Relative quantification by iTRAQ-labeling of in-gel digested proteins

A robust mass spectrometry-based quantification of spliceosomal proteins for characterization of the spliceosome’s protein composition at different intermediate states is required. Various methods for MS-based relative quantification exist (for an overview see Table 2.1 in the Introduction). iTRAQ reagents are chemical labels that specifically label amino termini and lysine side chains of peptides. iTRAQ quantification has some clear advantages: (i) iTRAQ reagents are multiplexing (i.e. up to four or eight samples can be compared in one experiment). (ii) As the iTRAQ reagents are amine specific, the peptides’ N-termini and lysine side chains are labeled thus providing many quantification data points per protein. (iii) Differentially labeled peptides are isobaric and the intensity in MS is thus enhanced. (iv) The different mass tags are completely cleaved during fragmentation leading to an enhanced fragment ion intensity. (v) iTRAQ-labeling offers the opportunity to create an internal standard by mixing aliquots of all samples to be compared, allowing comparison of even more samples than four (four-plex iTRAQ) or eight (eight-plex iTRAQ) samples.

4.2.1 Optimization of iTRAQ-labeling of in-gel digested proteins

The ABSciex iTRAQ labeling protocol comprises in-solution hydrolysis of the proteins, subsequent labeling of the peptides with iTRAQ reagents, and final removal of excess reagent by strong cation exchange chromatography (SCX). Here, we optimized iTRAQ labeling for in-gel digested proteins. This alteration has the major advantage that sample complexity is reduced before hydrolysis and even small quantities can be quantified as sample loss during SCX is avoided. For this purpose, the buffers used during in-gel hydrolysis of the proteins and the amount of iTRAQ reagents have been adjusted to the in-gel hydrolysis protocol. An additional reaction step to quench the excess of iTRAQ reagents by addition of glycine was included. Furthermore, an internal standard was prepared by

mixing equal amounts of all samples to be compared and labeling with one of the iTRAQ reagents. This allows controlling of the labeling reaction because the reporter ion intensity of the internal standard is related to the intensities of the reporter ions of the differently labeled samples. The optimized workflow thus comprises (i) the separation of the samples to be compared by gel electrophoresis, (ii) cutting entire gel lanes into gel slices of equal size, (iii) in-gel digestion of the proteins and extraction of the peptides, (iv) preparation of an internal standard from samples to be compared (i.e. from peptides extracted from gel slices of the same molecular weight region within the different samples), (v) iTRAQ-labeling of the internal standard and the samples with the different iTRAQ reagents and subsequent quenching of iTRAQ reagent excess, (vi) pooling of the samples and their respective internal standard, (vii) LC-MS/MS analysis, and (viii) identification (database search) and quantification of the peptides and finally the proteins (Figure 4.11).

Figure 4.11: Workflow for iTRAQ-labeling of in-gel digested proteins. Entire gel lanes of samples to be analyzed and quantified are cut into gel slices of equal size and gel slices are manually cut into smaller pieces.

Proteins are digested with trypsin within the gel and generated peptides are extracted. Extracted peptides are re-dissovled in 20 µl TEAB and an internal standard is prepared by pooling 5 µl of each sample. The internal standard and the samples to be compared are labeled with different iTRAQ reagents. After pooling, the samples are analyzed by LC-MS/MS and quantification is done by comparing the peak areas of individual reporter ions.

4.2.2 Relative quantification of different amounts of spliceosomal tri-snRNP proteins – a feasibility study

To validate the established iTRAQ workflow, different amounts of spliceosomal tri-snRNP proteins were quantified using iTRAQ labeling. To this end, 5.0 and 2.5 µg of purified human tri-snRNP were separated by gel electrophoresis (Figure 4.12) and entire gel lanes were cut into gel slices of equal size. In addition, an empty gel lane (blank) was cut and processed together with the other samples to show accuracy of the established iTRAQ protocol.

Figure 4.12: Separation of different amounts of spliceosomal tri-snRNP proteins by gel electrophoresis.

