Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Biochemical Fractionation and Stable Isotope Dilution Liquid Chromatography-mass Spectrometry for Targeted and Microdomain-specific Protein Quantification in Human Postmortem Brain Tissue

2012; Elsevier BV; Volume: 11; Issue: 12 Linguagem: Inglês

10.1074/mcp.m112.021766

ISSN

1535-9484

Autores

Matthew L. MacDonald, Eugene Ciccimaro, Amol Prakash, Anamika Banerjee, Steven H. Seeholzer, Ian A. Blair, Chang-Gyu Hahn,

Tópico(s)

Neuroscience and Neuropharmacology Research

Resumo

Synaptic architecture and its adaptive changes require numerous molecular events that are both highly ordered and complex. A majority of neuropsychiatric illnesses are complex trait disorders, in which multiple etiologic factors converge at the synapse via many signaling pathways. Investigating the protein composition of synaptic microdomains from human patient brain tissues will yield valuable insights into the interactions of risk genes in many disorders. These types of studies in postmortem tissues have been limited by the lack of proper study paradigms. Thus, it is necessary not only to develop strategies to quantify protein and post-translational modifications at the synapse, but also to rigorously validate them for use in postmortem human brain tissues. In this study we describe the development of a liquid chromatography-selected reaction monitoring method, using a stable isotope-labeled neuronal proteome standard prepared from the brain tissue of a stable isotope-labeled mouse, for the multiplexed quantification of target synaptic proteins in mammalian samples. Additionally, we report the use of this method to validate a biochemical approach for the preparation of synaptic microdomain enrichments from human postmortem prefrontal cortex. Our data demonstrate that a targeted mass spectrometry approach with a true neuronal proteome standard facilitates accurate and precise quantification of over 100 synaptic proteins in mammalian samples, with the potential to quantify over 1000 proteins. Using this method, we found that protein enrichments in subcellular fractions prepared from human postmortem brain tissue were strikingly similar to those prepared from fresh mouse brain tissue. These findings demonstrate that biochemical fractionation methods paired with targeted proteomic strategies can be used in human brain tissues, with important implications for the study of neuropsychiatric disease. Synaptic architecture and its adaptive changes require numerous molecular events that are both highly ordered and complex. A majority of neuropsychiatric illnesses are complex trait disorders, in which multiple etiologic factors converge at the synapse via many signaling pathways. Investigating the protein composition of synaptic microdomains from human patient brain tissues will yield valuable insights into the interactions of risk genes in many disorders. These types of studies in postmortem tissues have been limited by the lack of proper study paradigms. Thus, it is necessary not only to develop strategies to quantify protein and post-translational modifications at the synapse, but also to rigorously validate them for use in postmortem human brain tissues. In this study we describe the development of a liquid chromatography-selected reaction monitoring method, using a stable isotope-labeled neuronal proteome standard prepared from the brain tissue of a stable isotope-labeled mouse, for the multiplexed quantification of target synaptic proteins in mammalian samples. Additionally, we report the use of this method to validate a biochemical approach for the preparation of synaptic microdomain enrichments from human postmortem prefrontal cortex. Our data demonstrate that a targeted mass spectrometry approach with a true neuronal proteome standard facilitates accurate and precise quantification of over 100 synaptic proteins in mammalian samples, with the potential to quantify over 1000 proteins. Using this method, we found that protein enrichments in subcellular fractions prepared from human postmortem brain tissue were strikingly similar to those prepared from fresh mouse brain tissue. These findings demonstrate that biochemical fractionation methods paired with targeted proteomic strategies can be used in human brain tissues, with important implications for the study of neuropsychiatric disease. Synaptic architecture and its adaptive changes require numerous molecular events that are both highly ordered and complex (1Coba M.P. Pocklington A.J. Collins M.O. Kopanitsa M.V. Uren R.T. Swamy S. Croning M.D. Choudhary J.S. Grant S.G. Neurotransmitters drive combinatorial multistate postsynaptic density networks.Sci. Signal. 