Lys-N and trypsin cover complementary parts of the phosphoproteome in a refined SCX-based approach.

Article Details

Citation

Gauci S, Helbig AO, Slijper M, Krijgsveld J, Heck AJ, Mohammed S

Lys-N and trypsin cover complementary parts of the phosphoproteome in a refined SCX-based approach.

Anal Chem. 2009 Jun 1;81(11):4493-501. doi: 10.1021/ac9004309.

PubMed ID
19413330 [ View in PubMed
]
Abstract

The analysis of proteome-wide phosphorylation events is still a major analytical challenge because of the enormous complexity of protein phosphorylation networks. In this work, we evaluate the complementarity of Lys-N, Lys-C, and trypsin with regard to their ability to contribute to the global analysis of the phosphoproteome. A refined version of low-pH strong cation exchange was used to efficiently separate N-terminally acetylated, phosphorylated, and nonmodified peptides. A total of 5036 nonredundant phosphopeptides could be identified with a false discovery rate of <1% from 1 mg of protein using a combination of the three enzymes. Our data revealed that the overlap between the phosphopeptide data sets generated with different proteases was marginal, whereas the overlap between two similarly generated tryptic data sets was found to be at least 4 times higher. In this way, the parallel use of Lys-N and trypsin enabled a 72% increase in the number of detected phosphopeptides as compared to trypsin alone, whereas a trypsin replicate experiment only led to a 25% increase. Thus, when focusing solely on the trypsin and Lys-N data, we identified 4671 nonredundant phosphopeptides. Further analysis of the detected sites showed that the Lys-N and trypsin data sets were enriched in significantly different phosphorylation motifs, further evidencing that multiprotease approaches are very valuable in phosphoproteome analyses.

