Edited by: Rosanna Parlato, University of Ulm, Germany
Reviewed by: Victor Faundez, Emory University, United States; Francesca Ciccolini, Universität Heidelberg, Germany
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Type III adenylyl cyclase (AC3,
Primary cilia are tiny microtubule-based, membrane-ensheathed signaling devices present in most mammalian cells (Singla and Reiter,
AC3 represents a key enzyme mediating the cAMP signaling pathway in neuronal cilia (Bishop et al.,
Post-translational modifications (PTM) regulate signaling pathway and cellular processes, mediating intracellular communication and neuronal function. Protein phosphorylation is a major type of PTM, which can cause allosteric structure changes of proteins, activation, or inhibition of enzymes, alterations in protein's subcellular localization, and protein-protein interactions (Johnson,
To date, although phosphoproteomic profiling analyses have identified a high throughput of phosphorylation sites (p-sites) in a variety of tissues, mouse strains, and different brain regions (Huttlin et al.,
Prefrontal cortex tissues were isolated from 18 to 20 week old mice after euthanization, flash-frozen in liquid nitrogen, and stored in −80°C until analysis. Samples were then homogenized and lysed by grinding on ice in tissue lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM KCl, 1% TritonX-100, 0.5 mM PMSF, 0.5 mM EDTA) containing proteinase inhibitor cocktail (Cat. No. 04693159001, Roche, Germany) and phosphatase inhibitor cocktail (Cat. No. 04906837001, Roche, Germany). Lysates were cleared twice by centrifugation at 14K RPM for 20 min at 4°C. Protein centration was measured with Qubit fluorometer and ~3 mg of brain lysate from each sample was loaded in 12–20% gradient SDS-PAGE. In-gel digestion with reduction (final concentration 10 mM dithiothreitol, 56°C, 1 h) and alkylation (final concentration 55 mM iodoacetamide, 45 min in dark) were carried out at 37°C for 4 h. Phosphopeptides were enriched by MOAC (TitansphereTM Phos-TiO Kit; GL Sciences Inc., Tokyo, Japan). Briefly, 500 μl of Speed Vac enriched trypsin digested peptides (1 mg/ml) were mixed with 1,000 μl binding solution (25% lactose acid, 60% acetonitrile, 0.3% trifluoroacetic), loaded onto Phos-TiO tip with 3 mg titanium dioxide (TiO2) resin. The resin was washed with 80% acetonitrile and 0.4% trifluoroacetic and eluted with 50 μl 5% ammonium hydroxide followed by 50 μl 5% pyrrolidine. Enriched phosphopeptides were concentrated via Speed Vac for pyrrolidine removal and mass spectrometric analysis.
HPLC-MS/MS data was acquired on a LTQ Orbitrap Elite mass spectrometer (Thermo Fisher, CA) coupled to a NanoAccuity UPLC (Waters, MA) in Whitehead MS Facility at MIT (Boston, MA). Peptides were separated by a C18 column at 250 nL/min flow rate and 90-min gradient program. LC-MS data were acquired in an information-dependent acquisition mode, cycling between a MS scan (m/z 395–1,800, resolution 240,000) acquired in the Orbitrap, followed by 10 low-energy CID analysis in the linear ion trap. The centroided peak lists of the CID spectra were generated by PAVA (Guan and Burlingame,
Label-free quantification was performed using Skyline ver 4.1.0.18169 via MS1 full-scan filtering with the library generated by ProteinProspector (Cut-off score = 0.95; Precursor charge = 2, 3, 4, 5; Max Miss Cleavages = 1) and the SwissProt Mus Musculus protein FASTA file (Schilling et al.,
The phosphoprotein lists generated from ProteinProspector were analyzed by AmiGO 2 (Mi et al.,
Data analysis and figure constructs were performed with Origin Pro and Graphpad Prism 7 software for Student's
We used “Two Population Proportions” for comparison to set the “3 out of the
For an individual phosphopeptide, in control group,
We used two statistical methods “Two Population Proportions” comparison and “Student
AutDB collects a total of 1,053 ASD gene entries. The human genome is estimated to have 20,000 genes. Thus, the 1,053 ASD genes are estimated to represent 5.2% of all human genes in human genome. We have identified 204 sex-biased phosphorylation, among which 32 proteins (15.6%) were listed in the AutDB as ASD genes. The ASD gene percentage is 32/204 = 15.6%. We used “Two Population Proportions” method to compare two groups and produced a
We used Pearson's Chi-square test to carry out a cross comparison of four groups (Female KOs, Females WTs, Male KOs, Male WTs). Detailed method as well as the results of cross comparison of 4 groups were provided in the
Throughout this manuscript, we mostly used two population proportions comparison (3 out of
Because AC3 is associated with MDD (Wray et al.,
Immunofluorescence staining of AC3 in the mouse prefrontal cortex. AC3 was predominantly enriched in neuronal primary cilia in the prefrontal cortex, but not microglia or astrocyte cilia.
