2008
DOI: 10.1021/pr0705441
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Combining Protein-Based IMAC, Peptide-Based IMAC, and MudPIT for Efficient Phosphoproteomic Analysis

Abstract: 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… Show more

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Cited by 139 publications
(148 citation statements)
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References 26 publications
(56 reference statements)
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“…In some cases, definitive localization of the phosphorylation site could not be obtained (Figure 1a, sites marked in green). Some of these sites have previously been reported, including Ser21 and Ser1177, which have been identified in different largescale analyses of phosphoproteins (Beausoleil et al, 2004;Dai et al, 2007;Cantin et al, 2008;Daub et al, 2008;Dephoure et al, 2008;Zanivan et al, 2008). In addition, the phosphopeptides containing Ser1174, Ser1219, Thr1222, Thr1295, Ser1385, Tyr1386 and Ser1388, which have recently reported been, were also detected (Dephoure et al, 2008;Zanivan et al, 2008).…”
Section: Identification Of Phosphorylation Sites Within Rictormentioning
confidence: 70%
“…In some cases, definitive localization of the phosphorylation site could not be obtained (Figure 1a, sites marked in green). Some of these sites have previously been reported, including Ser21 and Ser1177, which have been identified in different largescale analyses of phosphoproteins (Beausoleil et al, 2004;Dai et al, 2007;Cantin et al, 2008;Daub et al, 2008;Dephoure et al, 2008;Zanivan et al, 2008). In addition, the phosphopeptides containing Ser1174, Ser1219, Thr1222, Thr1295, Ser1385, Tyr1386 and Ser1388, which have recently reported been, were also detected (Dephoure et al, 2008;Zanivan et al, 2008).…”
Section: Identification Of Phosphorylation Sites Within Rictormentioning
confidence: 70%
“…MYBBP1A is reported to be a heavily phosphorylated protein in cells, according to several recent large scale mass spectrometry-based phosphoproteomic studies (65)(66)(67)(68)(69)(70)(71)(72). The majority of the phosphorylation sites mapped in MYBBP1A in these studies (18 of a total of 21) reside within the ϳ200-amino acid-long C-terminal portion of the protein, which has been shown to be relevant for its nuclear and nucleolar localization (73).…”
Section: Discussionmentioning
confidence: 99%
“…EGF stimulates phosphorylation at Ser-54, Ser-58, Ser-171, and Ser-173) (106,107). S54F is a "special" EDMD-causing mutation that does not disrupt emerin expression or localization yet still causes EDMD (78).…”
Section: Discussionmentioning
confidence: 99%