DrugBank Data that Cites this Article

Polypeptides
NameUniProt ID
Glycogen phosphorylase, liver formP06737Details
Alcohol dehydrogenase class-3P11766Details
Tyrosine-protein kinase ABL1P00519Details
Phenylalanine--tRNA ligase alpha subunitQ9Y285Details
Serine--tRNA ligase, cytoplasmicP49591Details
Pyruvate kinase PKMP14618Details
Adenine phosphoribosyltransferaseP07741Details
Alpha-aminoadipic semialdehyde dehydrogenaseP49419Details
DNA ligase 3P49916Details
Monocarboxylate transporter 8P36021Details
Glutathione synthetaseP48637Details
Bifunctional glutamate/proline--tRNA ligaseP07814Details
Lysine--tRNA ligaseQ15046Details
DNA ligase 1P18858Details
Pyrroline-5-carboxylate reductase 1, mitochondrialP32322Details
Tyrosine--tRNA ligase, cytoplasmicP54577Details
Neutral amino acid transporter B(0)Q15758Details
Purine nucleoside phosphorylaseP00491Details
4-trimethylaminobutyraldehyde dehydrogenaseP49189Details
Alanine--tRNA ligase, cytoplasmicP49588Details
Sorbitol dehydrogenaseQ00796Details
Spermidine synthaseP19623Details
GMP synthase [glutamine-hydrolyzing]P49915Details
Cystathionine beta-synthaseP35520Details
Acetyl-coenzyme A synthetase, cytoplasmicQ9NR19Details
NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 6O95139Details
Vitamin K-dependent gamma-carboxylaseP38435Details
CalmodulinP0DP23Details
L-lactate dehydrogenase A chainP00338Details
Abelson tyrosine-protein kinase 2P42684Details
Isoleucine--tRNA ligase, cytoplasmicP41252Details
Branched-chain-amino-acid aminotransferase, cytosolicP54687Details
Dihydropteridine reductaseP09417Details
Glyceraldehyde-3-phosphate dehydrogenaseP04406Details
Methionine--tRNA ligase, cytoplasmicP56192Details
NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 2O43678Details
Pyruvate dehydrogenase E1 component subunit alpha, somatic form, mitochondrialP08559Details
Heme oxygenase 2P30519Details
Phosphoserine aminotransferaseQ9Y617Details
NADP-dependent malic enzymeP48163Details
Choline-phosphate cytidylyltransferase AP49585Details
C-1-tetrahydrofolate synthase, cytoplasmicP11586Details
ADP/ATP translocase 3P12236Details
Acetyl-CoA carboxylase 1Q13085Details
Interferon alpha/beta receptor 1P17181Details
Monocarboxylate transporter 1P53985Details
ADP/ATP translocase 2P05141Details
DNA polymerase alpha subunit BQ14181Details
Inosine-5'-monophosphate dehydrogenase 2P12268Details
Aspartate--tRNA ligase, cytoplasmicP14868Details
Pyrroline-5-carboxylate reductase 2Q96C36Details
Sodium/potassium-transporting ATPase subunit alpha-1P05023Details
Histidine--tRNA ligase, cytoplasmicP12081Details
Biliverdin reductase AP53004Details
DNA topoisomerase 2-alphaP11388Details
Aldose reductaseP15121Details
ATPase ASNA1O43681Details
N(G),N(G)-dimethylarginine dimethylaminohydrolase 1O94760Details
Epidermal growth factor receptorP00533Details
5'-AMP-activated protein kinase subunit beta-2O43741Details
Farnesyl pyrophosphate synthaseP14324Details
Multidrug resistance-associated protein 1P33527Details
Glutathione S-transferase omega-1P78417Details
Perilipin-3O60664Details
CTP synthase 1P17812Details
Trifunctional purine biosynthetic protein adenosine-3P22102Details
Cyclin-dependent-like kinase 5Q00535Details
DNA (cytosine-5)-methyltransferase 1P26358Details
CalpastatinP20810Details
Mitogen-activated protein kinase 1P28482Details
Eukaryotic translation initiation factor 4EP06730Details
Fatty acid synthaseP49327Details
Lactoylglutathione lyaseQ04760Details
Spermine synthaseP52788Details
DNA topoisomerase 1P11387Details
Peptidyl-prolyl cis-trans isomerase AP62937Details
Transcription factor AP-1P05412Details
Mitogen-activated protein kinase 3P27361Details
Tyrosine-protein phosphatase non-receptor type 11Q06124Details
Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2P59768Details
