Toward a comprehensive characterization of a human cancer cell phosphoproteome.

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Zhou H, Di Palma S, Preisinger C, Peng M, Polat AN, Heck AJ, Mohammed S

Toward a comprehensive characterization of a human cancer cell phosphoproteome.

J Proteome Res. 2013 Jan 4;12(1):260-71. doi: 10.1021/pr300630k. Epub 2012 Dec 18.

PubMed ID
23186163 [ View in PubMed
]
Abstract

Mass spectrometry (MS)-based phosphoproteomics has achieved extraordinary success in qualitative and quantitative analysis of cellular protein phosphorylation. Considering that an estimated level of phosphorylation in a cell is placed at well above 100,000 sites, there is still much room for improvement. Here, we attempt to extend the depth of phosphoproteome coverage while maintaining realistic aspirations in terms of available material, robustness, and instrument running time. We developed three strategies, where each provided a different balance between these three key parameters. The first strategy simply used enrichment by Ti(4+)-IMAC followed by reversed chromatography LC-MS (termed 1D). The second strategy incorporated an additional fractionation step through the use of HILIC (2D). Finally, a third strategy was designed employing first an SCX fractionation, followed by Ti(4+)-IMAC enrichment and additional fractionation by HILIC (3D). A preliminary evaluation was performed on the HeLa cell line. Detecting 3700 phosphopeptides in about 2 h, the 1D strategy was found to be the most sensitive but limited in comprehensivity, mainly due to issues with complexity and dynamic range. Overall, the best balance was achieved using the 2D based strategy, identifying close to 17,000 phosphopeptides with less than 1 mg of material in about 48 h. Subsequently, we confirmed the findings with the K562 cell sample. When sufficient material was available, the 3D strategy increased phosphoproteome allowing over 22,000 unique phosphopeptides to be identified. Unfortunately, the 3D strategy required more time and over 1 mg of material before it started to outperform 2D. Ultimately, combining all strategies, we were able to identify over 16,000 and nearly 24,000 unique phosphorylation sites from the cancer cell lines HeLa and K562, respectively. In summary, we demonstrate the need to carry out extensive fractionation for deep mining of the phosphoproteome and provide a guide for appropriate strategies depending on sample amount and/or analysis time.