Given the major target protein of cAMP in cells is PKA, which can phosphorylate numerous downstream proteins, we set out to determine if AC3 affects post-translational phosphorylation in neurons in the prefrontal cortex. To circumvent developmental complications, we utilized AC3 floxed:Ubc-Cre/ERT2 KO mouse strain (Chen et al.,
To efficiently identify phosphorylation differences in the prefrontal cortex between AC3 KO and WT mice, we utilized a MS-based phosphoproteomic approach to identify phosphopeptides in large scale. Proteins of the prefrontal cortex (isolated from WT, KO, male, and female mice, respectively) were extracted and digested with trypsin. Resultant phosphopeptides were enriched using TiO2 enrichment column and then subjected to HPLC-MS/MS analysis and database search for identification using UCSF Protein Prospector (
Workflow of phosphoproteomics analysis. Prefrontal cortex tissue homogenizations were separated via SDS-PAGE and cut into 3 fractions with equal protein amount (according to Coomassie blue staining intensity measured by ImageJ). Protein extraction via in-gel digestion with trypsin (1:300 enzyme/substrate) was performed on ~3 mg protein for each sample. Phosphopeptides from each fraction were enriched by TitansphereTM Phos-TiO2 Kit and analyzed with HPLC-MS/MS for 90 min, respectively. A MS2 spectra of Pde1b (p)Ser7 (p)Ser18 is shown as an example. MS spectra data were analyzed by ProteinProspector (ver. 5.21.2, UCSF, San Francisco, CA) and Skyline (ver. 4.1.0.18169). Among all MS-detected phosphopeptides (94%), 73% are (p)Ser, 19% (p)Thr, and 2% (p)Tyr.
In total, 4,655 different phosphopeptides were detected from 1,756 proteins (
Data quality of phosphoproteomic analysis.
For global phosphoproteomic profiling, the average MS1 peak area of KOs relative to WTs (x-axis), and the average MS1 peak area of males relative to females (y-axis) were constructed into a scatter plot (
To determine which classes of proteins were enriched in our MS-based phosphoproteomic analysis, the dataset of all phosphopeptides was subjected to Gene Ontology (GO) enrichment analysis (Mi et al.,
cAMP regulates many kinases' activity including PKA and ERK1/2 (Waltereit and Weller,
Proline-directed kinase recognized peptide abundance is decreased in AC3 KOs compared to WTs. The sequence logos for (p)Ser with a proline residue at the +1 position for 4 different sample groups.
To identify phosphopeptides that were differently expressed in AC3 KO and WT mice, we compared AC3 KO and WT datasets. Phosphopeptides that were expressed differently (
Phosphopeptides exclusively detected in AC3 WT dataset.
Acaca |
Acetyl-CoA carboxylase 1 | 3 | monophos-(18)FIIGSVSEDNSEDEISNLVK | Girirajan et al., |
Map1a |
Microtubule-associated protein 1A | 4 | monophos-(2586)AKPASPARR | Myers et al., |
Map2 | Microtubule-associated protein 2 | 3 | monophos-(1004)ELITTKDTSPEK | Mukaetova-Ladinska et al., |
Spast | Spastin | 3 | diphos-(89)SSGTAPAPASPSPPEPGPGGEAESVR | Talkowski et al., |
Ahsg | Alpha-2-HS-glycoprotein | 3 | monophos-(301)HAFSPVASVESASGETLHSPK | #N/A |
Bloc1s3 | Biogenesis of lysosome-related organelles complex 1 subunit 3 | 3 | diphos-(51)VAGEAAETDSEPEPEPTVVPVDLPPLVVQR | #N/A |
Ctps1 | CTP synthase 1 | 3 | diphos-(570)SGSSSPDSEITELKFPSISQD | #N/A |
Kcnip3 | Calsenilin | 3 | diphos-(49)WILSSAAPQGSDSSDSELELSTVR | #N/A |
Ppp1r14a | Protein phosphatase 1 regulatory subunit 14A | 4 | monophos-(19)ARGPGGSPSGLQK | #N/A |
Rabl6 | Rab-like protein 6 | 4 | monophos-(477)NISLSSEEEAEGLAGHPR | #N/A |
Sap30l | Histone deacetylase complex subunit SAP30L | 3 | diphos-(89)KASDDGGDSPEHDADIPEVDLFQLQVNTLR | #N/A |
Slc6a20b | Sodium- and chloride-dependent transporter XTRP3B | 3 | monophos-(0)MESPSAHAVSLPEDEELQPWGGAGGPGQHPGRPRSTECA |
#N/A |
Ube2v1 | Ubiquitin-conjugating enzyme E2 variant 1 | 3 | monophos-(135)LPQPPEGQCYSN | #N/A |
Znf281 | Zinc finger protein 281 | 3 | monophos-(0)MKIGSGFLSGGGGPSSSGGSGSGGSSGSASGGSGGGR | #N/A |
Phosphopeptides exclusively detected in AC3 KO dataset.
