Lys-N and trypsin cover complementary parts of the phosphoproteome in a refined SCX-based approach.
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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
Name UniProt ID Glycogen phosphorylase, liver form P06737 Details Alcohol dehydrogenase class-3 P11766 Details Tyrosine-protein kinase ABL1 P00519 Details Phenylalanine--tRNA ligase alpha subunit Q9Y285 Details Serine--tRNA ligase, cytoplasmic P49591 Details Pyruvate kinase PKM P14618 Details Adenine phosphoribosyltransferase P07741 Details Alpha-aminoadipic semialdehyde dehydrogenase P49419 Details DNA ligase 3 P49916 Details Monocarboxylate transporter 8 P36021 Details Glutathione synthetase P48637 Details Bifunctional glutamate/proline--tRNA ligase P07814 Details Lysine--tRNA ligase Q15046 Details DNA ligase 1 P18858 Details Pyrroline-5-carboxylate reductase 1, mitochondrial P32322 Details Tyrosine--tRNA ligase, cytoplasmic P54577 Details Neutral amino acid transporter B(0) Q15758 Details Purine nucleoside phosphorylase P00491 Details 4-trimethylaminobutyraldehyde dehydrogenase P49189 Details Alanine--tRNA ligase, cytoplasmic P49588 Details Sorbitol dehydrogenase Q00796 Details Spermidine synthase P19623 Details GMP synthase [glutamine-hydrolyzing] P49915 Details Cystathionine beta-synthase P35520 Details Acetyl-coenzyme A synthetase, cytoplasmic Q9NR19 Details NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 6 O95139 Details Vitamin K-dependent gamma-carboxylase P38435 Details Calmodulin P0DP23 Details L-lactate dehydrogenase A chain P00338 Details Abelson tyrosine-protein kinase 2 P42684 Details Isoleucine--tRNA ligase, cytoplasmic P41252 Details Branched-chain-amino-acid aminotransferase, cytosolic P54687 Details Dihydropteridine reductase P09417 Details Glyceraldehyde-3-phosphate dehydrogenase P04406 Details Methionine--tRNA ligase, cytoplasmic P56192 Details NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 2 O43678 Details Pyruvate dehydrogenase E1 component subunit alpha, somatic form, mitochondrial P08559 Details Heme oxygenase 2 P30519 Details Phosphoserine aminotransferase Q9Y617 Details NADP-dependent malic enzyme P48163 Details Choline-phosphate cytidylyltransferase A P49585 Details C-1-tetrahydrofolate synthase, cytoplasmic P11586 Details ADP/ATP translocase 3 P12236 Details Acetyl-CoA carboxylase 1 Q13085 Details Interferon alpha/beta receptor 1 P17181 Details Monocarboxylate transporter 1 P53985 Details ADP/ATP translocase 2 P05141 Details DNA polymerase alpha subunit B Q14181 Details Inosine-5'-monophosphate dehydrogenase 2 P12268 Details Aspartate--tRNA ligase, cytoplasmic P14868 Details Pyrroline-5-carboxylate reductase 2 Q96C36 Details Sodium/potassium-transporting ATPase subunit alpha-1 P05023 Details Histidine--tRNA ligase, cytoplasmic P12081 Details Biliverdin reductase A P53004 Details DNA topoisomerase 2-alpha P11388 Details Aldose reductase P15121 Details ATPase ASNA1 O43681 Details N(G),N(G)-dimethylarginine dimethylaminohydrolase 1 O94760 Details Epidermal growth factor receptor P00533 Details 5'-AMP-activated protein kinase subunit beta-2 O43741 Details Farnesyl pyrophosphate synthase P14324 Details Multidrug resistance-associated protein 1 P33527 Details Glutathione S-transferase omega-1 P78417 Details Perilipin-3 O60664 Details CTP synthase 1 P17812 Details Trifunctional purine biosynthetic protein adenosine-3 P22102 Details Cyclin-dependent-like kinase 5 Q00535 Details DNA (cytosine-5)-methyltransferase 1 P26358 Details Calpastatin P20810 Details Mitogen-activated protein kinase 1 P28482 Details Eukaryotic translation initiation factor 4E P06730 Details Fatty acid synthase P49327 Details Lactoylglutathione lyase Q04760 Details Spermine synthase P52788 Details DNA topoisomerase 1 P11387 Details Peptidyl-prolyl cis-trans isomerase A P62937 Details Transcription factor AP-1 P05412 Details Mitogen-activated protein kinase 3 P27361 Details Tyrosine-protein phosphatase non-receptor type 11 Q06124 Details Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 