2009; 2: ra19Crossref PubMed Scopus (101) Google Scholar, 2Bard L. Groc L. Glutamate receptor dynamics and protein interaction: lessons from the NMDA receptor.Mol. Cell. Neurosci. 2011; 48: 298-307Crossref PubMed Scopus (69) Google Scholar). Molecular and cellular research into these processes has, until recently, been limited to technologies that can examine one molecule to the next within a cascade. In the last decade, mass spectrometry (MS)-based proteomic methodologies, paired with biochemical fractionation techniques, have enabled us to begin cataloging the proteomes and signaling of mammalian synaptic microdomains (1Coba M.P. Pocklington A.J. Collins M.O. Kopanitsa M.V. Uren R.T. Swamy S. Croning M.D. Choudhary J.S. Grant S.G. Neurotransmitters drive combinatorial multistate postsynaptic density networks.Sci. Signal. 2009; 2: ra19Crossref PubMed Scopus (101) Google Scholar, 3Hahn C.G. A road less travelled: Unpacking the complex traits of schizophrenia.Brain Res. Bull. 2010; 83: 85Crossref PubMed Scopus (1) Google Scholar, 4Bayés A. van de Lagemaat L.N. Collins M.O. Croning M.D. Whittle I.R. Choudhary J.S. Grant S.G. Characterization of the proteome, diseases and evolution of the human postsynaptic density.Nat. Neurosci. 2011; 14: 19-21Crossref PubMed Scopus (324) Google Scholar, 5Cheng D. Hoogenraad C.C. Rush J. Ramm E. Schlager M.A. Duong D.M. Xu P. Wijayawardana S.R. Hanfelt J. Nakagawa T. Sheng M. Peng J. Relative and absolute quantification of postsynaptic density proteome isolated from rat forebrain and cerebellum.Mol. Cell. Proteomics. 2006; 5: 1158-1170Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar, 6English J.A. Dicker P. Föcking M. Dunn M.J. Cotter D.R. 2-D DIGE analysis implicates cytoskeletal abnormalities in psychiatric disease.Proteomics. 2009; 9: 3368-3382Crossref PubMed Scopus (127) Google Scholar, 7Peng J. Kim M.J. Cheng D. Duong D.M. Gygi S.P. Sheng M. Semiquantitative proteomic analysis of rat forebrain postsynaptic density fractions by mass spectrometry.J. Biol. Chem. 2004; 279: 21003-21011Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar) and microdomain specific protein complexes (8Husi H. Ward M.A. Choudhary J.S. Blackstock W.P. Grant S.G. Proteomic analysis of NMDA receptor-adhesion protein signaling complexes.Nat. Neurosci. 2000; 3: 661-669Crossref PubMed Scopus (1025) Google Scholar, 9Dosemeci A. Makusky A.J. Jankowska-Stephens E. Yang X. Slotta D.J. Markey S.P. Composition of the synaptic PSD-95 complex.Mol. Cell. Proteomics. 2007; 6: 1749-1760Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 10Fernández E. Collins M.O. Uren R.T. Kopanitsa M.V. Komiyama N.H. Croning M.D. Zografos L. Armstrong J.D. Choudhary J.S. Grant S.G. Targeted tandem affinity purification of PSD-95 recovers core postsynaptic complexes and schizophrenia susceptibility proteins.Mol. Syst. Biol. 2009; 5: 269Crossref PubMed Scopus (201) Google Scholar). These studies have revealed that synaptic architecture and plasticity involve numerous interactions between multiple signaling mechanisms with robust crosstalk and protein interactions (8Husi H. Ward M.A. Choudhary J.S. Blackstock W.P. Grant S.G. Proteomic analysis of NMDA receptor-adhesion protein signaling complexes.Nat. Neurosci. 2000; 3: 661-669Crossref PubMed Scopus (1025) Google Scholar, 9Dosemeci A. Makusky A.J. Jankowska-Stephens E. Yang X. Slotta D.J. Markey S.P. Composition of the synaptic PSD-95 complex.Mol. Cell. Proteomics. 2007; 6: 1749-1760Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 11Grant S.G. Targeted TAP tags, phosphoproteomes and the biology of thought.Expert Rev. Proteomics. 2010; 7: 169-171Crossref PubMed Scopus (1) Google Scholar, 12Trinidad J.C. Thalhammer A. Specht C.G. Lynn A.J. Baker P.R. Schoepfer R. Burlingame A.L. Quantitative analysis of synaptic phosphorylation and protein expression.Mol. Cell. Proteomics. 