Dlgap2 | Disks large-associated protein 2 | 3 | monophos-(717)CSSIGVQDSEFPDHQPYPR | Girirajan et al., |
Hepacam | Hepatocyte cell adhesion molecule | 5 | monophos-(316)DKDSSEPDENPATEPR | Myers et al., |
Lsm14a |
Protein LSM14 homolog A | 3 | monophos-(214)RSPVPARPLPPTSQK | Mukaetova-Ladinska et al., |
Map1a |
Microtubule-associated protein 1A | 3 | monophos-(457)KFSKPDLKPFTPEVR | Talkowski et al., |
Map2 | Microtubule-associated protein 2 | 4 | monophos-(1634)SGILVPSEK | Girirajan et al., |
Myh11 |
Myosin-11 | 4 | monophos-(1946)VIENTDGSEEEMDAR | Myers et al., |
Nav1 |
Neuron navigator 1 | 3 | monophos-(374)LELVESLDSDEVDLK | Mukaetova-Ladinska et al., |
Sptan1 |
Spectrin alpha chain, non-erythrocytic 1 | 4 | diphos-(1181)DEADSKTASPWK | Talkowski et al., |
Strip2 |
Striatin-interacting proteins 2 | 3 | monophos-(361)QDSLDIYNER | Girirajan et al., |
Agk | Acylglycerol kinase, mitochondrial | 3 | monophos-(281)LASFWAQPQDASSR | #N/A |
Arfgap2 | ADP-ribosylation factor GTPase-activating protein 2 | 4 | monophos-(428)AISSDMFFGR | #N/A |
Arhgap20 | Rho GTPase-activating protein 20 | 3 | diphos-(795)SKPVPISVASYSHGSSQDHPRK | #N/A |
Arhgap44 | Rho GTPase-activating protein 44 | 3 | monophos-(604)GSPGSIQGTPCPGTQLGPQPAASPSQLPADQSPHTLR | #N/A |
C2cd2l | C2 domain-containing protein 2-like | 3 | monophos-(413)NLGTPTSSTPRPSITPTK | #N/A |
Cdk14 | Cyclin-dependent kinase 14 | 3 | monophos-(92)VHSENNACINFK | #N/A |
Clip2 | CAP-Gly domain-containing linker protein 2 | 3 | monophos-(914)VLLLEANRHSPGPER | #N/A |
Cops5 | COP9 signalosome complex subunit 5 | 3 | monophos-(282)GSFMLGLETHDR | #N/A |
Cox4i1 | Cytochrome c oxidase subunit 4 isoform 1, mitochondrial | 3 | monophos-(42)DYPLPDVAHVTMLSASQK | #N/A |
F11r | Junctional adhesion molecule A | 3 | diphos-(278)VIYSQPSTRSEGEFK | #N/A |
Fam126a | Hyccin | 3 | monophos-(452)SFEQVSGAPVPR | #N/A |
Ggct | Gamma-glutamylcyclotransferase | 3 | monophos-(169)GKISDEMEDIIK | #N/A |
Itm2c | Integral membrane protein 2C | 3 | monophos-(20)AAASGPASASAPAAEILLTPAR | #N/A |
Kcnb2 | Potassium voltage-gated channel subfamily B member 2 | 3 | monophos-(460)SMELIDVAVEK | #N/A |
Kctd8 | BTB/POZ domain-containing protein KCTD8 | 3 | monophos-(410)RNSELFQSLISK | #N/A |
Lysmd2 | LysM and putative peptidoglycan-binding domain-containing protein 2 | 3 | monophos-(28)SRSTSEPEEAELSLSLAR | #N/A |
Mdh2 | Malate dehydrogenase, mitochondrial | 4 | monophos-(241)AGAGSATLSMAYAGAR | #N/A |
Nwd1 | NACHT and WD repeat domain-containing protein 1 | 3 | monophos-(935)LWSLLSGQEKVTILDGGSQNPTEPQSWDLHVDER | #N/A |
Pde1b | Calcium/calmodulin-dependent 3',5'-cyclic nucleotide phosphodiesterase 1B | 4 | diphos-(6)SPPEMLESDCPSPLELK | #N/A |
Pgam1 | Phosphoglycerate mutase 1 | 3 | monophos-(117)SYDVPPPPMEPDHPFYSNISK | #N/A |
Pja1 | E3 ubiquitin-protein ligase Praja-1 | 3 | monophos-(226)VFFDTDDDDDVPHSTSR | #N/A |
Serbp1 | Plasminogen activator inhibitor 1 RNA-binding protein | 3 | monophos-(240)QISYNCSDLDQSNVTEETPEGEEHPVADTENKENEVEEVK | #N/A |
Srcin1 | SRC kinase signaling inhibitor 1 | 3 | diphos-(1124)AVSEVVRPASTPPIMASAIKDEDDEER | #N/A |
Stac2 | SH3 and cysteine-rich domain-containing protein 2 | 3 | monophos-(45)SKSVENFFLR | #N/A |
