Titanium dioxide (TiO(2)) has been proven to be a highly efficient strategy and widely used for phosphopeptide enrichment. Many advances have been made recently, including online/offline mode and optimization of sample loading/elution buffer; however, beads usage has rarely been explored. In the current study, we found that the peptide-to-TiO(2) beads ratio was a significant factor for phosphopeptide enrichment, and insufficient or excessive beads could decrease the selectivity. Specifically, for HeLa total cell lysate, the optimum peptide-to-beads ratio is about 1:2-1:8 (mass/mass) to obtain the highest enrichment selectivity and the maximum phosphopeptides identification with single incubation. Pre-experiments are recommended to decide an optimum peptide-to-TiO(2) beads ratio when it comes to different samples. Interestingly, deficient beads can help identify much more multiphosphorylated peptides than the optimum peptide-to-beads ratio by consecutive incubations. Therefore, if multiphosphorylated peptides identification is desired, deficient beads amount is preferred. In addition, consecutive incubation using deficient beads could be used as a fractionation of phosphopeptides besides as an enrichment method.

Reversible phosphorylation of proteins is an important process modulating cellular activities from upstream, which mainly involves sequential phosphorylation of signaling molecules, to downstream where phosphorylation of transcription factors regulates gene expression. In this study, we combined quantitative labeling with multidimensional liquid chromatography-mass spectrometry to monitor the proteome and phosphoproteome changes in the initial period of adipocyte differentiation. The phosphorylation level of a specific protein may be regulated by a kinase or phosphatase without involvement of gene expression or as a phenomenon that accompanies the alteration of its gene expression. Concurrent quantification of phosphopeptides and nonphosphorylated peptides makes it possible to differentiate cellular phosphorylation changes at these two levels. Furthermore, on the system level, certain proteins were predicted as the targeted gene products regulated by identified transcription factors. Among them, several proteins showed significant expression changes along with the phosphorylation alteration of their transcription factors. This is to date the first work to concurrently quantify proteome and phosphoproteome changes during the initial period of adipocyte differentiation, providing an approach to reveal the system-wide association of protein phosphorylation and gene expression. Protein phosphorylation has been considered as a central role for cell regulation and signaling transduction. It is estimated that one-third of eukaryotic proteins are phosphorylated (1). In eukaryotes, the residues that undergo protein phosphorylation are mainly serine, threonine, and to a lesser extent tyrosine (2). Reversible phosphorylation of proteins is an important process for modulating cellular activities. At the upstream level, regulation of signaling transduction mainly relies on the appropriate phosphorylation events, such as sequential phosphorylation of various signaling molecules. At the downstream level, however, signaling transduction in the nucleus generally involves transcription of certain genes. Transcription factor activity, besides being regulated at the level of gene expression, is prominently regulated via post-translational events such as protein phosphorylation, processing, or localization (3). Phosphorylation or dephosphorylation of transcription-associated factors plays a crucial role in transcriptional regulation (see Fig. 1A).Most of the previous studies have focused on the initial period of signaling transduction, which mainly refers to the dynamic changes of phosphorylation events only. Recent developments in mass spectrometry promise to provide novel insights into the dynamics of protein expression and activities regulated by post-translational modifications (7,8). However, the isolation of phosphoproteins from complex mixtures and the determination of phosphorylation sites still remain a challenge. Different strategies have been applied for enrichment of phosphorylated peptides and proteins, including immuno...