DrugBank Data that Cites this Article

Polypeptides
NameUniProt ID
Histone deacetylase 5Q9UQL6Details
High affinity cAMP-specific and IBMX-insensitive 3',5'-cyclic phosphodiesterase 8BO95263Details
Inositol 1,4,5-trisphosphate receptor type 2Q14571Details
Inositol 1,4,5-trisphosphate receptor type 3Q14573Details
Jun dimerization protein 2Q8WYK2Details
Cyclic AMP-dependent transcription factor ATF-1P18846Details
Cyclic AMP-dependent transcription factor ATF-2P15336Details
Cyclic AMP-dependent transcription factor ATF-3P18847Details
Cyclic AMP-dependent transcription factor ATF-5Q9Y2D1Details
Cyclic AMP-dependent transcription factor ATF-7P17544Details
Metal regulatory transcription factor 1Q14872Details
Protein PMLP29590Details
Metal transporter CNNM2Q9H8M5Details
Tricarboxylate transport protein, mitochondrialP53007Details
Sodium-dependent phosphate transporter 2Q08357Details
Cytochrome c oxidase copper chaperoneQ14061Details
HephaestinQ9BQS7Details
Calcineurin B homologous protein 1Q99653Details
Calmodulin-2P0DP24Details
Calmodulin-3P0DP25Details
DNA-3-methyladenine glycosylaseP29372Details
60 kDa heat shock protein, mitochondrialP10809Details
40S ribosomal protein S2P15880Details
Heterogeneous nuclear ribonucleoproteins A2/B1P22626Details
Heterogeneous nuclear ribonucleoprotein HP31943Details
Heterogeneous nuclear ribonucleoprotein H3P31942Details
Heterogeneous nuclear ribonucleoprotein LP14866Details
Splicing factor, proline- and glutamine-richP23246Details
Splicing factor 3A subunit 2Q15428Details
Receptor of activated protein C kinase 1P63244Details
Stress-induced-phosphoprotein 1P31948Details
Eukaryotic initiation factor 4A-IP60842Details
14-3-3 protein beta/alphaP31946Details
Hepatoma-derived growth factorP51858Details
Phosphoglycerate mutase 1P18669Details
Ran-specific GTPase-activating proteinP43487Details
Kell blood group glycoproteinP23276Details
Integrin alpha-5P08648Details
HuntingtinP42858Details
Cap-specific mRNA (nucleoside-2'-O-)-methyltransferase 1Q8N1G2Details
Neural cell adhesion molecule L1P32004Details
Prohibitin-2Q99623Details
Flap endonuclease 1P39748Details
Beclin-1Q14457Details
Eukaryotic translation initiation factor 3 subunit FO00303Details
RuvB-like 2Q9Y230Details
Splicing factor 3B subunit 3Q15393Details
Heterogeneous nuclear ribonucleoprotein FP52597Details
Heterogeneous nuclear ribonucleoprotein KP61978Details
Heat shock 70 kDa protein 4P34932Details
Heat shock protein 105 kDaQ92598Details
Nuclear migration protein nudCQ9Y266Details
26S protease regulatory subunit 6AP17980Details
Splicing factor 3A subunit 3Q12874Details
SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily E member 1Q969G3Details
Tumor protein D54O43399Details
Tropomyosin alpha-3 chainP06753Details
Tropomyosin alpha-4 chainP67936Details
Ubiquitin carboxyl-terminal hydrolase 14P54578Details
Alpha-2-HS-glycoproteinP02765Details
Chromobox protein homolog 5P45973Details
Keratin, type I cytoskeletal 10P13645Details
Lys-63-specific deubiquitinase BRCC36P46736Details
DesmoplakinP15924Details
KAT8 regulatory NSL complex subunit 3Q9P2N6Details
Far upstream element-binding protein 2Q92945Details
Cysteine and glycine-rich protein 1P21291Details
Filamin-AP21333Details
Heterogeneous nuclear ribonucleoprotein D0Q14103Details
Heterochromatin protein 1-binding protein 3Q5SSJ5Details
Ras GTPase-activating-like protein IQGAP1P46940Details
NucleophosminP06748Details
60S ribosomal protein L14P50914Details
60S ribosomal protein L18Q07020Details
60S ribosomal protein L35P42766Details
60S ribosomal protein L4P36578Details
40S ribosomal protein S13P62277Details
40S ribosomal protein S17P08708Details
40S ribosomal protein S28P62857Details
40S ribosomal protein S5P46782Details
40S ribosomal protein S6P62753Details
40S ribosomal protein S8P62241Details
40S ribosomal protein S9P46781Details
Splicing factor 1Q15637Details