Tjp2 | Tight junction protein ZO-2 | 3 | monophos-(965)DASPPPAFKPEPPK | #N/A |
Tyro3 | Tyrosine-protein kinase receptor TYRO3 | 3 | monophos-(799)AEQPTESGSPEVHCGER | #N/A |
Comparison of phosphopeptide abundance in AC3 KO and WT datasets and their protein interaction.
In total, we identified 65 proteins either with phosphopeptides exclusively present in AC3 WTs or KOs (as listed in
To determine if some protein interaction network may be associated with the phosphoproteins differentially expressed in AC3 KOs and WTs, the 65 proteins were mapped onto the mouse Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database (string-db.org) of known protein interactions (
Males and females differ greatly in cognition, behaviors, and disease susceptibility (Zagni et al.,
Phosphopeptides exclusively detected in female dataset.
Apc | Adenomatous polyposis coli protein | 4 | monophos-(1434)SKTPPPPPQTVQAK | Girirajan et al., |
Atp1a1 | Sodium/potassium-transporting ATPase subunit alpha-1 | 6 | monophos-(707)QGAIVAVTGDGVNDSPALKK | Schlingmann et al., |
Atp1a3 | Sodium/potassium-transporting ATPase subunit alpha-3 | 6 |
monophos-(466)VAEIPFNSTNK |
Myers et al., |
Bin1 | Myc box-dependent-interacting protein 1 | 3 | monophos-(312)VNHEPEPASGASPGATIPK | Talkowski et al., |
Cnksr2 | Connector enhancer of kinase suppressor of ras 2 | 3 | monophos-(502)SNSPAHYSLLPSLQMDALR | Girirajan et al., |
Dlgap2 | Disks large-associated protein 2 | 3 | diphos-(1032)AASFRQNSATER | Myers et al., |
Jph3 |
Junctophilin-3 | 5 | monophos-(417)EFSPSFQHR | Mukaetova-Ladinska et al., |
Lrrc7 | Leucine-rich repeat-containing protein 7 | 3 | monophos-(1342)SREQQPYEGNINK | Talkowski et al., |
Magi2 |
Membrane-associated guanylate kinase, WW and PDZ domain-containing protein 2 | 4 | monophos-(1004)IIPQEELNSPTSAPSSEK | Girirajan et al., |
Map1b |
Microtubule-associated protein 1B | 4 | monophos-(1194)DYNASASTISPPSSMEEDKFSK | Myers et al., |
Map2 | Microtubule-associated protein 2 | 3 | monophos-(1004)ELITTKDTSPEK | Mukaetova-Ladinska et al., |
Map6 |
Microtubule-associated protein 6 | 3 | monophos-(293)SEGHEEKPLPPAQSQTQEGGPAAGK | Talkowski et al., |
Nav1 |
Neuron navigator 1 | 3 | monophos-(374)LELVESLDSDEVDLK | Girirajan et al., |
Neo1 | Neogenin | 3 | monophos-(1200)LELKPIDKSPDPNPVMTDTPIPR | Myers et al., |
Plcd3 |
1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase delta-3 | 3 | monophos-(489)ILSDREEEEEEEEEAEEALEAAEQR | Mukaetova-Ladinska et al., |
Prex1 | Phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 protein | 3 | monophos-(1178)SNSSYLGSDEMGSGDELPCDMR | Talkowski et al., |
Psmd4 |
26S proteasome non-ATPase regulatory subunit 4 | 4 | monophos-(237)AAAASAAEAGIATPGTEDSDDALLK | Girirajan et al., |
Ptpn1 |
Tyrosine-protein phosphatase non-receptor type 1 | 4 | monophos-(325)ELFSSHQWVSEETCGDEDSLAR | Myers et al., |
Slc12a6 |
Solute carrier family 12 member 6 | 3 | monophos-(21)IDDIPGLSDTSPDLSSR | Mukaetova-Ladinska et al., |
Smarcc1 |
SWI/SNF complex subunit SMARCC1 | 3 | diphos-(323)RKPSPSPPPPTATESR | Talkowski et al., |
Sorbs1 |
Sorbin and SH3 domain-containing protein 1 | 6 | monophos-(49)GTPSSSPVSPQESPKHESK | Girirajan et al., |