2008; 7: 684-696Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). These findings also dovetail with decades of genetic studies into neuropsychiatric disease, which have identified dozens of risk genes spread across multiple pathways at the synapse (13Keller M.A. Gwinn K. Nash J. Horsford J. Zhang R. Rich S.S. Corriveau R.A. Whole genome association studies of neuropsychiatric disease: An emerging era of collaborative genetic discovery.Neuropsychiatr. Dis. Treat. 2007; 3: 613-618PubMed Google Scholar, 14Uhl G.R. Grow R.W. The burden of complex genetics in brain disorders.Arch. Gen. Psychiatry. 2004; 61: 223-229Crossref PubMed Scopus (144) Google Scholar). Historically, signaling cascades and molecular disease models have been depicted as a string of molecular events that are connected in tandem. Such views were shaped in part by the methodologies that permitted the measure of a handful of protein events at a time. MS-based proteomic methodologies can monitor numerous proteins and post-translational modifications simultaneously, permitting us to examine signaling pathways in the context of many other intracellular molecular events. The next challenge in the field of neuroscience will be to establish methodologies for quantitative assessment of proteomes in specific microdomains of neural tissues, to monitor numerous molecular events simultaneously, and to understand them within the context of nonlinear intracellular trafficking, protein interactions and post-translational modifications. Postmortem brain studies are a critical component of neuropsychiatric research as brain tissues of patients may harbor pathophysiologic information of the illnesses. A majority of neuropsychiatric illnesses are complex trait disorders in which multiple etiologic factors converge at the synapse via many signaling pathways (13Keller M.A. Gwinn K. Nash J. Horsford J. Zhang R. Rich S.S. Corriveau R.A. Whole genome association studies of neuropsychiatric disease: An emerging era of collaborative genetic discovery.Neuropsychiatr. Dis. Treat. 2007; 3: 613-618PubMed Google Scholar, 14Uhl G.R. Grow R.W. The burden of complex genetics in brain disorders.Arch. Gen. Psychiatry. 2004; 61: 223-229Crossref PubMed Scopus (144) Google Scholar). The application of advanced molecular and cellular technologies to access synaptic microdomains, however, has been limited in postmortem brains by confounds such as therapeutic intervention, agonal state, and postmortem interval. Thus, there is a pressing need to validate and focus these biochemical fractionation and MS proteomic methods for use with human postmortem brain tissue. The vast majority of previous neuroproteomic studies have employed two dimensional separation/tandem MS approaches to perform qualitative analyses of mammalian synaptic preparations (3Hahn C.G. A road less travelled: Unpacking the complex traits of schizophrenia.Brain Res. Bull. 2010; 83: 85Crossref PubMed Scopus (1) Google Scholar, 7Peng J. Kim M.J. Cheng D. Duong D.M. Gygi S.P. Sheng M. Semiquantitative proteomic analysis of rat forebrain postsynaptic density fractions by mass spectrometry.J. Biol. Chem. 2004; 279: 21003-21011Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar, 9Dosemeci A. Makusky A.J. Jankowska-Stephens E. Yang X. Slotta D.J. Markey S.P. Composition of the synaptic PSD-95 complex.Mol. Cell. Proteomics. 2007; 6: 1749-1760Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Protein quantification has been accomplished using either synthetic stable isotope-labeled peptide standards (5Cheng D. Hoogenraad C.C. Rush J. Ramm E. Schlager M.A. Duong D.M. Xu P. Wijayawardana S.R. Hanfelt J. Nakagawa T. Sheng M. Peng J. Relative and absolute quantification of postsynaptic density proteome isolated from rat forebrain and cerebellum.Mol. Cell. Proteomics. 2006; 5: 1158-1170Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar) or isobaric peptide labeling methods, such as Isobaric tags for relative and absolute quantitation (12Trinidad J.C. Thalhammer A. Specht C.G. Lynn A.J. Baker P.R. Schoepfer R. Burlingame A.L. Quantitative analysis of synaptic phosphorylation and protein expression.Mol. Cell. Proteomics. 