Small nuclear ribonucleoprotein Sm D2P62316Details
Serine/arginine-rich splicing factor 4Q08170Details
Transgelin-2P37802Details
ZyxinQ15942Details
Serine/arginine-rich splicing factor 1Q07955Details
Survival motor neuron proteinB4DP61Details
Transient receptor potential cation channel subfamily V member 2Q9Y5S1Details
Myosin-9P35579Details
Probable ATP-dependent RNA helicase DDX5P17844Details
Splicing factor 3B subunit 1O75533Details
Sodium/myo-inositol cotransporterP53794Details
Elongation of very long chain fatty acids protein 5Q9NYP7Details
Serine/threonine-protein kinase 24Q9Y6E0Details
Serine/threonine-protein kinase 3Q13188Details
Serine/threonine-protein kinase 38Q15208Details
Serine/threonine-protein kinase 38-likeQ9Y2H1Details
STE20/SPS1-related proline-alanine-rich protein kinaseQ9UEW8Details
Serine/threonine-protein kinase TAO1Q7L7X3Details
Serine/threonine-protein kinase TAO3Q9H2K8Details
Serine/threonine-protein kinase TBK1Q9UHD2Details
Serine/threonine-protein kinase tousled-like 1Q9UKI8Details
Serine/threonine-protein kinase tousled-like 2Q86UE8Details
TRAF2 and NCK-interacting protein kinaseQ9UKE5Details
Non-receptor tyrosine-protein kinase TNK1Q13470Details
Tyrosine-protein kinase receptor TYRO3Q06418Details
Serine/threonine-protein kinase ULK1O75385Details
AP2-associated protein kinase 1Q2M2I8Details
BMP-2-inducible protein kinaseQ9NSY1Details
Calcium/calmodulin-dependent protein kinase kinase 1Q8N5S9Details
Calcium/calmodulin-dependent protein kinase kinase 2Q96RR4Details
Serine/threonine-protein kinase MRCK gammaQ6DT37Details
Citron Rho-interacting kinaseO14578Details
Dual specificity protein kinase CLK2P49760Details
Dual specificity protein kinase CLK4Q9HAZ1Details
Casein kinase I isoform alphaP48729Details
Death-associated protein kinase 2Q9UIK4Details
Serine/threonine-protein kinase DCLK2Q8N568Details
Interferon-induced, double-stranded RNA-activated protein kinaseP19525Details
eIF-2-alpha kinase GCN2Q9P2K8Details
Tyrosine-protein kinase FerP16591Details
Cyclin-G-associated kinaseO14976Details
Glycogen synthase kinase-3 alphaP49840Details
Interleukin-1 receptor-associated kinase 1P51617Details
Serine/threonine-protein kinase SIK3Q9Y2K2Details
Serine/threonine-protein kinase LATS1O95835Details
LIM domain kinase 2P53671Details
Dual specificity mitogen-activated protein kinase kinase 6P52564Details
Mitogen-activated protein kinase kinase kinase 11Q16584Details
Mitogen-activated protein kinase kinase kinase 15Q6ZN16Details
Mitogen-activated protein kinase kinase kinase 3Q99759Details
Mitogen-activated protein kinase kinase kinase 4Q9Y6R4Details
Mitogen-activated protein kinase kinase kinase 6O95382Details
Mitogen-activated protein kinase kinase kinase kinase 2Q12851Details
Mitogen-activated protein kinase kinase kinase kinase 4O95819Details
Mitogen-activated protein kinase kinase kinase kinase 5Q9Y4K4Details
Mitogen-activated protein kinase 4P31152Details
MAP kinase-activated protein kinase 5Q8IW41Details
Serine/threonine-protein kinase MARK1Q9P0L2Details
Serine/threonine-protein kinase MARK2Q7KZI7Details
MAP/microtubule affinity-regulating kinase 3P27448Details
MAP/microtubule affinity-regulating kinase 4Q96L34Details
CLIP-associating protein 1Q7Z460Details
Megakaryocyte-associated tyrosine-protein kinaseP42679Details
Maternal embryonic leucine zipper kinaseQ14680Details
Misshapen-like kinase 1Q8N4C8Details
MAP kinase-interacting serine/threonine-protein kinase 1Q9BUB5Details
MAP kinase-interacting serine/threonine-protein kinase 2Q9HBH9Details
Myosin light chain kinase 3Q32MK0Details
Serine/threonine-protein kinase Nek1Q96PY6Details
Serine/threonine-protein kinase Nek4P51957Details
Serine/threonine-protein kinase Nek9Q8TD19Details
Serine/threonine-protein kinase OSR1O95747Details
Serine/threonine-protein kinase PAK 1Q13153Details
Serine/threonine-protein kinase PAK 2Q13177Details