Glucose-6-phosphate 1-dehydrogenaseP11413Details
Hexokinase-1P19367Details
Heat shock protein HSP 90-alphaP07900Details
Protein kinase C alpha typeP17252Details
Inhibitor of nuclear factor kappa-B kinase subunit betaO14920Details
Caspase-3P42574Details
Microtubule-associated protein 1AP78559Details
14-3-3 protein zeta/deltaP63104Details
Glucose-6-phosphate isomeraseP06744Details
m7GpppX diphosphataseQ96C86Details
Thymidylate kinaseP23919Details
Bifunctional purine biosynthesis protein PURHP31939Details
Acetyl-CoA acetyltransferase, cytosolicQ9BWD1Details
Tyrosine-protein kinase CSKP41240Details
Tubulin alpha-1C chainQ9BQE3Details
Uridine-cytidine kinase 2Q9BZX2Details
Methionine aminopeptidase 2P50579Details
Uroporphyrinogen decarboxylaseP06132Details
Fructose-bisphosphate aldolase AP04075Details
Nicotinamide/nicotinic acid mononucleotide adenylyltransferase 1Q9HAN9Details
Caspase-7P55210Details
Peroxiredoxin-2P32119Details
Thioredoxin reductase 1, cytoplasmicQ16881Details
Fatty acid-binding protein, epidermalQ01469Details
Retinoblastoma-associated proteinP06400Details
Glycogen phosphorylase, brain formP11216Details
O-phosphoseryl-tRNA(Sec) selenium transferaseQ9HD40Details
Methylmalonic aciduria and homocystinuria type C proteinQ9Y4U1Details
Cyclin-dependent kinase 4P11802Details
Egl nine homolog 1Q9GZT9Details
Multifunctional protein ADE2P22234Details
CAD proteinP27708Details
Isoaspartyl peptidase/L-asparaginaseQ7L266Details
Phosphoribosylformylglycinamidine synthaseO15067Details
Glutamine synthetaseP15104Details
Glutamine--tRNA ligaseP47897Details
Glutaredoxin-1P35754Details
Proteasome subunit beta type-2P49721Details
Guanylate kinaseQ16774Details
Histone deacetylase 1Q13547Details
Histone deacetylase 2Q92769Details
Protein farnesyltransferase/geranylgeranyltransferase type-1 subunit alphaP49354Details
Protein kinase C delta typeQ05655Details
DNA topoisomerase 2-betaQ02880Details
Ribonucleoside-diphosphate reductase subunit M2P31350Details
Ephrin type-B receptor 4P54760Details
E3 ubiquitin-protein ligase UBR4Q5T4S7Details
NAD-dependent protein deacetylase sirtuin-1Q96EB6Details
Voltage-dependent anion-selective channel protein 1P21796Details
Voltage-dependent anion-selective channel protein 2P45880Details
Voltage-dependent anion-selective channel protein 3Q9Y277Details
Prostaglandin E synthase 3Q15185Details
Hypoxia-inducible factor 1-alpha inhibitorQ9NWT6Details
U1 small nuclear ribonucleoprotein AP09012Details
ATP-dependent Clp protease proteolytic subunit, mitochondrialQ16740Details
Carbonyl reductase [NADPH] 1P16152Details
Focal adhesion kinase 1Q05397Details
Nucleoside diphosphate kinase AP15531Details
Serine/threonine-protein phosphatase 5P53041Details
GDP-mannose 4,6 dehydrataseO60547Details
Nucleoside diphosphate kinase BP22392Details
Calpain-1 catalytic subunitP07384Details
Cellular tumor antigen p53P04637Details
Serine/threonine-protein kinase Chk1O14757Details
Bcl-2-like protein 2Q92843Details
Far upstream element-binding protein 1Q96AE4Details
cAMP-dependent protein kinase type II-alpha regulatory subunitP13861Details
cAMP-dependent protein kinase type I-alpha regulatory subunitP10644Details
Mitogen-activated protein kinase kinase kinase 1Q13233Details
Signal transducer and activator of transcription 3P40763Details
Microtubule-associated protein 4P27816Details
Casein kinase I isoform epsilonP49674Details
Heat shock factor protein 1Q00613Details
Histone deacetylase 7Q8WUI4Details
Adenosine kinaseP55263Details
NischarinQ9Y2I1Details
DNA polymerase epsilon subunit 4Q9NR33Details
DNA polymerase epsilon subunit 3Q9NRF9Details
Cation-independent mannose-6-phosphate receptorP11717Details
Nuclear receptor coactivator 1Q15788Details
Nuclear receptor coactivator 2Q15596Details
Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A alpha isoformP30153Details
Uracil-DNA glycosylaseP13051Details
Heat shock cognate 71 kDa proteinP11142Details
Dual specificity protein kinase TTKP33981Details
Inner centromere proteinQ9NQS7Details
Serine/threonine-protein phosphatase 2A 55 kDa regulatory subunit B alpha isoformP63151Details
ADP-ribosylation factor 1P84077Details
60S ribosomal protein L13aP40429Details
NHP2-like protein 1P55769Details
Triosephosphate isomeraseP60174Details
Myosin-14Q7Z406Details
Proteasome subunit alpha type-2P25787Details
Proteasome subunit alpha type-3P25788Details
Proteasome subunit alpha type-5P28066Details
Proteasome subunit beta type-3P49720Details
Calcium/calmodulin-dependent protein kinase type IVQ16566Details
Nuclear receptor coactivator 5Q9HCD5Details
MAP kinase-activated protein kinase 3Q16644Details
Hydroxymethylglutaryl-CoA synthase, cytoplasmicQ01581Details
Geranylgeranyl transferase type-2 subunit betaP53611Details
Cyclin-dependent kinase 16Q00536Details
Platelet-activating factor acetylhydrolase IB subunit gammaQ15102Details
Calcium/calmodulin-dependent protein kinase type II subunit deltaQ13557Details
Methionine aminopeptidase 1P53582Details
Profilin-1P07737Details
Calpain small subunit 1P04632Details
Annexin A5P08758Details
MKL/myocardin-like protein 1Q969V6Details
DestrinP60981Details
Cofilin-1P23528Details
Actin-related protein 3P61158Details
Elongation factor 2P13639Details
SRSF protein kinase 2P78362Details
AfadinP55196Details
Alcohol dehydrogenase [NADP(+)]P14550Details
CREB-binding proteinQ92793Details
Acyl-coenzyme A thioesterase 13Q9NPJ3Details
Mediator of RNA polymerase II transcription subunit 1Q15648Details
Mothers against decapentaplegic homolog 2Q15796Details
Protein arginine N-methyltransferase 3O60678Details
VimentinP08670Details
Uridine 5'-monophosphate synthaseP11172Details
Acyl-CoA-binding proteinP07108Details
Adenylate kinase 2, mitochondrialP54819Details
Myosin-11P35749Details
cAMP-dependent protein kinase type II-beta regulatory subunitP31323Details
Glycogenin-1P46976Details
T-complex protein 1 subunit gammaP49368Details
GTP-binding nuclear protein RanP62826Details
14-3-3 protein epsilonP62258Details
Transitional endoplasmic reticulum ATPaseP55072Details
Ubiquitin-like modifier-activating enzyme 1P22314Details
Sorting nexin-3O60493Details
SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5O60264Details
Phosphoserine phosphataseP78330Details
Spectrin beta chain, non-erythrocytic 1Q01082Details
Spliceosome RNA helicase DDX39BQ13838Details
Selenocysteine lyaseQ96I15Details
Transient receptor potential cation channel subfamily M member 7Q96QT4Details
Hepatocyte growth factor-regulated tyrosine kinase substrateO14964Details
Myc proto-oncogene proteinP01106Details
Equilibrative nucleoside transporter 2Q14542Details
Folate transporter 1P41440Details
Dual specificity mitogen-activated protein kinase kinase 2P36507Details
LIM domain kinase 1P53667Details
RalA-binding protein 1Q15311Details
Metallothionein-2P02795Details
Membrane-associated progesterone receptor component 1O00264Details
5'-AMP-activated protein kinase subunit gamma-2Q9UGJ0Details
Cyclic AMP-dependent transcription factor ATF-2P15336Details
Actin, cytoplasmic 1P60709Details
Actin, cytoplasmic 2P63261Details
Sodium-dependent phosphate transporter 1Q8WUM9Details
Histone H1.4P10412Details
Chloride intracellular channel protein 1O00299Details
CCAAT/enhancer-binding protein betaP17676Details
Mitogen-activated protein kinase kinase kinase MLTQ9NYL2Details
Dihydropyrimidinase-related protein 2Q16555Details
Nucleolar and coiled-body phosphoprotein 1Q14978Details
Protein O-GlcNAcaseO60502Details
Sodium-dependent phosphate transporter 2Q08357Details