2008; 7: 684-696Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 15Li K. Hornshaw M.P. van Minnen J. Smalla K.H. Gundelfinger E.D. Smit A.B. Organelle proteomics of rat synaptic proteins: Correlation-profiling by isotope-coded affinity tagging in conjunction with liquid chromatography-tandem mass spectrometry to reveal post-synaptic density specific proteins.J. Proteome Res. 2005; 4: 725-733Crossref PubMed Scopus (70) Google Scholar). Sample workup can complicate the application of isobaric tagging in an efficient and reproducible manner, (16Ishihama Y. Sato T. Tabata T. Miyamoto N. Sagane K. Nagasu T. Oda Y. Quantitative mouse brain proteomics using culture-derived isotope tags as internal standards.Nat. Biotechnol. 2005; 23: 617-621Crossref PubMed Scopus (195) Google Scholar) whereas use of synthetic peptide standards is costly and can lead to difficulty controlling for confounds in sample preparation, most notably protein digestion (17Brun V. Dupuis A. Adrait A. Marcellin M. Thomas D. Court M. Vandenesch F. Garin J. Isotope-labeled protein standards: Toward absolute quantitative proteomics.Mol. Cell. Proteomics. 2007; 6: 2139-2149Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar). Stable isotope labeling by amino acids in cell culture (SILAC) 1The abbreviations used are:SILACstable isotope labeling by amino acids in cell culture[13C6]brain ISTD[13C6]lysine-labeled brain proteome internal standard ([13C6]brain ISTD)ANOVAanalysis of varianceCVcoefficient of variationFWHMfull width at half maximumLC-SRM/MSliquid chromatography-selected reaction monitoring/mass spectrometryPMIpostmortem intervalPSDpostsynaptic densityRPMrotations per minuteSRMselected reaction monitoring. 1The abbreviations used are:SILACstable isotope labeling by amino acids in cell culture[13C6]brain ISTD[13C6]lysine-labeled brain proteome internal standard ([13C6]brain ISTD)ANOVAanalysis of varianceCVcoefficient of variationFWHMfull width at half maximumLC-SRM/MSliquid chromatography-selected reaction monitoring/mass spectrometryPMIpostmortem intervalPSDpostsynaptic densityRPMrotations per minuteSRMselected reaction monitoring. can produce only a partially labeled neural proteome (18Spellman D.S. Deinhardt K. Darie C.C. Chao M.V. Neubert T.A. Stable isotopic labeling by amino acids in cultured primary neurons: Application to brain-derived neurotrophic factor-dependent phosphotyrosine-associated signaling.Mol. Cell. Proteomics. 2008; 7: 1067-1076Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) and we have found that full expression levels and synaptic architecture of brain tissue are difficult to reproduce in fully labeled synaptic cultures. The recent availability of stable isotope labeling in mammals (SILAM) mouse tissue allows for the generation of a stable isotope labeled neuroproteome within the context of native tissues to serve as an internal standard for the quantification of a vast number of proteins (19Zanivan S. Krueger M. Mann M. In vivo quantitative proteomics: The SILAC mouse.Methods Mol. Biol. 2012; 757: 435-450Crossref PubMed Scopus (71) Google Scholar). Walther et al. recently used SILAM tissue to quantify over 4000 proteins in mouse hippocampus and cortex (20Walther D.M. Mann M. Accurate quantification of more than 4000 mouse tissue proteins reveals minimal proteome changes during aging.Mol. Cell. Proteomics. 2011; 10 (M110.004523)Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Considering the high level of homology between mammalian neuronal transcripts, we investigated the suitability of this stable isotope-labeled proteome to serve as a protein standard for human tissue as well as additional model organisms. stable isotope labeling by amino acids in cell culture [13C6]lysine-labeled brain proteome internal standard ([13C6]brain ISTD) analysis of variance coefficient of variation full width at half maximum liquid chromatography-selected reaction monitoring/mass spectrometry postmortem interval postsynaptic density rotations per minute selected reaction monitoring. stable isotope labeling by amino acids in cell culture [13C6]lysine-labeled brain proteome internal standard ([13C6]brain ISTD) analysis of variance coefficient of variation full width at half maximum liquid chromatography-selected reaction monitoring/mass spectrometry postmortem interval postsynaptic density rotations per minute selected reaction monitoring. In this study we first describe the development and validation of liquid chromatography-selected reaction monitoring (LC-SRM)/MS-based