Nearly 85 years after the isolation of phosphoserine, many analytical challenges remain in our pursuit of understanding how phosphorylation drives complex biological processes. As with all areas of science and natural history, the more we know, the more questions that will be raised. Phosphorylation is more widespread than could ever have been imagined. Is most of this activity noise, or is it, like intronic DNA, not yet fully understood? Uncovering the functional significance of these waves of phosphorylation is the major task at hand for at least the next decade. Confidently localizing specific sites of phosphorylation can still be problematic. The newly realized possibility of phosphate group migration during the CID process on certain types of mass spectrometers is troublesome and calls into question the validity of the database results posted from these instruments. Fortunately newer generations of mass spectrometers are less prone to these effects. Ultimately the assignment of specific phosphorylation sites must be confirmed by site-directed mutagenesis and biological testing. Strategies to enrich samples for their phosphopeptide content are now well established. However, in the absence of radioactive phosphate labeling, it is impossible to know whether all sites in a particular sample have been recovered. Localizing phosphorylation on multiply phosphorylated peptides is particularly difficult. Two phosphorylation events, differentially localized over three or more residues, with stoichiometry varying between the combinations is not an unusual circumstance.
Correlating a change in the degree of phosphosite occupancy with a biological response is the key to assigning functional significance to a given site. In practice this is less straightforward than simply measuring a change in the intensity of the phosphopeptide signal. For short duration signaling events, de novo protein synthesis can be ignored, but clearly both protein synthesis
and protein degradation can influence the abundance of a phosphopeptide, regardless of any real change at the site. For quantitative phosphoproteomics this means having to also analyze global protein expression in a separate set of experiments.
The occurrence of phosphorylation in close proximity with other PTMs is now well documented. Measuring changes in phosphosite occupancy will be confounded by any changes occurring on the other sites of modification at the same time. In fact, understanding how various PTMs work in opposition to each other or together to influence function is a major challenge for the study of cell signaling. While the global analysis of various PTMs has much to offer in terms of understanding how these modifications affect overall cell biology, a high-resolution picture of the intricacies of the process will likely need to result from a focus on simpler systems. As a universal quantitative tool, able in principle, to identify any type of PTM, MS has and will continue to have an important role to play in this investigation.