5.0 µg and 2.5 µg tri-snRNP were separated by gel electrophoresis and entire gel lanes were cut into slices of equal size. An empty gel lane (L1) was cut and processed with the other samples.

The proteins from two gel slices at different molecular weight (approximately 250 kDa and 100 kDa), showing proper stained protein bands at both concentrations, were hydrolyzed with trypsin in-gel. An internal standard was prepared from the samples to be compared (i.e.

L1, L2 (5.0 µg tri-snRNP) and L3 (2.5 µg tri-snRNP)) and the peptides of the different samples and the internal standard were labeled separately with iTRAQ reagents. The internal standard was labeled with iTRAQ reagent 114 and the different samples were labeled with iTRAQ reagents 115-117 (see also Figure 4.12). Excess iTRAQ reagents were quenched by adding glycine. Samples to be compared (i.e. L1-L3) and their corresponding internal standard were then pooled and subsequently analyzed by LC-MS/MS. Quantification was performed by comparing the peak areas of generated reporter ions after fragmentation.

Figure 4.13 shows a typical MS/MS spectrum.

Figure 4.13: Example MS/MS spectrum of an iTRAQ labeled peptide. During MS/MS KLPEEVVK (U5-200K) was sequenced and the reporter ions were released. The reporter region (m/z 114-117) is magnified and shows the different reporter ions that could be used for quantification.

The different tri-snRNP specific proteins within the two molecular weight regions were identified. For quantification, the protein ratios are calculated relatively to the internal standard, i.e. by dividing the peak area of reporter ions of the blank sample (iTRAQ-115), 5.0 µg (iTRAQ-116), and 2.5 µg (iTRAQ-117) tri-snRNP, respectively, by the peak area of the internal standard (iTRAQ-114). In addition, the protein ratio comparing the different amounts of tri-snRNP was also calculated (iTRAQ-116/iTRAQ-117).

A protein ratio of approximately 2.5 for the different amounts of tri-snRNP (iTRAQ-116/iTRAQ-117) was obtained for almost all proteins. The protein ratio of the blank sample (iTRAQ-115/iTRAQ-114) was in all cases approximately 0.1 or lower (Table 4.8). The obtained protein ratios are plotted in bar diagrams to visualize the relative protein amounts within the different gel lanes (Figure 4.14). As chemical labeling always risks being incomplete, the labeling efficiency (i.e. the percentage of labeled peptides) was calculated for all identified proteins. For this purpose, the obtained precursor masses (MS) and fragment ion masses (MS/MS) were searched against the database allowing for iTRAQ-labels as variable modifications. This yields labeled and non-labeled peptides and the labeling efficiency could be calculated from the number of identified iTRAQ-labeled peptides and the total number of identified peptides. In pilot experiments, the labeling efficiency was found to

be one of the major issues for reliable quantification, which was only achieved for labeling efficiencies > 90 %. For this proof of principle, a labeling efficiency of 92 % or higher was obtained (Table 4.8).

Figure 4.14: iTRAQ protein ratios for various identified tri-snRNP proteins. Ratios were calculated by dividing the peak area of reporter ions of the blank sample 115), 5.0 µg 116), and 2.5 µg (iTRAQ-117) tri-snRNP, respectively, by the peak area of the internal standard (iTRAQ-114).

Table 4.8: Protein ratios for the relative quantification of different amounts of spliceosomal tri-snRNP proteins. iTRAQ ratios relative to the internal standard and one iTRAQ ratio comparing the different tri-snRNP amounts were calculated. The labeling efficiency is indicated in percent.

Protein  iTRAQ ratios 

labeling efficiency [%] 

115/114  116/114  117/114  116/117 

U5‐220K  0.085  3.192  1.269  2.515  95.27 

U5‐200K  0.073  3.413  1.341  2.545  94.15 

U4/U6.U5‐110K  0.104  3.407  1.057  3.223  100.00 

U5‐102K  0.106  3.036  1.300  2.335  97.27 

U5‐100K  0.092  3.283  1.048  3.133  92.86 

4.3 Relative quantification of spliceosomal B and C complexes – a comparative