methodology for studies of mammalian synapses. Quantification of 189 neuronal proteins was conducted using a [13C6]lysine-labeled brain proteome internal standard ([13C6]-brain ISTD) prepared from [13C6]lysine SILAM mouse brain tissue. We also report the application of this method to further validate biochemical fractionation of human postmortem brain tissue for the study of synaptic biology in human disease. We observed highly reproducible enrichment of proteins into synaptic microdomains in a function- and family-specific manner. These protein enrichments were remarkably comparable between fresh-frozen mouse and human postmortem tissues. Additionally, bioinformatic analyses indicate that this method has the capability to quantify thousands of additional neuronal proteins in many model systems. These experiments demonstrate that the biochemical fractionation coupled with LC-SRM/MS and [13C6]-brain ISTDs provides a powerful tool for investigating the protein composition of synaptic microdomains in mammalian tissues, with broad applications to neuropathology and neurological studies in humans and model systems. Prefrontal cortex (Fig. 1B) tissue slices from three cognitively and neurpathologically normal subjects were obtained from the University of Pennsylvania (UPenn) Brain Bank. Consent for tissue collection was granted prospectively by patients. Autopsy consent was granted by the next-of-kin at time of death. Postmortem enrollment, brain autopsy, and neuropathological assessment were conducted under UPenn Conte Core A, IRB numbers 703835 and 188200. After death, the cases were stored at 2–4 °C until transport to UPenn, where all autopsies were performed. Upon sagittal bisection, a hemisphere from each case was cut into coronal slabs, which were frozen overnight at −80 °C and then sealed in plastic bags for long-term storage at −80 °C. Tissue samples from the other hemisphere were fixed and prepared for microscopic analysis. The cases were examined by trained neuropathologists for gross and microscopic abnormalities diagnostic of diverse neurodegenerative dementias. We selected fresh frozen tissue from the prefrontal cortex because its large volume allowed repeated sampling without depleting resources available for other studies on precious normal human brain tissue. Within this restriction, we selected tissues with a wide range in age, sex, and postmortem interval (PMI) (Fig. 1B). Mouse brain tissues consisted of the forebrain plus midbrain olfactory bulb, were dissected from three male C57/BL6 mice 8–12 months old. Synaptic fractions were obtained using a biochemical method we previously validated for use in human postmortem brain tissue (Fig. 1A).(3) 300–350 mg gray matter was homogenized in 1.5 ml solution A (0.32 m sucrose, 1 mm MgCl2, and 0.1 mm CaCl2) with a Teflon pestle. Approximately 100 μl of the homogenate was saved, solubilized with 1% SDS, and clarified by centrifugation. The remaining homogenate was centrifuged at 2500 rotations per minute (RPM) for 15 min. The pellet was discarded and 100 μl of the supernatant agitated on a rocker at 4 °C with a final concentration of 0.5% digitonin, 0.2% sodium cholate, and 0.5% Nonidet P-40 for 1 h, centrifuged at 14,000 RPM for 20 min, and the supernatant was saved as the synaptosomal fraction. The remainder of the supernatant prepared from the 2500 RPM spin was adjusted to 1.25 m sucrose with 2 m sucrose and 1 mm CaCl2 to a final volume of 5 ml. Five ml of 1 m sucrose was overlaid and the gradient ultracentrifuged at 28,000 rpm for 3 h in a SW 40 Ti rotor using a Beckmann L7 Ultracentrifuge. The band at the interface was collected, diluted 1:10 in 0.1 mm CaCl2 and centrifuged at 12,000 RPM for 20 min. The supernatant was discarded and the pellet, the intermediate membrane fraction, was dissolved in 1 ml 20 mm Tris pH 6.0 and sonicated with three 10 s pulses. One ml of 20 mm Tris pH 6.0 with 2% Triton X-100 (Sigma) was added to the solution and agitated on a rocker at 4 °C for 30 min, followed by centrifugation at 18,000 RPM for 30 min. The supernatant, the Vesicular fraction (V), was precipitated in acetone at –20°C overnight. The pellet was air dried, dissolved in 1 ml 20 mm Tris pH 8.0, sonicated with three 10-s pulses, mixed with 1 ml of 20 mm Tris pH 8.0 with 2% Triton X-100, agitated on a rocker at 4 °C