Spast | Spastin | 3 | diphos-(89)SSGTAPAPASPSPPEPGPGGEAESVR | Myers et al., |
Spry2 |
Protein sprouty homolog 2 | 3 | monophos-(108)SISTVSSGSR | Mukaetova-Ladinska et al., |
Srgap2 |
SLIT-ROBO Rho GTPase-activating protein 2 | 5 |
monophos-(496)KQDSSQAIPLVVESCIR |
Talkowski et al., |
Srpk1 |
SRSF protein kinase 1 | 44 | monophos-(31)GSAPHSESDIPEQEEEILGSDDDEQEDPNDYCK |
Myers et al., |
Strip1 |
Striatin-interacting protein 1 | 3 | monophos-(56)KDSEGYSESPDLEFEYADTDK | Talkowski et al., |
Trio | Triple functional domain protein | 3 | monophos-(2274)NFLNALTSPIEYQR | Girirajan et al., |
Aagab | Alpha- and gamma-adaptin-binding protein p34 | 4 | monophos-(196)VASAESCHSEQQEPSPTAER | #N/A |
Aak1 | AP2-associated protein kinase 1 | 4 | monophos-(678)TSQQNVSNASEGSTWNPFDDDNFSK | #N/A |
Acot11 | Acyl-coenzyme A thioesterase 11 | 3 | monophos-(24)SISHPESGDPPTMAEGEGYR | #N/A |
Akap12 | A-kinase anchor protein 12 | 4 | diphos-(260)EKEPTKPLESPTSPVSNETTSSFK | #N/A |
Amer2 | APC membrane recruitment protein 2 | 4 | monophos-(551)DSDSGDALCDLYVEPEASPATLPATEDPPCLSR | #N/A |
Arfgef3 | Brefeldin A-inhibited guanine nucleotide-exchange protein 3 | 3 | monophos-(2050)GPDSPLLQRPQHLIDQGQMR | #N/A |
Arhgef12 | Rho guanine nucleotide exchange factor 12 | 3 | monophos-(326)SEGVQDAEPQSLVGSPSTR | #N/A |
Atg4c | Cysteine protease ATG4C | 5 | monophos-(424)DFDFTSTAASEEDLFSEDERK | #N/A |
Atp1a2 | Sodium/potassium-transporting ATPase subunit alpha-2 | 6 | monophos-(474)VAEIPFNSTNK | #N/A |
6 | monophos-(704)QGAIVAVTGDGVNDSPALKK | |||
Bsn | Protein bassoon | 3 | monophos-(1038)SHGPLLPTIEDSSEEEELREEEELLR | #N/A |
Camkv | CaM kinase-like vesicle-associated protein | 4 | monophos-(395)SATPATDGSATPATDGSVTPATDGSITPATDGSVTPATDR | #N/A |
Cir1 | Corepressor interacting with RBPJ 1 | 3 | monophos-(188)NLTANDPSQDYVASDCEEDPEVEFLK | #N/A |
Cmtm4 | CKLF-like MARVEL transmembrane domain-containing protein 4 | 4 | monophos-(191)TESRDVDSRPEIQR | #N/A |
Cpsf7 | Cleavage and polyadenylation specificity factor subunit 7 | 3 | monophos-(192)DSSDSADGRATPSENLVPSSAR | #N/A |
Csnk1a1 | Casein kinase I isoform alpha | 3 | monophos-(304)AAQQAASSSGQGQQAQTPTGK | #N/A |
Dos | Protein Dos | 3 | monophos-(613)RGDSVDCPPEGR | #N/A |
Epb41l1 | Band 4.1-like protein 1 | 5 | diphos-(465)SEAEEGEVRTPTK | #N/A |
Epb41l3 | Band 4.1-like protein 3 | 4 | monophos-(48)QQPALEQFPEAAAHSTPVKR | #N/A |
Evl | Ena/VASP-like protein | 4 | monophos-(232)VQRPEDASGGSSPSGTSK | #N/A |
Farp1 | FERM, RhoGEF and pleckstrin domain-containing protein 1 | 3 | monophos-(387)QSPQSASLTFGEGTESPGGQSCQQAK | #N/A |
Gpalpp1 | GPALPP motifs-containing protein 1 | 3 | monophos-(127)GREDPGQVSSFFNSEEAESGEDEDIVGPMPAK | #N/A |
Hid1 | Protein HID1 | 3 | diphos-(583)TPEPLSRTGSQEGTSMEGSRPAAPAEPGTLK | #N/A |
Hook3 | Protein Hook homolog 3 | 4 | monophos-(226)LNQSDSIEDPNSPAGR | #N/A |
Hspa4 | Heat shock 70 kDa protein 4 | 5 | monophos-(521)MQVDQEEPHTEEQQQQPQTPAENKAESEEMETSQAGSK | #N/A |
Ipo5 | Importin-5 | 3 | monophos-(814)RQDEDYDEQVEESLQDEDDNDVYILTK | #N/A |
Kctd16 | BTB/POZ domain-containing protein KCTD16 | 4 | monophos-(282)WSSSHCDCCCK | #N/A |
