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Achieving Reliable Protein Quantitation Results Using ICAT Reagents, MALDITOF Mass Spectrometry, and Advanced Data Interpretation Tool

Overview

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.

Key Feature

ICAT reagent experiments can be performed on a MALDI-TOF instrument to achieve reliable quantitative results.

Experimental Conditions

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 reage
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