Combining protein-based IMAC, peptide-based IMAC, and MudPIT for efficient phosphoproteomic analysis

Departments of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Journal of Proteome Research (Impact Factor: 4.25). 04/2008; 7(3):1346-51. DOI: 10.1021/pr0705441
Source: PubMed


Immobilized metal affinity chromatography (IMAC) is a common strategy used for the enrichment of phosphopeptides from digested protein mixtures. However, this strategy by itself is inefficient when analyzing complex protein mixtures. Here, we assess the effectiveness of using protein-based IMAC as a pre-enrichment step prior to peptide-based IMAC. Ultimately, we couple the two IMAC-based enrichments and MudPIT in a quantitative phosphoproteomic analysis of the epidermal growth factor pathway in mammalian cells identifying 4470 unique phosphopeptides containing 4729 phosphorylation sites.

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    • "I in early publication) phosphorylated proteins were essential for the elongation of body segments, production of hooded hooks, and preparation of juvenile tissues during larval metamorphosis (Chandramouli et al., 2011a). Protein enrichment methods have enabled the separation of proteins according to their PTM (e.g., phosphorylation) (Gilchrist et al., 2006; Puente et al., 2006; Cantin et al., 2008). This approach was used successfully by Chandramouli et al. (2011b) to improve the detection and identification of phosphoproteins during larval metamorphosis of P. vexillosa, revealing that competent larvae exhibit a higher degree of phosphorylation than precompetent larvae and juveniles. "
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    • "Since the initial release of P3DB, high-quality phosphorylation sites in this database have accumulated at a rapid pace due to improvements in enrichment techniques and mass spectrometry [Supplementary Figure S1a and b]. Most of the datasets in the database came from large-scale experiments (MS/MS) (3), although several smaller datasets were also deposited. To help users analyze the proteome-wide phosphorylation data more systematically, the new P3DB 3.0 provides more information and annotations about phosphoproteins such as gene ontology, homolog, 3D structures, kinase and phosphatase families, protein–protein interactions (PPIs) and protein domains, together with protein–protein networks, kinase-substrate or phosphatase-substrate networks and domain co-occurrence networks (4). "
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    • "Some isoforms decreased in abundance but new isoforms accumulated in the S phase samples (Figure 5C, compare lane 1 with lanes 2 and 3). Of note, the hnRNPA3 protein has been reported to be heavily phosphorylated, raising the possibility that the decrease observed by mass spectrometry was due to cell cycle regulated post-translational modifications [52], [53], [54], [55], [56], [57], [58], [59]. Indeed, a number of hnRNPs, including hnRNPD0, were identified as Cyclin A/Cdk2 substrates [17]. "
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