Phosphoproteomic Analysis of the Developing Mouse Brain

Abstract

Proper development of the mammalian brain requires the precise integration of numerous temporally and spatially regulated stimuli. Many of these signals transduce their cues via the reversible phosphorylation of downstream effector molecules. Neuronal stimuli acting in concert have the potential of generating enormous arrays of regulatory phosphoproteins. Toward the global profiling of phosphoproteins in the developing brain, we report here the use of a mass spectrometry-based methodology permitting the first proteomic-scale phosphorylation site analysis of primary animal tissue, identifying over 500 protein phosphorylation sites in the developing mouse brain. Proper development of the mammalian brain requires the precise integration of numerous temporally and spatially regulated stimuli. Many of these signals transduce their cues via the reversible phosphorylation of downstream effector molecules. Neuronal stimuli acting in concert have the potential of generating enormous arrays of regulatory phosphoproteins. Toward the global profiling of phosphoproteins in the developing brain, we report here the use of a mass spectrometry-based methodology permitting the first proteomic-scale phosphorylation site analysis of primary animal tissue, identifying over 500 protein phosphorylation sites in the developing mouse brain. Phosphorylation can dramatically change a protein’s biological location or activity. The profound influence of protein phosphorylation on mammalian brain development has strong genetic support. This is exemplified by the important brain phenotypes observed in mice with loss of function mutations in genes encoding kinases such as p35/cyclin-dependent kinase 5 (Cdk5) 1The abbreviations used are: Cdk5, cyclin-dependent kinase 5; SCX, strong cation exchange; SH2, Src homology 2; SH3, Src homology 3; NCBI, National Center for Biotechnology Information; FA, formic acid. (1Chae T. Kwon Y.T. Bronson R. Dikkes P. Li E. Tsai L.H. Mice lacking p35, a neuronal specific activator of Cdk5, display cortical lamination defects, seizures, and adult lethality..Neuron. 1997; 18: 29-42Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar, 2Ohshima T. Ward J.M. Huh C.G. Longenecker G. Veeranna Pant H.C. Brady R.O. Martin L.J. Kulkarni A.B. Targeted disruption of the cyclin-dependent kinase 5 gene results in abnormal corticogenesis, neuronal pathology and perinatal death..Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11173-11178Crossref PubMed Scopus (810) Google Scholar), loss of tyrosine phosphorylation sites in signaling molecules such as the Reelin-stimulated adaptor protein Disabled-1 (3Howell B.W. Herrick T.M. Hildebrand J.D. Zhang Y. Cooper J.A. Dab1 tyrosine phosphorylation sites relay positional signals during mouse brain development..Curr. Biol. 2000; 10: 877-885Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar), or loss of genes encoding proteins that interact with phosphorylated protein motifs such as 14-3-3ε (4Toyo-oka K. Shionoya A. Gambello M.J. Cardoso C. Leventer R. Ward H.L. Ayala R. Tsai L.H. Dobyns W. Ledbetter D. Hirotsune S. Wynshaw-Boris A. 14-3-3ε is important for neuronal migration by binding to NUDEL: A molecular explanation for Miller-Dieker syndrome..Nat. Genet. 2003; 34: 274-285Crossref PubMed Scopus (343) Google Scholar). Historically, the analysis of protein phosphorylation sites has been restricted to studies at the single-protein level. Recently, larger-scale MS-based analyses have emerged. However, such studies have been challenging due to a) the immaturity of methods to enrich for low-abundance phosphoproteins or phosphopeptides and b) the reduction in quality of informative tandem mass spectra obtained from phosphopeptides subjected to CID (5DeGnore J.P. Qin J. Fragmentation of phosphopeptides in an ion trap mass spectrometer..J. Am. Soc. Mass Spectrom. 1998; 9: 1175-1188Crossref PubMed Scopus (235) Google Scholar). The latter challenge is due primarily to the propensity for precursor ions containing phosphoserine or phosphothreonine to undergo β-elimination of phosphoric acid with an accompanied reduction of structurally informative ions from peptide backbone fragmentation. Recent advances in metal ion affinity chromatography have permitted large-scale phosphorylation analysis (200–400 sites identified) in yeast (6Ficarro S.B. McCleland M.L. Stukenberg P.T. Burke D.J. Ross M.M. Shabanowitz J. Hunt D.F. White F.M. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae..Nat. Biotechnol. 2002; 20: 301-305Crossref PubMed Scopus (1499) Google Scholar) and plants (7Nuhse T.S. Stensballe A. Jensen O.N. Peck S.C. Large-scale analysis of in vivo phosphorylated membrane proteins by immobilized metal ion affinity chromatography and mass spectrometry..Mol. Cell. Proteomics. 2003; 2: 1234-1243Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar). Here, using strong cation exchange (SCX) chromatography at low pH to enrich for tryptic phosphopeptides (8Beausoleil S.A. Jedrychowski M. Schwartz D. Elias J. Villen J. Li J. Cohn M. Cantely L.C. Gygi S.P. Large-scale characterization of HeLa cell nuclear phosphoproteins..Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 12130-12135Crossref PubMed Scopus (1239) Google Scholar), we show the first large-scale proteomic profiling of phosphorylation sites from primary animal tissue. These methods promise to greatly enrich our global view of the dynamic changes of phosphoproteins during brain development and may be applied to a variety of primary tissues or comparative states in cultured cells. A timed pregnant Swiss Webster mouse was obtained from Taconic (Germantown, New York). Developing forebrains and midbrains were dissected from embryos at day 16.5 (E16.5). The tissue from four brains (10 mg) was dounce homogenized in 25 mm Tris pH 7.2, 137 mm NaCl, 10% glycerol, 1% Nonidet P-40, 25 mm NaF, 10 mm Na2P2O7, 1 mm Na3VO4, 1 mm DTT, 1 mm PMSF, 10 μg/ml leupeptin, and 1% aprotinin. Cleared extracts were boiled in bromphenol blue sample buffer (150 mm Tris pH 6.8, 2% SDS, 5% β-mercaptoethanol, 7.8% glycerol), and 6 mg of extract was loaded onto a hand-poured, 7.5–20% gradient SDS-polyacrylamide (37.5:1 acrylamide:bis-acrylamide) preparative gel (see Fig. 2A). The Coomassie blue-stained gel was cut into four regions and then diced into 1-mm cubes. The gel pieces were washed with water and further destained with 50% ACN, 50 mm NH4HCO3 pH 8.5. Gel slices were dehydrated with ACN, dried, and subjected to in-gel digestion with sequencing-grade modified trypsin (12.5 ng/μl; Promega, Madison, WI) in 50 mm NH4HCO3 overnight at 37 °C. Peptides were extracted with 50% ACN, 5% formic acid (FA) and dried. Peptides from each gel region were resuspended in 500 μl of SCX solvent A (5 mm KH2PO4 pH 2.7, 33% ACN). Then 400 μl of each peptide mixture were resolved by SCX chromatography on a polysulphoethyl aspartimide (5 μm, 200 Å) column (3 × 200 mm; PolyLC Inc., Columbia, MD), against an increasing gradient of SCX solvent B (5 mm KH2PO4 pH 2.7, 33% ACN, 350 mm KCl) as shown in Fig. 2, B and C. Fractions were collected every minute with a flow rate of 400 μl/min. Each fraction was lyophilized and then desalted by resuspending peptides in 0.5% TFA and loading them on a gel-loading tip column packed with 2 cm of OLIGO R3 (Applied Biosystems, Foster City, CA). After washing with 0.1% TFA, peptides were eluted from the column with 95% ACN and 0.4% FA and dried. For SCX chromatography of peptide standards shown in Fig. 1B, 200 pmol of synthetic peptides IGEGTYGVVYK with or without one phosphorylated residue at the underlined threonine or tyrosine residue (kind gifts of J. Rush, Cell Signaling Technology, Beverly MA) were analyzed using SCX chromatography. Note that “solution charge state” is used throughout when referencing peptide separation using SCX chromatography and should not be confused with the charge state of ions in the gas phase. Following desalting, individual SCX fractions from each gel region were resuspended in 0.5% FA and subjected to LC-MS/MS runs. Samples were loaded using an autosampler onto a microcapillary column (100 μm × 12 cm) packed with reverse phase MagicC18 material (5 μm, 200 Å; Michrom Bioresources, Inc., Auburn, CA). Elution was achieved with a 5–35% ACN (0.1% FA) gradient over 100 min, after a 20-min isocratic loading at 0% ACN, 0.5% FA. Mass spectra were acquired on a LCQ-Deca XP (Thermo Electron, Woburn, MA) over the entire