P59768 Details Glucose-6-phosphate 1-dehydrogenase P11413 Details Hexokinase-1 P19367 Details Heat shock protein HSP 90-alpha P07900 Details Protein kinase C alpha type P17252 Details Inhibitor of nuclear factor kappa-B kinase subunit beta O14920 Details Caspase-3 P42574 Details Microtubule-associated protein 1A P78559 Details 14-3-3 protein zeta/delta P63104 Details Glucose-6-phosphate isomerase P06744 Details m7GpppX diphosphatase Q96C86 Details Thymidylate kinase P23919 Details Bifunctional purine biosynthesis protein PURH P31939 Details Acetyl-CoA acetyltransferase, cytosolic Q9BWD1 Details Tyrosine-protein kinase CSK P41240 Details Tubulin alpha-1C chain Q9BQE3 Details Uridine-cytidine kinase 2 Q9BZX2 Details Methionine aminopeptidase 2 P50579 Details Uroporphyrinogen decarboxylase P06132 Details Fructose-bisphosphate aldolase A P04075 Details Nicotinamide/nicotinic acid mononucleotide adenylyltransferase 1 Q9HAN9 Details Caspase-7 P55210 Details Peroxiredoxin-2 P32119 Details Thioredoxin reductase 1, cytoplasmic Q16881 Details Fatty acid-binding protein, epidermal Q01469 Details Retinoblastoma-associated protein P06400 Details Glycogen phosphorylase, brain form P11216 Details O-phosphoseryl-tRNA(Sec) selenium transferase Q9HD40 Details Methylmalonic aciduria and homocystinuria type C protein Q9Y4U1 Details Cyclin-dependent kinase 4 P11802 Details Egl nine homolog 1 Q9GZT9 Details Multifunctional protein ADE2 P22234 Details CAD protein P27708 Details Isoaspartyl peptidase/L-asparaginase Q7L266 Details Phosphoribosylformylglycinamidine synthase O15067 Details Glutamine synthetase P15104 Details Glutamine--tRNA ligase P47897 Details Glutaredoxin-1 P35754 Details Proteasome subunit beta type-2 P49721 Details Guanylate kinase Q16774 Details Histone deacetylase 1 Q13547 Details Histone deacetylase 2 Q92769 Details Protein farnesyltransferase/geranylgeranyltransferase type-1 subunit alpha P49354 Details Protein kinase C delta type Q05655 Details DNA topoisomerase 2-beta Q02880 Details Ribonucleoside-diphosphate reductase subunit M2 P31350 Details Ephrin type-B receptor 4 P54760 Details E3 ubiquitin-protein ligase UBR4 Q5T4S7 Details NAD-dependent protein deacetylase sirtuin-1 Q96EB6 Details Voltage-dependent anion-selective channel protein 1 P21796 Details Voltage-dependent anion-selective channel protein 2 P45880 Details Voltage-dependent anion-selective channel protein 3 Q9Y277 Details Prostaglandin E synthase 3 Q15185 Details Hypoxia-inducible factor 1-alpha inhibitor Q9NWT6 Details U1 small nuclear ribonucleoprotein A P09012 Details ATP-dependent Clp protease proteolytic subunit, mitochondrial Q16740 Details Carbonyl reductase [NADPH] 1 P16152 Details Focal adhesion kinase 1 Q05397 Details Nucleoside diphosphate kinase A P15531 Details Serine/threonine-protein phosphatase 5 P53041 Details GDP-mannose 4,6 dehydratase O60547 Details Nucleoside diphosphate kinase B P22392 Details Calpain-1 catalytic subunit P07384 Details Cellular tumor antigen p53 P04637 Details Serine/threonine-protein kinase Chk1 O14757 Details Bcl-2-like protein 2 Q92843 Details Far upstream element-binding protein 1 Q96AE4 Details cAMP-dependent protein kinase type II-alpha regulatory subunit P13861 Details cAMP-dependent protein kinase type I-alpha regulatory subunit P10644 Details Mitogen-activated protein kinase kinase kinase 1 Q13233 Details Signal transducer and activator of transcription 3 P40763 Details Microtubule-associated protein 4 P27816 Details Casein kinase I isoform epsilon P49674 Details Heat shock factor protein 1 Q00613 Details Histone deacetylase 7 Q8WUI4 Details Adenosine kinase P55263 Details Nischarin Q9Y2I1 Details DNA polymerase epsilon subunit 4 Q9NR33 Details DNA polymerase epsilon subunit 3 Q9NRF9 Details Cation-independent mannose-6-phosphate receptor P11717 Details Nuclear receptor coactivator 1 Q15788 Details Nuclear receptor coactivator 2 Q15596 Details Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A alpha isoform P30153 Details Uracil-DNA glycosylase P13051 