for 45min, and centrifuged at 36,000 RPM for 1 h. The resulting supernatant, the parasynaptic membrane fraction, was precipitated in acetone at –20°C overnight. The pellet, the postsynaptic density (PSD) fraction, was air dried, washed three times with 20 mm Tris pH 8.0 and dissolved in 300 μl 20 mm Tris pH 7.4 with 1% SDS. Following acetone precipitation, the vesicular and parasynaptic fractions were centrifuged at 3000 RPM for 30 min and dissolved in 100 μl 20 mm Tris pH 7.4 with 1% SDS. To prepare the [13C6]brain ISTD, 400 mg labeled MouseExpress brain tissue (Cambridge Isotopes, Cambridge, MA) was homogenized in 4 ml buffer A as described above, centrifuged at 1000 × g for 10 min to clarify, agitated on a rocker at 4 °C for 30 min, and centrifuged at 10,000 × g for 20 min. The pellet, a crude membrane fraction, was dissolved in 2 ml 20 mm Tris pH 7.4 with 1% SDS. Total protein in all preparations was quantified with the micro BCA assay (Pierce, Waltham, MA) and all solutions were supplemented with protease and phosphate inhibitor cocktails (Sigma) as well as 1 mm sodium fluoride and sodium orthovanadate (Sigma). Synaptic preparations were mixed with the [13C6]brain ISTD (1 μg/μl) at a ratio of 2:1(μg/μg) and processed for MS analysis as described in Cheng et al. 2006. Briefly, 40 μl preparations were heated with lithium dodecyl sulfate (LDS) (Invitrogen, Carlsbad, CA) buffer at 95 °C for 20 min, separated on a 1.5 mm 4–12% Bis-Tris Gel (Invitrogen), cut into three fractions for LC-SRM/MS and five for LC-MS/MS (Figs. 1C and 1D), chopped into ∼2 mm cubes, washed in 200 μl 50% acetonitrile (ACN) containing 25 mm NH4HCO3, reduced in 300 μl 10 mm dithiothreitol, alkylated in 300 μl 55 mm iodoacetamide (Sigma), and digested with 80–120 μl trypsin (.025 μg/μl) (Promega) overnight at 37 °C, so that the amount of trypsin was 1:5 of the total protein to be digested by mass. Peptides were recovered from gel cubes into 200 μl 50/50 H2O/ACN with 3% formic acid by vortex-mixing and sonication for 20 min each, twice. Samples were then evaporated to a 100 μl volume, brought to 1 ml in H20 with 0.1% formic acid, desalted with Oasis® HLB cartridges (Waters, Milford, MA), evaporated almost to completion and suspended in ∼45 μl H2O with 0.1% formic acid, and filtered with 0.22 μm Ultra free-MC filter cartridges (Millipore). To confirm that light/heavy peptide SRM measures were within linear range, a dilution curve was prepared by varying the ratio of a human total homogenate fraction/[13C6]brain ISTD from 0.05 to 19. Five mixtures were prepared, (μg homogenate/μg [13C6]brain ISTD): 1/19, 3/17. 10/10, 17/3 and 19/1. These preparations were analyzed by LC-SRM/MS in duplicate. For LC-SRM/MS analyses, all peptide preparations were run on a TSQ Vantage triple stage quadrupole mass spectrometer (ThermoFisher Scientific) with an Eksigent 2Dnano LC (Eksigent) and a CaptiveSpray source (Michrom). Five μl (∼2.5 μg protein) sample was loaded on to a 3μ 200A 1 × 150 mm Magic C18 column (Michrom) at 1 μl/min for 12 min, and eluted at 750 nl/min over a 25 min gradient from 3–35% mobile phase B (ACN containing 0.1% formic acid). SRM transitions were timed using 1–1.5 min retention windows, depending on the number of SRMs to be assayed. Transitions were monitored, allowing for a cycle time of 1 s, resulting in a dynamic dwell time, never falling below 10 msec. The MS instrument parameters were as follows: capillary temperature 275οC, spray voltage 1100 V, and a collision gas of 1.4 mTorr (argon). The resolving power of the instrument was set to 0.7 Da full width at half maximum (FWHM) for Q1 and Q3. Data were acquired using a chrom filter peak width of 4.0 s. LC-data dependant -MS/MS analyses were conducted on a Q Exactive (ThermoFisher Scientific) quadrupole orbitrap hybrid instrument with an Easy nLC-II (ThermoFisher Scientific) nano-pump/autosampler. Three μl peptide sample (∼1 ug) was loaded and resolved on a 20 cm × 75 μm proteopepII C18 packed tip column over a 90 min gradient at a flow rate of 350 nl/min, 0–35% mobile phase B (ACN, 0.1 formic acid). A data-dependent top 10 method was used to acquire a 70,000 resolution full scan to trigger ten 17,500 resolution HCD scans. Ions were isolated for MS/MS analysis with a 2.0 Da window on the quadrupole. Average cycle times were 1.2 