run using eight MS/MS scans following each survey scan. Approximately 3,000 sequencing events were performed for each run. Raw data were searched for fully tryptic peptides against the NCBI nonredundant mouse database using Sequest software, permitting a dynamic modification of 80 Da on serine, threonine, and tyrosine residues and a mass allowance of 2 Da. Database matches were filtered for XCorr values higher than 2.5 and 3.3 for doubly and triply charged ions, respectively. Resulting sequences were inspected manually and validated with the assistance of in-house software to assign prominent peaks unmatched by Sequest such as doubly charged ions, neutral losses, and the same with accompanying water losses (see Supplemental Fig. 1). As the vast majority of phosphopeptides identified showed significant loss of phosphoric acid, they can be distinguished from sulfonation, which also imparts a mass addition of 80 Da to serine, threonine, and tyrosine residues (9Medzihradszky K.F. Darula Z. Perlson E. Fainzilber M. Chalkley R.J. Ball H. Greenbaum D. Bogyo M. Tyson D.R. Bradshaw R.A. Burlingame A.L. O-sulfonation of serine and threonine: Mass spectrometric detection and characterization of a new posttranslational modification in diverse proteins throughout the eukaryotes..Mol. Cell. Proteomics. 2004; 3: 429-440Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). When the exact site of phosphorylation could not be assigned for a given phosphopeptide, it was tabulated as ambiguous. Motif analysis of identified phosphorylation sites was performed using “phosphomotif” software (deer.med.harvard.edu/dan/phosmotif.html). SCX chromatography separates peptide ions based on solution charge state (resulting from protonation/deprotanation of basic and acidic groups). As much as 68% of an in silico tryptic digest of the human NCBI protein database generates peptides with a predicted solution charge state of (+)2 at pH 2.7, and fewer than 3% with a solution charge state of less than (+)2 (8Beausoleil S.A. Jedrychowski M. Schwartz D. Elias J. Villen J. Li J. Cohn M. Cantely L.C. Gygi S.P. Large-scale characterization of HeLa cell nuclear phosphoproteins..Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 12130-12135Crossref PubMed Scopus (1239) Google Scholar). Phosphorylation at serine, threonine, or tyrosine reduces the solution charge state of peptides at pH 2.7, allowing (+)2 phosphopeptides to be enriched into a far less complex fraction (Fig. 1). This greatly increases the probability of their favorable analysis by MS. These properties were applied toward the identification of phosphorylation events occurring during mouse brain development. As the relative abundance of rare mRNA species in mammalian cells represents as much as 95% of unique message expressed per cell (10Hastie N.D. Bishop J.O. The expression of three abundance classes of messenger RNA in mouse tissues..Cell. 1976; 9: 761-774Abstract Full Text PDF PubMed Scopus (302) Google Scholar), we reasoned that milligram amounts of protein were required to successfully identify phosphorylation events achieving as high as 10% stoichiometry (Supplemental Table I). Six milligrams of embryonic brain extract were separated on a preparative polyacrylamide gel. The gel was cut into four large regions, and each region was subjected to in-gel digestion with trypsin (Fig. 2A). Extracted peptides from each region were separated by SCX chromatography at pH 2.7 (Fig. 2, B and C), desalted, and analyzed by reverse-phase LC-MS/MS. More than 250,000 MS/MS spectra were acquired while analyzing the first 40% of SCX fractions from all four gel regions. Following interrogation of the nonredundant NCBI murine database using the Sequest algorithm, results were conservatively filtered by requiring top-hit phosphopeptides to have XCorr values of more than 2.5 and 3.3 for doubly and triply charged ions, respectively. All spectra passing these criteria were manually examined to explain intense (>15% of the most intense peak) ions left unexplained by conventional b- and y-type ions. We found manual evaluation an essential step for proper identification of phosphorylation sites due to the frequent unassigned loss of phosphoric acid and ambiguity found between top-hit