically remove sodium and potassium adducts and user-defined common contaminants from ICAT reagent experiments. Data processing algorithms such as advanced baseline correction, noise reduction, and de-isotoping could be applied to data automatically prior to quantitative analysis. Because of trace amounts of D7 and D6 present in the heavy ICAT reagent, the software allows for a correction factor for these species.
A key element in the software is the flexibility to identify the ICAT reagent pairs based on a theoretical peptide mass list from known protein digests, which could lead to accurate identification of ICAT reagent pairs and therefore reliable quantitative results. For example, the quantitative results from the mass spectrum of the labeled(D8) vs. non-labeled(D0) digested BSA yielded a ratio of 0.91 (vs. theoretical value of 1.0) with a standard deviation of 0.12 (Figure 2 and Table 1).
Alternatively, the VBA software automatically looks for all possible ICAT reagent pairs if the protein digests are not previously identified. False positives could be deleted by applying statistics or re-evaluating the spectrum. For MALDI-TOF analysis, this suppression is often a concern for quantitative analysis because of the complex nature of the sample. Reverse phase separation can improve the dynamic range, as is demonstrated in Figure 3. In this case, the dynamic range for a peptide of interest from a 10-protein mixture is demonstrated by the software to achieve 0.11 (vs. theoretical ratio of 10:1) and 0.03 (vs. theoretical value of 30:1) accurately.
Using MALDI-TOF and advanced data interpretation software, users can achieve reliable quantitative results of affinity-purified ICAT reagent-labeled protein digests. This method can be used as a potential assay to rapidly screen peptides derived from the upand down-regulated proteins that are potential bio-marker candidates in complex mixtures.