Details Heat shock cognate 71 kDa protein P11142 Details Dual specificity protein kinase TTK P33981 Details Inner centromere protein Q9NQS7 Details Serine/threonine-protein phosphatase 2A 55 kDa regulatory subunit B alpha isoform P63151 Details ADP-ribosylation factor 1 P84077 Details 60S ribosomal protein L13a P40429 Details NHP2-like protein 1 P55769 Details Triosephosphate isomerase P60174 Details Myosin-14 Q7Z406 Details Proteasome subunit alpha type-2 P25787 Details Proteasome subunit alpha type-3 P25788 Details Proteasome subunit alpha type-5 P28066 Details Proteasome subunit beta type-3 P49720 Details Calcium/calmodulin-dependent protein kinase type IV Q16566 Details Nuclear receptor coactivator 5 Q9HCD5 Details MAP kinase-activated protein kinase 3 Q16644 Details Hydroxymethylglutaryl-CoA synthase, cytoplasmic Q01581 Details Geranylgeranyl transferase type-2 subunit beta P53611 Details Cyclin-dependent kinase 16 Q00536 Details Platelet-activating factor acetylhydrolase IB subunit gamma Q15102 Details Calcium/calmodulin-dependent protein kinase type II subunit delta Q13557 Details Methionine aminopeptidase 1 P53582 Details Profilin-1 P07737 Details Calpain small subunit 1 P04632 Details Annexin A5 P08758 Details MKL/myocardin-like protein 1 Q969V6 Details Destrin P60981 Details Cofilin-1 P23528 Details Actin-related protein 3 P61158 Details Elongation factor 2 P13639 Details SRSF protein kinase 2 P78362 Details Afadin P55196 Details Alcohol dehydrogenase [NADP(+)] P14550 Details CREB-binding protein Q92793 Details Acyl-coenzyme A thioesterase 13 Q9NPJ3 Details Mediator of RNA polymerase II transcription subunit 1 Q15648 Details Mothers against decapentaplegic homolog 2 Q15796 Details Protein arginine N-methyltransferase 3 O60678 Details Vimentin P08670 Details Uridine 5'-monophosphate synthase P11172 Details Acyl-CoA-binding protein P07108 Details Adenylate kinase 2, mitochondrial P54819 Details Myosin-11 P35749 Details cAMP-dependent protein kinase type II-beta regulatory subunit P31323 Details Glycogenin-1 P46976 Details T-complex protein 1 subunit gamma P49368 Details GTP-binding nuclear protein Ran P62826 Details 14-3-3 protein epsilon P62258 Details Transitional endoplasmic reticulum ATPase P55072 Details Ubiquitin-like modifier-activating enzyme 1 P22314 Details Sorting nexin-3 O60493 Details SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5 O60264 Details Phosphoserine phosphatase P78330 Details Spectrin beta chain, non-erythrocytic 1 Q01082 Details Spliceosome RNA helicase DDX39B Q13838 Details Selenocysteine lyase Q96I15 Details Transient receptor potential cation channel subfamily M member 7 Q96QT4 Details Hepatocyte growth factor-regulated tyrosine kinase substrate O14964 Details Myc proto-oncogene protein P01106 Details Equilibrative nucleoside transporter 2 Q14542 Details Folate transporter 1 P41440 Details Dual specificity mitogen-activated protein kinase kinase 2 P36507 Details LIM domain kinase 1 P53667 Details RalA-binding protein 1 Q15311 Details Metallothionein-2 P02795 Details Membrane-associated progesterone receptor component 1 O00264 Details 5'-AMP-activated protein kinase subunit gamma-2 Q9UGJ0 Details Cyclic AMP-dependent transcription factor ATF-2 P15336 Details Actin, cytoplasmic 1 P60709 Details Actin, cytoplasmic 2 P63261 Details Sodium-dependent phosphate transporter 1 Q8WUM9 Details Histone H1.4 P10412 Details Chloride intracellular channel protein 1 O00299 Details CCAAT/enhancer-binding protein beta P17676 Details Mitogen-activated protein kinase kinase kinase MLT Q9NYL2 Details Dihydropyrimidinase-related protein 2 Q16555 Details Nucleolar and coiled-body phosphoprotein 1 Q14978 Details Protein O-GlcNAcase O60502 Details Sodium-dependent phosphate transporter 2 Q08357 Details Metallothionein-1X P80297 Details Calmodulin-2 P0DP24 Details Calmodulin-3 P0DP25 Details Heterogeneous nuclear ribonucleoproteins A2/B1 P22626 Details Heterogeneous nuclear ribonucleoprotein H P31943 Details Splicing factor, proline- and glutamine-rich P23246 Details