Lrrc47 | Leucine-rich repeat-containing protein 47 | 3 | monophos-(507)STSENKEEDMLSGTEADAGCGLSDPNLTLSSGK | #N/A |
Lrsam1 | E3 ubiquitin-protein ligase LRSAM1 | 6 | monophos-(206)ESGLDYYPPSQYLLPVLEQDGAENTQDSPDGPASR | #N/A |
Mvb12b | Multivesicular body subunit 12B | 3 | monophos-(194)NHDSSQPTTPSQSSASSTPAPNLPR | #N/A |
Nckipsd | NCK-interacting protein with SH3 domain | 5 | monophos-(257)APSPEPPTEEVAAETNSTPDDLEAQDALSPETTEEK | #N/A |
Ndel1 | Nuclear distribution protein nudE-like 1 | 5 | monophos-(223)GTENSFPSPK | #N/A |
Nsf | Vesicle-fusing ATPase | 3 | monophos-(202)ENRQSIINPDWNFEK | #N/A |
Nufip2 | Nuclear fragile X mental retardation-interacting protein 2 | 4 | monophos-(614)DYEIENQNPLASPTNTLLGSAK | #N/A |
Ogfrl1 | Opioid growth factor receptor-like protein 1 | 3 | monophos-(372)EPGEEADKPSPEPGSGDPKPR | #N/A |
Oxr1 | Oxidation resistance protein 1 | 3 | diphos-(358)QEKSSDASSESVQTVSQMEVQSLTATSEAANVPDR | #N/A |
Pacsin1 | Protein kinase C and casein kinase substrate in neurons protein 1 | 4 | monophos-(388)ALYDYDGQEQDELSFK | #N/A |
Pacsin3 | Protein kinase C and casein kinase II substrate protein 3 | 6 | monophos-(332)DGTAPPPQSPSSPGSGQDEDWSDEESPRK | #N/A |
PAGR1 | PAXIP1-associated glutamate-rich protein 1 | 3 | monophos-(222)DLFSLDSEGPSPTSPPLR | #N/A |
Pds5b | Sister chromatid cohesion protein PDS5 homolog B | 3 | monophos-(1353)AESPETSAVESTQSTPQK | #N/A |
Pitpnc1 | Cytoplasmic phosphatidylinositol transfer protein 1 | 3 | diphos-(112)YEDNKGSNDSIFDSEAK | #N/A |
Pnmal1 | PNMA-like protein 1 | 3 | monophos-(319)SALPAADSPGNLEDSDQDGGPENPAK | #N/A |
Ppp1r7 | Protein phosphatase 1 regulatory subunit 7 | 3 | diphos-(20)RVESEESGDEEGK | #N/A |
Prkce | Protein kinase C epsilon type | 4 | monophos-(343)SKSAPTSPCDQELK | #N/A |
Psen1 | Presenilin-1 | 3 | monophos-(344)DSHLGPHRSTPESR | #N/A |
Ptrf | Polymerase I and transcript release factor | 4 | diphos-(175)ESEALPEKEGDELGEGER |
#N/A |
Rad23a | UV excision repair protein RAD23 homolog A | 4 | diphos-(119)EDKSPSEESTTTTSPESISGSVPSSGSSGR | #N/A |
Rap1gap2 | Rap1 GTPase-activating protein 2 | 3 | monophos-(8)KQELANSSDVTLPDRPLSPPLTAPPTMK | #N/A |
Rasgrf2 | Ras-specific guanine nucleotide-releasing factor 2 | 3 | monophos-(722)KFSSPPPLAVSR | #N/A |
Rps6kc1 | Ribosomal protein S6 kinase delta-1 | 5 | monophos-(646)ESEAQDSVSRGSDDSVPVISFK | #N/A |
Rragc | Ras-related GTP-binding protein C | 3 | monophos-(83)MSPNETLFLESTNK | #N/A |
Rtn1 | Reticulon-1 | 5 | diphos-(303)QDLCLKPSPDTVPTVTVSEPEDDSPGSVTPPSSG |
#N/A |
Serbp1 | Plasminogen activator inhibitor 1 RNA-binding protein | 3 | monophos-(240)QISYNCSDLDQSNVTEETPEGEEHPVAD |
#N/A |
Snx16 | Sorting nexin-16 | 3 | monophos-(91)EAEEQHPEAVNWEDRPSTPTILGYEVMEER | #N/A |
Ssbp3 | Single-stranded DNA-binding protein 3 | 5 | monophos-(345)NSPNNISGISNPPGTPR | #N/A |
Stambpl1 | AMSH-like protease | 4 | monophos-(232)SDGSNFANYSPPVNR | #N/A |
Synpo | Synaptopodin | 5 | monophos-(760)VASLSPAR | #N/A |
Tacc1 | Transforming acidic coiled-coil-containing protein 1 | 3 | monophos-(549)APVSVACGGESPLDGICLSEADK | #N/A |
Tmf1 | TATA element modulatory factor | 4 | monophos-(333)SVSEINSDDELPGK | #N/A |
Tmpo | Lamina-associated polypeptide 2, isoforms beta/delta/epsilon/gamma | 3 | monophos-(60)GPPDFSSDEEREPTPVLGSGASVGR | #N/A |
