A probability-based approach for high-throughput protein phosphorylation analysis and site localization.

Article Details


Beausoleil SA, Villen J, Gerber SA, Rush J, Gygi SP

A probability-based approach for high-throughput protein phosphorylation analysis and site localization.

Nat Biotechnol. 2006 Oct;24(10):1285-92. Epub 2006 Sep 10.

PubMed ID
16964243 [ View in PubMed

Data analysis and interpretation remain major logistical challenges when attempting to identify large numbers of protein phosphorylation sites by nanoscale reverse-phase liquid chromatography/tandem mass spectrometry (LC-MS/MS) (Supplementary Figure 1 online). In this report we address challenges that are often only addressable by laborious manual validation, including data set error, data set sensitivity and phosphorylation site localization. We provide a large-scale phosphorylation data set with a measured error rate as determined by the target-decoy approach, we demonstrate an approach to maximize data set sensitivity by efficiently distracting incorrect peptide spectral matches (PSMs), and we present a probability-based score, the Ascore, that measures the probability of correct phosphorylation site localization based on the presence and intensity of site-determining ions in MS/MS spectra. We applied our methods in a fully automated fashion to nocodazole-arrested HeLa cell lysate where we identified 1,761 nonredundant phosphorylation sites from 491 proteins with a peptide false-positive rate of 1.3%.

DrugBank Data that Cites this Article

NameUniProt ID
Pyruvate kinase PKMP14618Details
DNA ligase 3P49916Details
Bifunctional glutamate/proline--tRNA ligaseP07814Details
DNA ligase 1P18858Details
Serine/threonine-protein kinase A-RafP10398Details
Abelson tyrosine-protein kinase 2P42684Details
cGMP-inhibited 3',5'-cyclic phosphodiesterase AQ14432Details
Chromodomain-helicase-DNA-binding protein 1O14646Details
Choline-phosphate cytidylyltransferase AP49585Details
Serine/threonine-protein kinase B-rafP15056Details
Cytosolic phospholipase A2P47712Details
Pyrroline-5-carboxylate reductase 2Q96C36Details
DNA topoisomerase 2-alphaP11388Details
Glucocorticoid receptorP04150Details
CTP synthase 1P17812Details
Signal transducer and activator of transcription 5BP51692Details
Fatty acid synthaseP49327Details
Deoxyuridine 5'-triphosphate nucleotidohydrolase, mitochondrialP33316Details
Thymidine kinase, cytosolicP04183Details
Kinesin-like protein KIF11P52732Details
Keratin, type II cytoskeletal 8P05787Details
Pyridoxal-dependent decarboxylase domain-containing protein 1Q6P996Details
Molybdenum cofactor sulfuraseQ96EN8Details
Multifunctional protein ADE2P22234Details
CAD proteinP27708Details
Protein farnesyltransferase/geranylgeranyltransferase type-1 subunit alphaP49354Details
DNA topoisomerase 2-betaQ02880Details
Ribonucleoside-diphosphate reductase subunit M2P31350Details
NAD-dependent protein deacetylase sirtuin-1Q96EB6Details
Focal adhesion kinase 1Q05397Details
Integrin beta-4P16144Details
Far upstream element-binding protein 1Q96AE4Details
Heat shock protein beta-1P04792Details
Ribosome-binding protein 1Q9P2E9Details
Microtubule-associated protein 4P27816Details
Nuclear receptor coactivator 2Q15596Details
Proteasome subunit alpha type-3P25788Details
Proteasome subunit alpha type-5P28066Details
Ribosomal protein S6 kinase alpha-1Q15418Details
SRSF protein kinase 2P78362Details
Glycylpeptide N-tetradecanoyltransferase 1P30419Details
Mediator of RNA polymerase II transcription subunit 1Q15648Details
Uridine-cytidine kinase-like 1Q9NWZ5Details
Hepatocyte growth factor-regulated tyrosine kinase substrateO14964Details
Ribosomal protein S6 kinase alpha-3P51812Details
Dihydropyrimidinase-related protein 2Q16555Details
Nucleolar and coiled-body phosphoprotein 1Q14978Details
60 kDa heat shock protein, mitochondrialP10809Details
Hepatoma-derived growth factorP51858Details
Cyclin-dependent kinase inhibitor 1P38936Details
Heterogeneous nuclear ribonucleoprotein KP61978Details
Far upstream element-binding protein 2Q92945Details
Cysteine and glycine-rich protein 1P21291Details
60S ribosomal protein L18Q07020Details
Splicing factor 1Q15637Details
Splicing factor 3B subunit 1O75533Details
Serine/threonine-protein kinase TAO1Q7L7X3Details
AP2-associated protein kinase 1Q2M2I8Details
Casein kinase I isoform alphaP48729Details
Mitogen-activated protein kinase kinase kinase 11Q16584Details
Serine/threonine-protein kinase MARK2Q7KZI7Details
Serine/threonine-protein kinase Nek9Q8TD19Details
Serine/threonine-protein kinase PAK 1Q13153Details
Serine/threonine-protein kinase N1Q16512Details
Serine/threonine-protein kinase N2Q16513Details
Serine/threonine-protein kinase 10O94804Details
Histone-lysine N-methyltransferase EZH2Q15910Details
POU domain, class 2, transcription factor 1P14859Details
Tyrosine-protein phosphatase non-receptor type 12Q05209Details
Molybdopterin synthase catalytic subunitO96007Details
Proliferation marker protein Ki-67P46013Details