Receptor of activated protein C kinase 1 P63244 Details Stress-induced-phosphoprotein 1 P31948 Details Hepatoma-derived growth factor P51858 Details Ran-specific GTPase-activating protein P43487 Details Cystatin-B P04080 Details Huntingtin P42858 Details Cap-specific mRNA (nucleoside-2'-O-)-methyltransferase 1 Q8N1G2 Details RuvB-like 2 Q9Y230 Details ATP synthase subunit g, mitochondrial O75964 Details Heterogeneous nuclear ribonucleoprotein F P52597 Details Heterogeneous nuclear ribonucleoprotein K P61978 Details Heat shock protein 105 kDa Q92598 Details Nuclear migration protein nudC Q9Y266 Details 26S protease regulatory subunit 6A P17980 Details Splicing factor 3A subunit 3 Q12874 Details Tumor protein D54 O43399 Details Tropomyosin alpha-4 chain P67936 Details Desmoplakin P15924 Details KAT8 regulatory NSL complex subunit 3 Q9P2N6 Details Far upstream element-binding protein 2 Q92945 Details Filamin-A P21333 Details Heterogeneous nuclear ribonucleoprotein D0 Q14103 Details Nucleophosmin P06748 Details 60S ribosomal protein L4 P36578 Details 40S ribosomal protein S18 P62269 Details 40S ribosomal protein S5 P46782 Details 40S ribosomal protein S8 P62241 Details Splicing factor 1 Q15637 Details Transgelin-2 P37802 Details Zyxin Q15942 Details Survival motor neuron protein B4DP61 Details Myosin-9 P35579 Details Splicing factor 3B subunit 1 O75533 Details Serine/threonine-protein kinase 3 Q13188 Details Serine/threonine-protein kinase 33 Q9BYT3 Details STE20/SPS1-related proline-alanine-rich protein kinase Q9UEW8 Details Serine/threonine-protein kinase TAO1 Q7L7X3 Details Serine/threonine-protein kinase tousled-like 2 Q86UE8 Details TRAF2 and NCK-interacting protein kinase Q9UKE5 Details Serine/threonine-protein kinase ULK1 O75385 Details AP2-associated protein kinase 1 Q2M2I8 Details BMP-2-inducible protein kinase Q9NSY1 Details Citron Rho-interacting kinase O14578 Details Casein kinase I isoform alpha P48729 Details Cyclin-G-associated kinase O14976 Details Glycogen synthase kinase-3 alpha P49840 Details Serine/threonine-protein kinase LATS1 O95835 Details Dual specificity mitogen-activated protein kinase kinase 3 P46734 Details Mitogen-activated protein kinase kinase kinase 4 Q9Y6R4 Details MAP kinase-activated protein kinase 5 Q8IW41 Details Serine/threonine-protein kinase MARK2 Q7KZI7 Details MAP/microtubule affinity-regulating kinase 3 P27448 Details CLIP-associating protein 1 Q7Z460 Details Megakaryocyte-associated tyrosine-protein kinase P42679 Details Misshapen-like kinase 1 Q8N4C8 Details Myosin light chain kinase, smooth muscle Q15746 Details Serine/threonine-protein kinase OSR1 O95747 Details Serine/threonine-protein kinase PAK 1 Q13153 Details Phosphatidylinositol 5-phosphate 4-kinase type-2 gamma Q8TBX8 Details Serine/threonine-protein kinase N1 Q16512 Details Serine/threonine-protein kinase N2 Q16513 Details Serine/threonine-protein kinase D1 Q15139 Details Serine/threonine-protein kinase PRP4 homolog Q13523 Details Serine/threonine-protein kinase RIO1 Q9BRS2 Details Serine/threonine-protein kinase RIO2 Q9BVS4 Details SNF-related serine/threonine-protein kinase Q9NRH2 Details Serine/threonine-protein kinase 10 O94804 Details Histone-lysine N-methyltransferase EZH2 Q15910 Details POU domain, class 2, transcription factor 1 P14859 Details NAD-dependent protein deacetylase sirtuin-2 Q8IXJ6 Details Dystrophin P11532 Details Zinc transporter ZIP6 Q13433 Details V-type proton ATPase subunit E 1 P36543 Details Receptor protein-tyrosine kinase Q504U8 Details UbiA prenyltransferase domain-containing protein 1 Q9Y5Z9 Details Serine/threonine-protein kinase ATR Q13535 Details Eukaryotic translation initiation factor 4E-binding protein 1 Q13541 Details Lysine-specific histone demethylase 1A O60341 Details Proliferation marker protein Ki-67 P46013 Details Histone acetyltransferase p300 Q09472 Details Dual specificity mitogen-activated protein kinase kinase 4 P45985 Details Dual specificity mitogen-activated protein kinase kinase 7 O14733 Details Apoptosis regulator BAX Q07812 Details