Scaffold has recently been updated to the Sc+ version with enhanced features for iTRAQ quantitation. Although iTRAQ-labeling has been widely applied, there is an ongoing discussion about the accuracy of the deduced protein quantitations, particularly when sample mixtures are highly complex .
Robust expression changes were also observed for 17 cellular proteins in HAdV-B3-infected cells (Figure 4(c)), while three proteins showed inconsistent patterns in iTRAQ-label and label-free quantitations. These included cellular nucleic acid-binding protein (CNBP1), ubiquitin-conjugating enzyme E2L3 (UbcH7), and nuclease-sensitive element-binding protein 1 (YBX1).
Western blot analyses confirmed downregulation of AG2 in HAdV-B3-infected cells, even more pronounced in labelfree quantitation than iTRAQ quantitations (Figure 5(c)).
Relative quantitation of two or more samples for studies of differential protein expression is of particular importance.
There are two general approaches for label-free quantitation, measurement of spectral peak intensities  and spectral counting .
Only Mascot was utilized to identify proteins from data acquired on the LTQ-FT-ICR instrument for label-free quantitation. For the analysis with PP v3.0 (Applied Biosystems), the acquired data was directly fetched from the Oracle database and searched with the Paragon algorithm.
Software Used for Protein Quantitation. PP and Sc+ (Proteome Software) were used for protein quantitation of iTRAQ-labeled samples.
The idea of this strategy termed ProgenesisF-T2PQ (PF2) or ProgenesisF-T3PQ (PF3)  is that irrespective of how many peptides are found for one particular protein; only the most intense (n) precursor signals are used for protein quantitation. We adapted this method referred to as PF2 or PF3, respectively, based on the aligned Progenesis feature map by averaging the top N normalized volumes of features from the same protein.
Signal detection was performed in the chemiluminescence scanning mode of Image reader LAS 3000 (FUJIFILM Science Lab), and signal quantitation was performed using Image Gauge version 3 (FUJIFILM Science Lab).
Correlation Analyses for iTRAQ Quantitation Using PP and Sc+ Software.
In the Sc+, both ion precursors and their MS/MS fragmentations are used for protein quantitation while in the PP only one ion precursor and its MS/MS fragmentation are considered.