Improved Immobilized Metal Affinity Chromatography for Large-Scale Phosphoproteomics Applications

Dysregulated protein phosphorylation is a primary culprit in multiple physiopathological states. Hence, although analysis of signaling cascades on a proteome-wide scale would provide significant insight into both normal and aberrant cellular function, such studies are simultaneously limited by sheer...

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Bibliographic Details
Published in:Journal of proteome research 2006-10, Vol.5 (10), p.2789-2799
Main Authors: Ndassa, Yasmine M, Orsi, Chris, Marto, Jarrod A, Chen, She, Ross, Mark M
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Buffers
Chromatography, Affinity - methods
Humans
Metals - chemistry
Molecular Sequence Data
Phosphopeptides - analysis
Phosphorylation
Proteomics - methods
Sequence Analysis, Protein
Tumor Cells, Cultured
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Summary:Dysregulated protein phosphorylation is a primary culprit in multiple physiopathological states. Hence, although analysis of signaling cascades on a proteome-wide scale would provide significant insight into both normal and aberrant cellular function, such studies are simultaneously limited by sheer biological complexity and concentration dynamic range. In principle, immobilized metal affinity chromatography (IMAC) represents an ideal enrichment method for phosphoproteomics. However, anecdotal evidence suggests that this technique is not widely and successfully applied beyond analysis of simple standards, gel bands, and targeted protein immunoprecipitations. Here, we report significant improvements in IMAC-based methodology for enrichment of phosphopeptides from complex biological mixtures. Moreover, we provide detailed explanation for key variables that in our hands most influenced the outcome of these experiments. Our results indicate 5- to 10-fold improvement in recovery of singly- and multiply phosphorylated peptide standards in addition to significant improvement in the number of high-confidence phosphopeptide sequence assignments from global analysis of cellular lysate. In addition, we quantitatively track phosphopeptide recovery as a function of phosphorylation state, and provide guidance for impedance-matching IMAC column capacity with anticipated phosphopeptide content of complex mixtures. Finally, we demonstrate that our improved methodology provides for identification of phosphopeptide distributions that closely mimic physiological conditions. Keywords: immobilized metal affinity chromatography • phosphoproteomics
ISSN:1535-3893
1535-3907
DOI:10.1021/pr0602803