Reversible phosphorylation of proteins regulates the majority of all cellular processes, e.g. proliferation, differentiation, and apoptosis. A fundamental understanding of these biological processes at the molecular level requires characterization of the phosphorylated proteins. Phosphorylation is often substoichiometric, and an enrichment procedure of phosphorylated peptides derived from phosphorylated proteins is a necessary prerequisite for the characterization of such peptides by modern mass spectrometric methods. We report a highly selective enrichment procedure for phosphorylated peptides based on TiO 2 microcolumns and peptide loading in 2,5-dihydroxybenzoic acid (DHB). The effect of DHB was a very efficient reduction in the binding of nonphosphorylated peptides to TiO 2 while retaining its high binding affinity for phosphorylated peptides. Thus, inclusion of DHB dramatically increased the selectivity of the enrichment of phosphorylated peptides by TiO 2 . We demonstrated that this new procedure was more selective for binding phosphorylated peptides than IMAC using MALDI mass spectrometry. In addition, we showed that LC-ESI-MSMS was biased toward monophosphorylated peptides, whereas MALDI MS was not. Other substituted aromatic carboxylic acids were also capable of specifically reducing binding of nonphosphorylated peptides, whereas phosphoric acid reduced binding of both phosphorylated and nonphosphorylated peptides. A putative mechanism for this intriguing effect is presented. Molecular & Cellular Proteomics 4: 873-886, 2005.Phosphorylation is among the most widespread post-translational modifications in nature, and it has been estimated that more than 30% of the proteins in a given mammalian cell at some point during their expression are phosphorylated (1). Phosphorylation and dephosphorylation of proteins regulates a large number of biological processes such as signal transduction (2), molecular recognition and interaction, and other cellular events. A fundamental understanding of these biological processes at the molecular level thus requires a characterization of the phosphorylated sites in the proteins. It is therefore essential to develop sensitive and selective methods for this task.A wide variety of methods are known for characterization of phosphorylated proteins. The most widely used have been peptide sequencing using Edman degradation combined with 32 P labeling. This method is well established and very robust but has several limitations. For example, in Edman degradation the peptides have to be separated before the analysis using liquid chromatography. This decreases the overall sensitivity and increases analysis time, and it is therefore not well suited for analysis of complex samples.Recently a number of MS-based strategies have been developed that are relatively sensitive and in many cases easier to perform than Edman degradation with respect to handling complex mixtures (e.g. Ref.3). The increased sensitivity is especially needed for low stoichiometric phosphorylation. However, presently ...

Despite decades of accumulated knowledge about proteins and their post-translational modifications (PTMs), numerous questions remain regarding their molecular composition and biological function. One of the most fundamental queries is the extent to which the combinations of DNA-, RNA- and PTM-level variations explode the complexity of the human proteome. Here, we outline what we know from current databases and measurement strategies including mass spectrometry-based proteomics. In doing so, we examine prevailing notions about the number of modifications displayed on human proteins and how they combine to generate the protein diversity underlying health and disease. We frame central issues regarding determination of protein-level variation and PTMs, including some paradoxes present in the field today. We use this framework to assess existing data and to ask the question, "How many distinct primary structures of proteins (proteoforms) are created from the 20,300 human genes?" We also explore prospects for improving measurements to better regularize protein-level biology and efficiently associate PTMs to function and phenotype.

The characterization of phosphorylated proteins is a challenging analytical task since many of the proteins targeted for phosphorylation are low in abundance and phosphorylation is typically substoichiometric. Highly efficient enrichment procedures are therefore required. Here we describe a protocol for selective phosphopeptide enrichment using titanium dioxide (TiO2) chromatography. The selectivity toward phosphopeptides is obtained by loading the sample in a 2,5-dihydroxybenzoic acid (DHB) or phthalic acid solution containing acetonitrile and trifluoroacetic acid (TFA) onto a TiO2 micro-column. Although phosphopeptide enrichment can be achieved by using TFA and acetonitrile alone, the selectivity is dramatically enhanced by adding DHB or phthalic acid since these compounds, in conjunction with the low pH caused by TFA, prevent binding of nonphosphorylated peptides to TiO2. Using an alkaline solution (pH > or = 10.5) both monophosphorylated and multiphosphorylated peptides are eluted from the TiO2 beads. This highly efficient method for purification of phosphopeptides is well suited for the characterization of phosphoproteins from both in vitro and in vivo studies in combination with mass spectrometry (MS). It is a very easy and fast method. The entire protocol requires less than 15 min per sample if the buffers have been prepared in advance (not including lyophilization).

The complete analysis of phosphoproteomes has been hampered by the lack of methods for efficient purification, detection, and characterization of phosphorylated peptides from complex biological samples. Despite several strategies for affinity enrichment of phosphorylated peptides prior to mass spectrometric analysis, such as immobilized metal affinity chromatography or titanium dioxide, the coverage of the phosphoproteome of a given sample is limited. Here we report a simple and rapid strategy, SIMAC (sequential elution from IMAC), for sequential separation of monophosphorylated peptides and multiply phosphorylated peptides from highly complex biological samples. This allows individual analysis of the two pools of phosphorylated peptides using mass spectrometric parameters differentially optimized for their unique properties. We compared the phosphoproteome identified from 120 g of human mesenchymal stem cells using SIMAC and an optimized titanium dioxide chromatographic method. More than double the total number of identified phosphorylation sites was obtained with SI-

Protein phosphorylation is a key regulator of cellular signaling pathways. It is involved in most cellular events in which the complex interplay between protein kinases and protein phosphatases strictly controls biological processes such as proliferation, differentiation, and apoptosis. Defective or altered signaling pathways often result in abnormalities leading to various diseases, emphasizing the importance of understanding protein phosphorylation. Phosphorylation is a transient modification, and phosphoproteins are often very low abundant. Consequently, phosphoproteome analysis requires highly sensitive and specific strategies. Today, most phosphoproteomic studies are conducted by mass spectrometric strategies in combination with phosphospecific enrichment methods. This review presents an overview of different analytical strategies for the characterization of phosphoproteins. Emphasis will be on the affinity methods utilized specifically for phosphoprotein and phosphopeptide enrichment prior to MS analysis, and on recent applications of these methods in cell biological applications.