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Reproducible isolation of distinct, overlapping segments of the phosphoproteome


The ability to routinely analyze and quantitatively measure changes in protein phosphorylation on a proteome-wide scale is essential for biological and clinical research. We assessed the ability of three common phosphopeptide isolation methods (phosphoramidate chemistry (PAC), immobilized metal affinity chromatography (IMAC) and titanium dioxide) to reproducibly, specifically and comprehensively isolate phosphopeptides from complex mixtures. Phosphopeptides were isolated from aliquots of a tryptic digest of the cytosolic fraction of Drosophila melanogaster Kc167 cells and analyzed by liquid chromatography–electrospray ionization tandem mass spectrometry. Each method reproducibly isolated phosphopeptides. The methods, however, differed in their specificity of isolation and, notably, in the set of phosphopeptides isolated. The results suggest that the three methods detect different, partially overlapping segments of the phosphoproteome and that, at present, no single method is sufficient for a comprehensive phosphoproteome analysis.

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Figure 1: Strategy used to analyze the reproducibility, overlap and specificity of the different phosphopeptide isolation methods.
Figure 2: Similarity and overlap within and between isolates of the investigated phosphopeptide isolation methods.
Figure 3: Comparison of LC-MS base peak chromatograms for isolation products from the investigated methods.
Figure 4: Overlap between phosphopeptide isolation methods on the level of identified phosphorylation sites.
Figure 5: Characteristics of phosphopeptides identified from PAC, IMAC and TiO2 isolates.


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We thank O. Rinner, J. Malmström, P. Picotti and M. Larsen for fruitful discussions. This project has been funded in part by ETH Zurich and by federal funds from the US National Heart, Lung, and Blood Institute of the National Institutes of Health (N01-HV-28179). B.B. is the recipient of a fellowship by the Boehringer Ingelheim Fonds.

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Correspondence to Ruedi Aebersold.

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Supplementary information

Supplementary Fig. 1

Amino acid usage of the identified (phospho)peptides compared to the starting material and the used protein database. (PDF 88 kb)

Supplementary Fig. 2

The influence of the peptide to resin ratio on the selectivity and percentage of singly phosphorylated peptides in the case of pTiO2. (PDF 66 kb)

Supplementary Fig. 3

The influence of the peptide to resin ratio on the selectivity and percentage of singly phosphorylated peptides in the case of IMAC. (PDF 21 kb)

Supplementary Table 1

Values of the similarity and overlap between any two LC-MS runs as computed by Superhirn. (PDF 99 kb)

Supplementary Table 2

Phosphopeptides identified with PAC, IMAC, pTiO2 and dhbTiO2. (PDF 214 kb)

Supplementary Table 3

Influence of methylation prior and/or after phosphopeptide isolation using IMAC. (PDF 156 kb)

Supplementary Methods (PDF 93 kb)

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Bodenmiller, B., Mueller, L., Mueller, M. et al. Reproducible isolation of distinct, overlapping segments of the phosphoproteome. Nat Methods 4, 231–237 (2007).

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