Results and Discussion
The iTRAQ-labeled peptide digest of six proteins showed many intense peaks in the full MS, MS/MS, and MS3 modes (Figure 1). The latter mode was only triggered when the iTRAQ-labeled lysine y-1 ion at m/z 291 was detected. Many iTRAQ peptides were found in the Data Dependent MS3 scans. The data was searched using TurboSEQUEST in BioWorks 3.2, selecting two differential modifications with an increase of 144 Da at the N-terminus of the peptide and an increase of 144 Da at any lysine residue.
Using these parameters, BioWorks identified all six of the expected proteins. Coverage was typically excellent, e.g., 36% coverage for bovine serum albumin from 13 peptides distributed throughout the protein. The iTRAQ label confers 144 Da to the N-terminal residue of the peptide so that the b-1 ion in the MS/MS scan will also be increased by this mass.
The spectrum in Figure 2 of the labeled peptide (LVNELTEFAK) from BSA shows a b-1 ion at 258 due to the iTRAQ label instead of the expected 114 ion for leucine. Likewise, lysine (y-1 ion) has been increased to 291 instead of the normal 147 for the lysine y-1 ion. The MS3 spectrum of m/z 291 from the peptide LVNELTEFAK revealed all four iTRAQ labels with equal intensity, as expected.
The relative standard deviation from theoretical was 2.4% based on the expected ratio of 1:1:1:1. These ratios were determined by normalizing the intensity of the 115, 116, and 117 mass tags to the intensity for the 114 tag. Figure 3 shows the results of the MS3 scan for a peptide where iTRAQ labels are at the low end of the intensity range.
The carboxypeptidase peptide revealed nearly equivalent labeling by iTRAQ with all the ratios falling within 10% of the theoretical ratios of 1:1:1:1. Producing a significant y-1 lysine ion was not a problem with this large, 15 residue peptide.
The results for many iTRAQ labeled peptides in the digest of the six proteins in the mixture is shown in the Table. Peptides were selected that had iTRAQ label intensities of at least 200, that had no missed cleavages, and that terminated in lysine. The intensities of the iTRAQ labels for all peptides varied by about 100-fold, and the length of the peptides varied from 7 to 22 residues. For a given peptide, the raw intensity of the iTRAQ labels was relatively consistent for all four tags.
The standard deviation of the raw intensities of the four tags from each peptide ranged from 317%, relative standard deviation. Four proteins had multiple peptides that met the selection criteria, and the results for each peptide were averaged and listed in red below the last peptide belonging to that protein in the Table.
The iTRAQ ratios for all four proteins with multiple peptides were closer to unity and had a lower standard deviation than the two proteins with just one peptide that qualified. The average ratio for all 24 peptides in the Table is 1.026 with a relative standard deviation of 6.0%.