s. Each sample was analyzed in triplicate. Raw files from triplicate injections were searched together within Proteome Discoverer 1.3 (ThermoFisher Scientific) using SEQUEST and the human refseq. database (release 47, ftp://ftp.ncbi.nih.gov/refseq/H_sapiens/mRNA_Prot/) with 34,340 entries. Search parameters allowed trypsin to cleave after Lysine and Arginine and have two missed cleavages. Precursor ion mass tolerance was set to 15 ppm and fragment ion mass tolerance was 20 mmu. The dynamic modification of methionine (oxidation = 15.995 Da) and lysine (13C6 = 6.020 Da), in addition to the static modification of cysteine (carbamidomethylation = 57.021 Da) was accepted on up to four residues per peptide. Within the Proteome Discoverer Software, SILAC pairs were identified using the "2-plex" workflow node, and all peptides were rescored using the Percolator (21Käll L. Canterbury J.D. Weston J. Noble W.S. MacCoss M.J. Semi-supervised learning for peptide identification from shotgun proteomics datasets.Nat. Methods. 2007; 4: 923-925Crossref PubMed Scopus (1367) Google Scholar) algorithm node. Finally, peptides were filtered at 1% false discovery rate (FDR). Method development began with the selection of ∼300 synaptic proteins of interest from published multidimensional MS/MS analyses of mouse and human brain tissue biochemical fractions (27English J.A. Pennington K. Dunn M.J. Cotter D.R. The neuroproteomics of schizophrenia.Biol. Psychiatry. 2011; 69: 163-172Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 12Trinidad J.C. Thalhammer A. Specht C.G. Lynn A.J. Baker P.R. Schoepfer R. Burlingame A.L. Quantitative analysis of synaptic phosphorylation and protein expression.Mol. Cell. Proteomics. 2008; 7: 684-696Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar) as well as the LC-data dependant MS/MS analysis described in this study. RAW data from the two analysis of human tissue were searched in Proteome Discoverer™ 1.1 (ThermoFisher Scientific) using SEQUEST. Proteome Discoverer™ files were loaded into ProteinCenter™ (ThermoFisher Scientific) and sorted by function and subcellular location using Gene Ontology (GO) terms. Targets for inclusion in the LC-SRM/MS assay were selected with a bias toward well annotated synaptic proteins, such as glutamate receptors, kinases, phosphatases, vesicular fusion, amino acid metabolism, protein trafficking and scaffolding. Peptides for proteins of interest were then filtered based on the following criteria: 1) presence of lysine, 2) nonredundant to a selected protein or protein group (determined by BLAST search) and 3) 100% homology across mouse and human sequences (determined by BLAST search). Acceptable peptide sequences, along with MS2 spectra, were imported into Pinpoint™ (Thermo-Scientific). Initially, five mass transitions were selected for each target peptide and its "heavy" counter-part. Validation experiments were performed in 1:1 mixtures of human intermediate membrane fraction and the [13C6]brain ISTD. Pinpoint™ was used to visually and statistically validate SRM transitions. Ten male 8–12 month old C57/B6 mice were sacrificed by CO2 and cervical dislocation, per protocol, and placed at 4 °C. The brains were removed from the crania at 0, 4, 8, 12, and 16 h postsacrifice, separated from the cerebellum and olfactory bulbs, frozen on dry ice, stored at −80 °C and subject to biochemical fractionation. The PSD fractions were analyzed by LC-SRM/MS as described above. Raw files generated by LC-SRM/MS analysis were loaded into Pinpoint files containing target proteins/peptides/transitions. All individual SRM transitions and integration areas were inspected both manually and by Pinpoint. Pinpoint first identifies the peak based on co-elution of transitions, and then calculates the peak start and peak stop. This helps calculate the area under the curve for that transition. Summing this area for all transitions from the light gives the total area for the light peptides, and taking the ratio against the total area of the heavy peptide calculates the relative intensity with respect to the heavy internal standard. After computing the area under the peak for each transition for both the light and the heavy peptides, Pinpoint computes three scores, each of which help the user evaluate the quantitation fidelity. The

Referência(s)