Alcance

As highlighted in Chapter 1, protein kinases are essential regulators of cell signalling that modulate each other’s functions and activities through site-specific phosphorylation events (Manning et al., 2002b; Shi et al., 2006). Their low abundances make it difficult to identify them from complete lysates. Thus, to increase the analytical sensitivity for protein kinases, they have to be enriched from total cell extracts prior to MS analysis. We applied an experimental strategy dedicated to enrich phosphorylated peptides from kinases to analyze protein kinase regulation in cell cycle progression (Daub et al., 2008).

The cell cycle comprises the progression of events leading to the replication of the eukaryotic cell. It can be divided into mitosis (M phase) including the nuclear and cytoplasmic division followed by interphase consisting of four phases: During the G1 phase the cell starts to grow

and synthesis of enzymes required for the next phasis – the S phase – is initated. In S-phase DNA is replicated while rates of protein synthesis are slow except for histones, which are

needed for packaging of the DNA. During the G2 phase, the cell prepares for mitosis by

producing microtubules, for instance.

Figure 4.22: Schematic illustration of the cell cycle

In our study, we combined efficient kinase enrichment with quantitative mass spectrometry using SILAC. The basic experimental design is similar to the one applied for the identification of the human phosphoproteome upon EGF stimulation (Chapter 4.6.1.1.1), as it is also based on the same cell type and mass spectrometric technologies including SCX chromatography, TiO2 peptide enrichment, and the SILAC labelling technique (Daub et al., 2008; Gruhler et

al., 2005; Larsen et al., 2005; Ong et al., 2002). The statistical analysis of detected peptides and quantitation were also analogous.

Two populations of HeLa cells were quantitatively labelled by growing them in medium containing either normal arginine and lysine or their heavy isotopic variants. The cells were synchronized in early S phase by a double thymidine block in suspension culture. One of the populations was harvested at this point, whereas cells of the second population were released into a mitotic arrest. Then, pooled lysates from M and S phase cells were loaded onto a series of affinity columns displaying different immobilized kinase inhibitors with distinct kinase binding profiles to enrich protein kinases. We applied both gel electrophoresis followed by tryptic digestion on one of the kinase enriched subfractions and SCX chromatography to the kinase enriched fractions. The resulting peptide fractions were then subjects to phosphopeptide enrichment on TiO2 beads. The combination of gel-based and gel-free MS

separation strategies with phosphopeptide enrichment increases the overall number of detected phosphorylated peptides.

The statistical analysis of assigned peptide sequences and quantitation similar as described above (Chapter 4.6.1.1.1) and employed MASCOT, MSQuant, and various methods provided by the PHOSIDA administration tools (Chapter 4.2.6). This proteomic approach enabled us to quantify protein kinases from S and M phase arrested human cells and to elucidate cell-cycle dependent protein kinase regulation.

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We uniquely identified and quantified phosphorylated peptides from 1377 proteins (Daub et al., 2008). The identified peptides harbored 3144 phosphorylation sites (83.5% pS, 14.2% pT, 2.3% pY) (Figure 4.23A). About 14% of all analyzed proteins were protein kinases: 219 different members of the human protein kinase superfamily were detected to be phosphorylated in our study. The phosphorylated kinases embraced 1007 phosphosites that could be assigned to serine (77.5%), threonine (17.2%), and tyrosine (5.3%) residues with high confidence. The vast majority of these detected phosphorylation sites in protein kinases have not been reported earlier.

Figure 4.23: (A) Distribution of phosphorylation sites by amino acid. (B) Overlapping phosphoproteins between this study (green) and the previously reported study (blue) (Chapter 4.6.1.1.1). (C) Identified protein kinases marked in the kinome tree as illustrated in Chapter 1. The identification of at least two- fold differentially regulated phosphopeptides (PPs) in M versus S phase derived protein kinases is indicated by different colors.

We determined the overlap between this study and the investigation of the human phosphoproteome upon EGF stimulation (Chapter 4.6.1.1.1) and found that 508 (37%) out of 1377 phosphoproteins were also identified in the other large scale phosphoproteome analysis (Figure 4.23B). An even lower overlap was observed on the site level, as 546 (17%) out of 3144 phosphosites had also been measured in the EGF study. Interestingly, more than half of all kinase phosphopeptides were upregulated at least two-fold in mitotically arrested HeLa cells. In comparison, only 10% showed increased S phase abundance. At the protein level,

regulation by factor two or more was observed for less than 10% of all protein kinases. If only SILAC phosphopeptide ratios are considered, apparent changes in phosphorylation could actually be due to a change in protein amount. Therefore, for each phosphorylated peptide the online application of PHOSIDA shows whether the given quantitative data describing the phosphorylation regulation during cell cycle could be normalized by the corresponding protein ratios or not. This information was stored as a special ‘feature’ attribute in the ‘peptides_sub’ relation (Chapter 4.2.1). Overall, 75% of all detected protein kinases contained at least one cell cycle regulated phosphopeptide (greather than two-fold upregulated in S phase or M phase). Strikingly, even for intensely studied cell cycle kinases including PLK1 and CDC2, a large number of new phosphorylation sites were found, demonstrating the high analytical sensitivity of our experimental approach. However, our study also covered a large number of other proteins that were quantitatively evaluated. For example, several regulatory kinase subunits such as different members of the cyclin family showed cell cycle dependent phosphorylation patterns.

In this study the spectra of measured phosphopeptides were also integrated into PHOSIDA because the applied MSQuant version enabled to create and automically save an image file of each spectrum. The corresponding file names can be derived from the MSQuant result files. The corresponding image file names are another ‘feature’ tuple of each entry (peptide object) stored in the ‘peptides_sub’ database relation.

The database storage of associated spectra and the visualization of these spectra enable web users to validate both identification and quantitation of each identified peptide. In PHOSIDA, the corresponding ‘spectrum’ buttons appear at the result page listing all detected peptides that contain the selected phosphorylation site (Figure 4.24). Besides the linkages to spectra, the cell cycle dependent phosphorylation regulation, Mascot scores, PTM scores, and further information are illustrated as discussed in Chapter 4.2.5.

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Figure 4.24: For large scale phosphorylation studies using MSQuant, PHOSIDA provides linkages (A) to an integrated online spectrum visualizer (B).