peptides containing multiple serine and threonine residues, represented by low ΔCorr values (see Supplemental Fig. 1). After validation we identified 460 unique phosphorylation sites and 86 more for which the precise site of phosphorylation was ambiguous. This entire dataset is provided as Supplemental Table II and as well on our laboratory website with interactive Sequest links at gygi.med.harvard.edu/pubs/brain/PhosphoBrain.xls. To evaluate the relative enrichment of phosphopeptides afforded by SCX chromatography, we plotted the number of phosphopeptides and nonphosphorylated peptides (many of which were carboxyl termini) identified from gel region 1. Intriguingly, we noticed two distinct peaks of phosphopeptides eluting in the early SCX fractions. These peaks represented phosphopeptides to nonphosphorylated peptides ratios of 8:1 and 5:1, respectively, and this ratio tapered quickly to 0:260 in the last SCX fractions analyzed by MS (Fig. 3A). To determine if the two peaks of phosphopeptides were the result of a distinct separation of solution charge states by SCX, we plotted the expected solution charge states for the phosphopeptides from the entire dataset as a function of the SCX fraction from which they were derived. In excellent agreement with theoretical elution patterns, we determined that the two peaks represent phosphopeptides with a predicted net solution charge of 0 and (+)1, respectively. Additionally, a minor peak with a net solution charge of (–)1 was observed in the earliest fractions. All phosphopeptides with net solution charges of less than (+)1 were the result of multiple phosphorylation events and/or phosphorylation of carboxyl termini. These peptides had little to no retention on the SCX column, owing to disproportionate charge distribution along the lengths of the peptides. Protein kinases target substrates based on primary sequence proximal to the serine, threonine, or tyrosine residue(s) they phosphorylate (11Songyang Z. Lu K.P. Kwon Y.T. Tsai L.H. Filhol O. Cochet C. Brickey D.A. Soderling T.R. Bartleson C. Graves D.J. DeMaggio A.J. Hoekstra M.F. Blenis J. Hunter T. Cantley L.C. A structural basis for substrate specificities of protein Ser/Thr kinases: Primary sequence preference of casein kinases I and II, NIMA, phosphorylase kinase, calmodulin-dependent kinase II, CDK5, and Erk1..Mol. Cell. Biol. 1996; 16: 6486-6493Crossref PubMed Scopus (490) Google Scholar). There are over 500 protein kinases (12Manning G. Whyte D.B. Martinez R. Hunter T. Sudarsanam S. The protein kinase complement of the human genome..Science. 2002; 298: 1912-1934Crossref PubMed Scopus (6315) Google Scholar), many of which share overlapping sequence specificities. To provide a preliminary affiliation between kinases with known target sequences and the phosphorylation sites in our database, we queried our dataset with 11 different phosphorylation motifs and tallied the results (Fig. 4A). In addition to proline-directed (p(S/T)P) phosphorylation sites, both phosphorylation sites upstream of acidic or downstream of basic residues constituted a large fraction of the dataset (Fig. 4, A–C). Basophilic kinases capable of phosphorylating a serine residue lying three or four residues downstream of arginine and two residues upstream of proline (RXXpSXP or RXXXpSXP) a potential binding site for the of K. S. A. H. Cantley L.C. The structural basis for binding 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). This is of as loss of a of this to in neuronal migration during brain development in mice (4Toyo-oka K. Shionoya A. Gambello M.J. Cardoso C. Leventer R. Ward H.L. Ayala R. Tsai L.H. Dobyns W. Ledbetter D. Hirotsune S. Wynshaw-Boris A. 14-3-3ε is important for neuronal migration by binding to NUDEL: A molecular explanation for Miller-Dieker syndrome..Nat. Genet. 2003; 34: 274-285Crossref PubMed Scopus (343) Google Scholar) and is to the of from genetic loss of gene encoding in (4Toyo-oka K. Shionoya A. Gambello M.J. Cardoso C. Leventer R. Ward H.L. Ayala R. Tsai L.H. Dobyns W. Ledbetter D. Hirotsune S. Wynshaw-Boris A. 14-3-3ε is important for neuronal migration by binding to NUDEL: A molecular explanation for Miller-Dieker syndrome..Nat. Genet. 