Trappc10 | Trafficking protein particle complex subunit 10 | 4 | monophos-(704)RQESGSSLEPPSGLALEDGAHVLR | #N/A |
Trim28 | Transcription intermediary factor 1-beta | 3 | monophos-(435)QGSGSSQPMEVQEGYGFGSDDPYSSAEPHVSGMK | #N/A |
4 | diphos-(591)LASPSGSTSSGLEVVAPEVTSAPVSGPGILDDSATICR | |||
Tyro3 | Tyrosine-protein kinase receptor TYRO3 | 3 | monophos-(799)AEQPTESGSPEVHCGER | #N/A |
Zfyve20 | Rabenosyn-5 | 6 | monophos-(206)DSLSTHTSPSQSPNSVHGSR | #N/A |
Phosphopeptides exclusively detected in male dataset.
Abi1 |
Abl interactor 1 | 3 | monophos-(173)TNPPTQKPPSPPVSGR | Girirajan et al., |
Apc | Adenomatous polyposis coli protein | 3 | diphos-(1856)NDSLSSLDFDDDDVDLSR | Myers et al., |
Caskin1 |
Caskin-1 | 3 | monophos-(727)SQEYLLDEGMAPGTPPK | Mukaetova-Ladinska et al., |
Cspg5 |
Chondroitin sulfate proteoglycan 5 | 3 | monophos-(529)LKEEESFNIQNSMSPK | Talkowski et al., |
Ctnnd1 |
Catenin delta-1 | 3 | monophos-(344)GSLASLDSLRK | Girirajan et al., |
Ctnnd2 | Catenin delta-2 | 6 | monophos-(4)KQSGAAPFGAMPVPDQPPSASEK | Myers et al., |
Efnb3 |
Ephrin-B3 | 3 | monophos-(271)GGSLGLGGGGGMGPR | Mukaetova-Ladinska et al., |
Irf2bpl | Interferon regulatory factor 2-binding protein-like | 3 | diphos-(633)RNSSSPVSPASVPGQR | Talkowski et al., |
Map1b |
Microtubule-associated protein 1B | 3 | diphos-(1290)SVSPGVTQAVVEEHCASPEEK | Girirajan et al., |
Srgap2 |
SLIT-ROBO Rho GTPase-activating protein 2 | 3 | diphos-(981)TSPVVAPTSEPSSPLHTQLLKDPEPAFQR | Myers et al., |
Tbc1d5 | TBC1 domain family member 5 | 3 | monophos-(543)SESMPVQLNK | Mukaetova-Ladinska et al., |
Atat1 | Alpha-tubulin N-acetyltransferase 1 | 3 | diphos-(269)SSSLGNSPDRGPLRPFVPEQELLR | #N/A |
Camsap1 | Calmodulin-regulated spectrin-associated protein 1 | 4 | diphos-(546)TDVSPPSPQMPR | #N/A |
Cryab | Alpha-crystallin B chain | 4 | monophos-(56)APSWIDTGLSEMR | #N/A |
Fam103a1 | RNMT-activating mini protein | 3 | monophos-(31)RPPESPPIVEEWNSR | #N/A |
Fam134a | Protein FAM134A | 3 | diphos-(293)TALALAITDSELSDEEASILESGGFSVSR | #N/A |
Kbtbd11 | Kelch repeat and BTB domain-containing protein 11 | 4 | monophos-(60)ASAAEGSEASPPSLR | #N/A |
Kiaa1467 | Uncharacterized protein KIAA1467 | 6 | monophos-(15)SPDLGEYDPLTQADSDESEDDLVLNLQQK | #N/A |
Map4 | Microtubule-associated protein 4 | 5 | monophos-(514)DMSPSAETEAPLAK | #N/A |
Mbp | Myelin basic protein | 3 | monophos-(145)YLATASTMDHAR | #N/A |
Mrpl23 | 39S ribosomal protein L23, mitochondrial | 5 | monophos-(117)SPEPLEEELPQQR | #N/A |
Phactr1 | Phosphatase and actin regulator 1 | 4 | diphos-(326)LESSEQRVPCSTSYHSSGLHSSDGITK | #N/A |
Rbm5 | RNA-binding protein 5 | 4 | monophos-(614)GLVAAYSGDSDNEEELVER | #N/A |
Sik3 | Serine/threonine-protein kinase SIK3 | 3 | monophos-(490)RASDGGANIQLHAQQLLK | #N/A |
Slc6a17 | Sodium-dependent neutral amino acid transporter SLC6A17 | 3 | diphos-(679)VPSEAPSPMPTHR | #N/A |
Sptbn1 | Spectrin beta chain, non-erythrocytic 1 | 3 | monophos-(2122)GDQVSQNGLPAEQGSPR | #N/A |