Metallothionein-1XP80297Details
Calmodulin-2P0DP24Details
Calmodulin-3P0DP25Details
Heterogeneous nuclear ribonucleoproteins A2/B1P22626Details
Heterogeneous nuclear ribonucleoprotein HP31943Details
Splicing factor, proline- and glutamine-richP23246Details
Receptor of activated protein C kinase 1P63244Details
Stress-induced-phosphoprotein 1P31948Details
Hepatoma-derived growth factorP51858Details
Ran-specific GTPase-activating proteinP43487Details
Cystatin-BP04080Details
HuntingtinP42858Details
Cap-specific mRNA (nucleoside-2'-O-)-methyltransferase 1Q8N1G2Details
RuvB-like 2Q9Y230Details
ATP synthase subunit g, mitochondrialO75964Details
Heterogeneous nuclear ribonucleoprotein FP52597Details
Heterogeneous nuclear ribonucleoprotein KP61978Details
Heat shock protein 105 kDaQ92598Details
Nuclear migration protein nudCQ9Y266Details
26S protease regulatory subunit 6AP17980Details
Splicing factor 3A subunit 3Q12874Details
Tumor protein D54O43399Details
Tropomyosin alpha-4 chainP67936Details
DesmoplakinP15924Details
KAT8 regulatory NSL complex subunit 3Q9P2N6Details
Far upstream element-binding protein 2Q92945Details
Filamin-AP21333Details
Heterogeneous nuclear ribonucleoprotein D0Q14103Details
NucleophosminP06748Details
60S ribosomal protein L4P36578Details
40S ribosomal protein S18P62269Details
40S ribosomal protein S5P46782Details
40S ribosomal protein S8P62241Details
Splicing factor 1Q15637Details
Transgelin-2P37802Details
ZyxinQ15942Details
Survival motor neuron proteinB4DP61Details
Myosin-9P35579Details
Splicing factor 3B subunit 1O75533Details
Serine/threonine-protein kinase 3Q13188Details
Serine/threonine-protein kinase 33Q9BYT3Details
STE20/SPS1-related proline-alanine-rich protein kinaseQ9UEW8Details
Serine/threonine-protein kinase TAO1Q7L7X3Details
Serine/threonine-protein kinase tousled-like 2Q86UE8Details
TRAF2 and NCK-interacting protein kinaseQ9UKE5Details
Serine/threonine-protein kinase ULK1O75385Details
AP2-associated protein kinase 1Q2M2I8Details
BMP-2-inducible protein kinaseQ9NSY1Details
Citron Rho-interacting kinaseO14578Details
Casein kinase I isoform alphaP48729Details
Cyclin-G-associated kinaseO14976Details
Glycogen synthase kinase-3 alphaP49840Details
Serine/threonine-protein kinase LATS1O95835Details
Dual specificity mitogen-activated protein kinase kinase 3P46734Details
Mitogen-activated protein kinase kinase kinase 4Q9Y6R4Details
MAP kinase-activated protein kinase 5Q8IW41Details
Serine/threonine-protein kinase MARK2Q7KZI7Details
MAP/microtubule affinity-regulating kinase 3P27448Details
CLIP-associating protein 1Q7Z460Details
Megakaryocyte-associated tyrosine-protein kinaseP42679Details
Misshapen-like kinase 1Q8N4C8Details
Myosin light chain kinase, smooth muscleQ15746Details
Serine/threonine-protein kinase OSR1O95747Details
Serine/threonine-protein kinase PAK 1Q13153Details
Phosphatidylinositol 5-phosphate 4-kinase type-2 gammaQ8TBX8Details
Serine/threonine-protein kinase N1Q16512Details
Serine/threonine-protein kinase N2Q16513Details
Serine/threonine-protein kinase D1Q15139Details
Serine/threonine-protein kinase PRP4 homologQ13523Details
Serine/threonine-protein kinase RIO1Q9BRS2Details
Serine/threonine-protein kinase RIO2Q9BVS4Details
SNF-related serine/threonine-protein kinaseQ9NRH2Details
Serine/threonine-protein kinase 10O94804Details
Histone-lysine N-methyltransferase EZH2Q15910Details
POU domain, class 2, transcription factor 1P14859Details
NAD-dependent protein deacetylase sirtuin-2Q8IXJ6Details
DystrophinP11532Details
Zinc transporter ZIP6Q13433Details
V-type proton ATPase subunit E 1P36543Details
Receptor protein-tyrosine kinaseQ504U8Details
UbiA prenyltransferase domain-containing protein 1Q9Y5Z9Details
Serine/threonine-protein kinase ATRQ13535Details