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 ...

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.

Protein activity and turnover is tightly and dynamically regulated in living cells. Whereas the three-dimensional protein structure is predominantly determined by the amino acid sequence, posttranslational modification (PTM) of proteins modulates their molecular function and the spatial-temporal distribution in cells and tissues. Most PTMs can be detected by protein and peptide analysis by mass spectrometry (MS), either as a mass increment or a mass deficit relative to the nascent unmodified protein. Tandem mass spectrometry (MS/MS) provides a series of analytical features that are highly useful for the characterization of modified proteins via amino acid sequencing and specific detection of posttranslationally modified amino acid residues. Large-scale, quantitative analysis of proteins by MS/MS is beginning to reveal novel patterns and functions of PTMs in cellular signaling networks and biomolecular structures.

Cell–cell and intracellular signaling are critical mechanisms by which an organism can respond quickly and appropriately to internal or environmental stimuli. Transmission of the stimulus to effector proteins must be coordinated, rapid and transient such that the response is not exaggerated and the overall balance of the cell or tissue is retained. Proteomics technology has traditionally been adept at analyzing effector proteins (such as cytoskeletal and heat shock proteins, and those involved in metabolic processes) in studies examining the effects of altered environmental or nutritional conditions, drugs, or genetic manipulation, since these proteins are often highly abundant, soluble and therefore amenable to analysis. Conversely, the proteins mediating the transmission of the signal have been generally under‐represented, typically because of their low abundance. One mechanism that has overcome this to some extent is the advent of very high‐resolution phosphoproteomics techniques, which have enabled temporal profiling of intracellular signal pathways via quantitative assessment of peptide phosphorylation sites. One group of proteins, however, that still remains under‐represented in proteomics studies are those found in the plasma membrane (PM). Such proteins are crucial in sensing changes in the external environment and in stimulating the transmission of the signal intracellularly. This review examines PM proteins and appraises the proteomics approaches currently available for providing a comprehensive analysis of these crucial mediators of signal pathways. We discuss different strategies for enrichment and solubilization of these proteins and include discussion on cross‐linking of PM complexes and glycoproteomics as the basis for purification prior to proteomic analyses.

The study of cellular dynamics by proteomics using mass spectrometry requires a quantitation strategy that is robust, sensitive, and of sufficient resolution to deal with subtle changes in protein expression or post-translational modification. The major quantitation strategies are stable isotopic labeling of proteins and peptides for in vitro cell culture systems (stable isotope labeling using amino acids in cell culture, SILAC) or isobaric peptide labels such as isobaric tags for relative and absolute quantitation (iTRAQ) and tandem mass tags (TMT) for both in vitro and in vivo systems. These quantitation strategies have also been successfully applied to phosphoproteomics studies for the investigation of signal transduction pathways. Here we describe major drawbacks associated with isobaric labeling for the identification and quantitation of phosphopeptides using electrospray tandem mass spectrometry. Phosphopeptide derivatization with isobaric tags results in significantly greater charging in electrospray ionization. This reduces phosphopeptide identification efficiency with multistage activation and HCD MS/MS by more than 50% and may contribute to the discrepancy observed between identifications observed for large cell- or tissue-based data sets from labeled and nonlabeled peptide mixtures. Ammonia vapor sprayed perpendicular to the electrospray needle during ionization resulted in an overall decrease in the average charge states and a concomitant increase in phosphopeptide identifications.

Phosphorylation of plasma membrane proteins frequently initiates signal transduction pathways or attenuate plasma membrane transport processes. Because of the low abundance and hydrophobic features of many plasma membrane proteins and the low stoichiometry of protein phosphorylation, studies of the plasma membrane phosphoproteome are challenging. We present an optimized analytical strategy for plasma membrane phosphoproteomics that combines efficient plasma membrane protein preparation with TiO(2)-based phosphopeptide enrichment and high-performance mass spectrometry for phosphopeptide sequencing. We used sucrose centrifugation in combination with sodium carbonate extraction to achieve efficient and reproducible purification of low microgram levels of plasma membrane proteins from human mesenchymal stem cells (hMSCs, 10(7) cells), achieving more than 70% yield of membrane proteins. Phosphopeptide enrichment by titanium dioxide chromatography followed by capillary liquid chromatography-tandem mass spectrometry allowed us to assign 703 unique phosphorylation sites in 376 phosphoproteins. Our experiments revealed that treatment of cell cultures with three different types of protein phosphatase inhibitors produces distinct phosphopeptide populations and an increase of 10-40% of the number of detected and sequenced phosphoserine, phosphothreonine and phosphotyrosine containing peptides. In summary, our analytical strategy enables functional phosphoproteomic analysis of stem cell differentiation and cell surface biomarker discovery using very low amounts of starting material.