Serine/threonine-protein kinase PAK 4O96013Details
Cyclin-dependent kinase 17Q00537Details
Phosphatidylinositol 4-kinase betaQ9UBF8Details
Membrane-associated tyrosine- and threonine-specific cdc2-inhibitory kinaseQ99640Details
Serine/threonine-protein kinase N1Q16512Details
Serine/threonine-protein kinase N2Q16513Details
Serine/threonine-protein kinase PLK4O00444Details
Serine/threonine-protein kinase PRP4 homologQ13523Details
Serine/threonine-protein kinase RIO1Q9BRS2Details
Serine/threonine-protein kinase RIO2Q9BVS4Details
Serine/threonine-protein kinase RIO3O14730Details
Receptor-interacting serine/threonine-protein kinase 1Q13546Details
Receptor-interacting serine/threonine-protein kinase 2O43353Details
Serine/threonine-protein kinase SIK2Q9H0K1Details
SNF-related serine/threonine-protein kinaseQ9NRH2Details
Serine/threonine-protein kinase 10O94804Details
Serine/threonine-protein kinase 17AQ9UEE5Details
Histone-lysine N-methyltransferase EZH2Q15910Details
Exportin-1O14980Details
POU domain, class 2, transcription factor 1P14859Details
Mitogen-activated protein kinase 6Q16659Details
NAD-dependent protein deacetylase sirtuin-2Q8IXJ6Details
DystrophinP11532Details
Solute carrier family 43 member 3Q8NBI5Details
Peroxisome proliferator-activated receptor gamma coactivator 1-betaQ86YN6Details
Voltage-gated potassium channel subunit beta-2Q13303Details
Cytochrome b reductase 1Q53TN4Details
ATP-citrate synthaseP53396Details
Very long-chain acyl-CoA synthetaseO14975Details
Solute carrier family 23 member 2Q9UGH3Details
Sodium-coupled neutral amino acid transporter 1Q9H2H9Details
Sodium-coupled neutral amino acid transporter 2Q96QD8Details
DNA (cytosine-5)-methyltransferase 3AQ9Y6K1Details
Sphingosine 1-phosphate receptor 3Q99500Details
Zinc transporter ZIP10Q9ULF5Details
Zinc transporter ZIP6Q13433Details
Zinc transporter 1Q9Y6M5Details
Zinc transporter ZIP3Q9BRY0Details
Zinc transporter 6Q6NXT4Details
Receptor tyrosine-protein kinase erbB-3P21860Details
Diacylglycerol kinase zetaQ13574Details
V-type proton ATPase subunit S1Q15904Details
Tyrosine-protein phosphatase non-receptor type 12Q05209Details
Tyrosine-protein phosphatase non-receptor type 6P29350Details
V-type proton ATPase 116 kDa subunit a isoform 1Q93050Details
V-type proton ATPase 116 kDa subunit a isoform 2Q9Y487Details
Mitochondrial potassium channelQ96ER9Details
Plasma membrane calcium-transporting ATPase 1P20020Details
Plasma membrane calcium-transporting ATPase 4P23634Details
Caspase-9P55211Details
DNA (cytosine-5)-methyltransferase 3BQ9UBC3Details
Interleukin-6 receptor subunit betaP40189Details
Sphingosine kinase 2Q9NRA0Details
Leucyl-cystinyl aminopeptidaseQ9UIQ6Details
Equilibrative nucleoside transporter 3Q9BZD2Details
Molybdopterin synthase catalytic subunitO96007Details
Receptor protein-tyrosine kinaseQ504U8Details
Serine/threonine-protein kinase ATRQ13535Details
P-selectin glycoprotein ligand 1Q14242Details
Microphthalmia-associated transcription factorO75030Details
Eukaryotic translation initiation factor 4E-binding protein 1Q13541Details
Lysine-specific histone demethylase 1AO60341Details
Signal transducer and activator of transcription 1-alpha/betaP42224Details
Proliferation marker protein Ki-67P46013Details
G1/S-specific cyclin-D3P30281Details
G1/S-specific cyclin-D2P30279Details
Histone acetyltransferase p300Q09472Details
ELAV-like protein 1Q15717Details
Nuclear factor erythroid 2-related factor 2Q16236Details
Dual specificity mitogen-activated protein kinase kinase 7O14733Details
cAMP-dependent protein kinase type I-beta regulatory subunitP31321Details
Hemoglobin subunit gamma-1P69891Details
Hemoglobin subunit gamma-2P69892Details
Bcl-2-binding component 3, isoforms 1/2Q9BXH1Details
Bcl-2-like protein 11O43521Details
Adenylate cyclase type 3O60266Details
Adenylate cyclase type 6O43306Details
Adenylate cyclase type 9O60503Details