2003; 34: 274-285Crossref PubMed Scopus (343) Google Scholar). A of our dataset for phosphorylation sites these two of binding identified 10 phosphopeptides (Fig. one of these was in a protein to interact with the sites of were not C. of over 200 human phosphoproteins new links to of and J. 2004; PubMed Scopus Google Scholar). was in signaling downstream of D. A. Cooper J.A. of a in Reelin-stimulated Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), a essential for proper brain development G. S.C. T. A protein to proteins in the mouse PubMed Scopus Google Scholar). these phosphoproteins were to interact with in a the binding be expected to show high in the of from the As an of the of the protein sequences containing these phosphopeptides were analyzed by against both the nonredundant protein and NCBI were sequence homology to of the animal could be of the binding motifs are shown for 2, and (Fig. for all potential proteins are provided in Supplemental Fig. CID spectra are shown for the more phosphorylation sites identified in 2 and (Fig. Large-scale studies have been by the to methods to enrich for low-abundance phosphoproteins and Recent advances in enrichment are large-scale phosphorylation site Here, using SCX chromatography to enrich for tryptic we the identification of over 500 phosphorylation sites from the developing mouse brain. enrichment promise toward to an global profiling of phosphorylation sites in cells and their with enrichment by SCX to a global phosphopeptides an solution charge at low pH (Fig. their enrichment by SCX chromatography. Additionally, enrichment is when using tryptic and a number of tryptic peptides are or for MS/MS SCX enrichment of phosphopeptides may also be by that a charge at low pH or trypsin due to the relative of basic residues to proline or modified of the enrichment the significant in abundance generates the known of tyrosine phosphorylation in numerous brain one tyrosine phosphorylation site was identified in this (see 1 in Supplemental Table As are also enriched in early SCX fractions (see Fig. this that phosphopeptides are the of detection when using mg of These results are with the of Hunter T. K. W. that the phosphorylation of tyrosine is essential for by 20: Full Text PDF PubMed Scopus Google Scholar) or its low phosphorylation stoichiometry Cooper J.A. of protein tyrosine kinase signaling by substrate during brain Cell. Biol. 2003; PubMed Scopus Google Scholar). studies may higher amounts of material and/or with the important of 14-3-3ε in the developing brain (4Toyo-oka K. Shionoya A. Gambello M.J. Cardoso C. Leventer R. Ward H.L. Ayala R. Tsai L.H. Dobyns W. Ledbetter D. Hirotsune S. Wynshaw-Boris A. 14-3-3ε is important for neuronal migration by binding to NUDEL: A molecular explanation for Miller-Dieker syndrome..Nat. Genet. 2003; 34: 274-285Crossref PubMed Scopus (343) Google Scholar), we searched our dataset for phosphorylation events potential We identified eight 1 and two 2 motifs K. S. A. H. Cantley L.C. The structural basis for binding 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) (Fig. of these motifs and their animal B and Supplemental Fig. showed to of potential biological For the identified in to the R. J. phosphorylation of murine Google Scholar), strong is for all residues from to with the of the phosphorylated serine residue to in The identified in 2, in the of the in J.A. H. P. The a of proteins that interact with of the and a new protein Biol. Full Text Full Text PDF PubMed Scopus Google Scholar), was and showed much higher than The found in the is and is to a region M. K. A with the Src homology of protein via a binding Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, S. C. T. a human J. 1998; PubMed Scopus Google Scholar). the in 2, it is with much less analyses performed on phosphorylation be important when phosphorylation site in specific classes of The and complex regulatory of protein phosphorylation an and challenging for proteomic Here, using for the enrichment of we the first large-scale analysis of primary animal tissue. The further of these and the use of high and provide an global profiling of the unique of phosphorylation sites occurring mammalian brain development. We and D. for to mice and and and of the Gygi laboratory for important and with