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

Article Details

Citation
Copy to clipboard

Cantin GT, Yi W, Lu B, Park SK, Xu T, Lee JD, Yates JR 3rd

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

J Proteome Res. 2008 Mar;7(3):1346-51. doi: 10.1021/pr0705441. Epub 2008 Jan 26.

PubMed ID
18220336 [ View in PubMed
]
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 identifying 4470 unique phosphopeptides containing 4729 phosphorylation sites.

DrugBank Data that Cites this Article

Polypeptides
NameUniProt ID
RAF proto-oncogene serine/threonine-protein kinaseP04049Details
Bifunctional glutamate/proline--tRNA ligaseP07814Details
Solute carrier family 12 member 2P55011Details
Serine/threonine-protein kinase A-RafP10398Details
Acetyl-CoA carboxylase 1Q13085Details
5'-AMP-activated protein kinase catalytic subunit alpha-1Q13131Details
DNA topoisomerase 2-alphaP11388Details
Epidermal growth factor receptorP00533Details
CTP synthase 1P17812Details
DNA (cytosine-5)-methyltransferase 1P26358Details
C-X-C chemokine receptor type 4P61073Details
Insulin receptor substrate 1P35568Details
Catenin beta-1P35222Details
Cyclin-dependent kinase 1P06493Details
Phosphatidylserine synthase 1P48651Details
Microtubule-associated protein 2P11137Details
cGMP-inhibited 3',5'-cyclic phosphodiesterase BQ13370Details
Cyclin-dependent kinase 7P50613Details
Retinoblastoma-associated proteinP06400Details
DNA topoisomerase 2-betaQ02880Details
Wee1-like protein kinaseP30291Details
Mitogen-activated protein kinase kinase kinase 1Q13233Details
Heat shock factor protein 1Q00613Details
Cation-independent mannose-6-phosphate receptorP11717Details
Multidrug resistance-associated protein 7Q5T3U5Details
Uracil-DNA glycosylaseP13051Details
Inner centromere proteinQ9NQS7Details
Protein kinase C iota typeP41743Details
AfadinP55196Details
VimentinP08670Details
[3-methyl-2-oxobutanoate dehydrogenase [lipoamide]] kinase, mitochondrialO14874Details
Sodium bicarbonate cotransporter 3Q9Y6M7Details
Mitogen-activated protein kinase kinase kinase 2Q9Y2U5Details
RalA-binding protein 1Q15311Details
Cyclic AMP-dependent transcription factor ATF-2P15336Details
TGF-beta-activated kinase 1 and MAP3K7-binding protein 1Q15750Details
Heterogeneous nuclear ribonucleoproteins A2/B1P22626Details
Splicing factor, proline- and glutamine-richP23246Details
DesmoplakinP15924Details
Cysteine and glycine-rich protein 1P21291Details
Filamin-AP21333Details
NucleophosminP06748Details
Splicing factor 1Q15637Details
ZyxinQ15942Details
Splicing factor 3B subunit 1O75533Details
AP2-associated protein kinase 1Q2M2I8Details
Mitogen-activated protein kinase kinase kinase kinase 4O95819Details
Serine/threonine-protein kinase MARK1Q9P0L2Details
Serine/threonine-protein kinase MARK2Q7KZI7Details
MAP/microtubule affinity-regulating kinase 3P27448Details
CLIP-associating protein 1Q7Z460Details
Misshapen-like kinase 1Q8N4C8Details
Phosphatidylinositol 4-kinase betaQ9UBF8Details
Serine/threonine-protein kinase N1Q16512Details
Serine/threonine-protein kinase N2Q16513Details
Histone-lysine N-methyltransferase EZH2Q15910Details
Inositol polyphosphate 1-phosphataseP49441Details
Tyrosine-protein phosphatase non-receptor type 12Q05209Details
Receptor protein-tyrosine kinaseQ504U8Details
Microphthalmia-associated transcription factorO75030Details
Eukaryotic translation initiation factor 4E-binding protein 1Q13541Details
Runt-related transcription factor 2Q13950Details
Lysine-specific histone demethylase 1AO60341Details
Proliferation marker protein Ki-67P46013Details
ELAV-like protein 1Q15717Details