Similarly, to compare phosphorylation level differences between females and males, Skyline-based label-free quantification was also performed on female and male datasets. We further found 96 phosphopeptides (out of 88 proteins) having increased phosphorylation levels in females relative to males, whereas only 11 peptides had significantly decreased phosphorylation levels in females (
Comparison of phosphopeptide abundance in female and male datasets.
It is common that one protein activity can up- or down-regulate the activity of its interacting proteins in shared signal transduction pathways (von Mering et al.,
Protein-protein interaction analysis using phosphoproteins differentially expressed in females and males.
Malfunctions of primary cilia cause a broad spectrum of diseases in humans, particularly developmental disorders. AC3 is highly enriched in neuronal primary cilia, and not well-expressed in mature astrocyte cilia or microglia in the frontal cortex (
We utilized a high-efficiency method to conduct comparative phosphoproteomics analyses combining TiO2 phosphopeptide enrichment with HPLC-MS/MS analysis. This approach allows for large-scale identification of phosphopeptides. In our assay, more than 1,500 phosphorylated peptides and 30,000 spectra were detected from each sample (
To compare phosphorylation levels in proteins that are involved in cAMP signaling pathway, phosphopeptides identified from G-proteins, adenylyl cyclases, PKA, phosphodiesterases (PDE), phosphatases, as well as their regulating proteins were summarized in
Male and female brains differ in many aspects including connectivity (Ingalhalikar et al.,
Among all differentially modified proteins in genders, one protein family is of particular interest. That is the delta catenin family, which contains ARVCF (encoded by
In summary, this comparative phosphoproteomic profiling has generated several interesting findings: (1) AC3 ablation leads to decreased activity of proline-directed kinases in the frontal cortex; (2) There is a gender-biased phosphorylation in 204 proteins, 31% of which are associated with ASD; (3) Four delta catenin family members, all associated with autism, contain gender-biased phosphorylation sites. Hence, although future work is warranted, this study provides useful phosphoproteomic clues to elucidate the function of AC3 in the CNS. It also presents the first proteomic evidence suggesting that sex-biased post-translational phosphorylation is implicated in the sexual dimorphism of autism.
Proteome raw and complete datasets (4 groups: AC3 controls, KOs, males and females, 16 mice) have been submitted to ProteomeXchange via the PRIDE-EBI database (
All animal-related procedures were approved and conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of the University of New Hampshire.
YZ and XC designed experiments. YZ conducted phosphoproteomic assays, immunostaining, and data analysis. LQ maintained mouse colony and provided support for phosphoproteomic assay. AS contributed to immunostaining. FC provided expertise in phosphoproteomic experiments and data analysis. HW provided expertise in statistical methods. YZ and XC interpreted data and wrote the paper.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The Supplementary Material for this article can be found online at: