unbiased proteomic study of the axons of cultured rat cortical neurons

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UNBIASED PROTEOMIC STUDY OF THE AXONS OF CULTURED RAT CORTICAL NEURONS Chih-Fan Chuang, Chih-En King, Bo-Wei Ho, Kun-Yi Chien, and Yen-Chung Chang J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00069 • Publication Date (Web): 10 Apr 2018 Downloaded from http://pubs.acs.org on April 11, 2018

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Journal of Proteome Research

UNBIASED PROTEOMIC STUDY OF THE AXONS OF CULTURED RAT CORTICAL NEURONS Chih-Fan Chuang1, Chih-En King2, Bo-Wei Ho2, Kun-Yi Chien3,4,* and Yen-Chung Chang2,5,* 1

Institute of Molecular Medicine, National Tsing Hua University, Hsinchu, Taiwan, Republic of China 2 Institute of Systems Neuroscience, National Tsing Hua University, Hsinchu, Taiwan, Republic of China 3 Department of Biochemistry and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan City, 33302, Taiwan 4

Clinical Proteomics Core Laboratory, Linkou Chang Gung Memorial Hospital, Taoyuan City, 33305, Taiwan 5 Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China *Corresponding authors

*Corresponding author: Dr. Yen-Chung Chang, Department of Life Science, National Tsing Hua University, 101 Kung-Fu Rd. Sec. 2, Hsinchu 300, Taiwan; telephone: 886-3-574-2754; e-mail: [email protected] *Corresponding author for MS analysis: Dr. Kun-Yi Chien, Department of Biochemistry and Molecular Biology, College of Medicine, Chang Gung University, 259 Wenhua 1st Rd., Guishan Dist., Taoyuan City 33302, Taiwan; telephone: 886-3-2118800; e-mail: [email protected]

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Abbreviations: CaMKIIα, Calcium, calmodulin-dependent protein kinase II α-subunit; DAPI, 4', 6-diamidino-2-phenylindole; DAVID, Database for annotation, visualization and integrated discovery; DIV, Days in vitro; ECM, Extracellular matrix; ER, Endoplasmic reticulum; FDR, False discovery rate; GO, Gene ontology; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; iBAQ, Intensity-based absolute quantification; LTQ, Linear trap quadrupole; MEM, Minimum essential medium; NB, Neurobasal; PLL, Poly-L-lysine; RP, Reversed phase; SCX, Strong cation exchange; SDS-PAGE, Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

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ABSTRACT The axon is a long projection connecting a neuron to its targets. Here, the axons of cultured rat cortical neurons were isolated with micro-patterned chips that enable the separation of axons from their cell bodies. Proteins extracted from isolated axons and whole neurons were subjected to analyses using two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) analyses without and with stable isotope dimethyl labeling, resulting in the identification of >2,500 axonal proteins and 103 axon-enriched proteins. A strong correlation exists between the abundances of axonal proteins and their counterparts in whole neurons. The proteomic results confirm the axonal protein constituents of the subcellular structures documented in earlier electron microscopic studies. Cortical axons have proteins that are components of machineries for protein degradation and the synthesis of soluble, membrane, and secretory proteins, although axons lack conventional Golgi apparatus. Despite the fact that axons lack nucleus, nuclear proteins were identified, and 67 of them were found enriched in axons. Some of the results obtained by the MS-based studies were validated by quantitative Western blotting and immunofluorescence staining analyses. The results represent the first comprehensive description of the axonal protein landscape. The MS proteomics data are available via ProteomeXchange with identifier PXD005527.

Keywords: Axon; Label-free quantification; Dimethyl-labeling quantitative proteomics

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INTRODUCTION The structure of a typical neuron consists of three major compartments, namely the cell body, the dendrites, and the axon. The axon is a long, slender process emitting from the cell body or the base of the dendrite of a neuron. The axon serves as a cable whereon action potentials propagate and a conduit wherein various bio-molecules and organelles travel bidirectionally.1 In early development, a highly dynamic growth cone at the tip leads an axon to migrate toward its target area under the guidance of various cues.2 Upon entering the target area, part of the axon differentiates into presynaptic terminals which form synapses with opposed postsynaptic processes derived from target cells. At synapses, signals between the neuron and its target are transmitted. Upon further developmental progress, the structure and transmission efficiency of synapses can be modulated in an activity-dependent fashion.3 The structure and function of the axon are sustained by the protein repertoire of this cellular compartment. Proteins residing in the axon can be synthesized in the cell body and in the axon.4-12 Most of the studies of proteins residing in the axon were performed by immunostaining using specific antibodies or by examining the expression of specific proteins with fluorescent tags. The proteome of axonal growth cones, a subdomain of the axon, was reported.13,14 However, a more complete study of the axon proteome and an understanding of the differences between the proteomes of the axon and the whole neuron are still lacking. Mass spectrometry (MS)-based proteomics technology has evolved into a powerful tool

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for global profiling and targeted analyses of proteins in a sample.15,16 MS-based proteomic technology has already been used widely in neuroscience research.17-21 To gain a systemic understanding of the protein constituents of axons, we performed proteomic analyses of the axons of cultured rat cortical neurons by two dimensional-liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS). Proteins of axons and whole neurons were further labeled with stable isotopic dimethyl groups before 2D-LC-MS/MS analysis (Fig. 1).22 The results identify proteins that can be found and a subset of these proteins that are enriched in the axon. In addition to axonal proteins constituting various organelles and cellular structures documented in earlier electron microscopic (EM) studies, proteins that are known to associate with the Golgi apparatus and nucleus, which are absent in the axon,23 were detected. Selected proteins that were identified with the MS-based studies were further validated by Western blotting and immunofluorescence staining analyses. Possible functions of axon-residing Golgi and nuclear proteins and the mechanisms shaping the axon proteome will also be discussed.

MATERIALS AND METHODS Preparation of axon and whole-cell samples from cultured rat cortical neurons

Pregnant

Sprague-Dawley (SD) rats were purchased from LASCO (BioLASCO Taiwan Co., Ltd, Taipei, Taiwan). All experimental procedures with rats in this study were performed according to the animal guidelines of the National Tsing Hua University Institutional Animal Care and Use

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Committee. Cells dissociated from the cortices of rat fetuses at embryonic day 18 (E18) were grown on micro-patterned glass chips (Fig. 1) at a density of 1,280 cells/mm2 on Regions 1 according to the procedures of Wu et al.24 The glass chips used had a poly-L-lysine (PLL) coated micro-pattern on the surface as shown in Fig. 1. The micro-pattern consisted of three parts: Regions 1 and 2 and lines connecting Regions 1 and 2. The cell bodies of neurons were confined to grow in Region 1. At DIV (day in vitro) 15, Regions 2 were fully occupied by axons migrated from Region 1 along the PLL-coated lines. After washing three times with phosphate-buffered saline (PBS) at 37°C, the chip was cleaved along the grooves pre-fabricated on the backside, resulting in the isolation of the cell body-occupied Region 1 and axonoccupied Region 2. The cells and axons in these regions were harvested separately in HEPES buffer (0.01% (w/v) SDS in 2 mM HEPES at pH 7.5) with a rubber scraper. Chips that were not seeded with cells were also subjected to the same cell-culture and cleavage procedures. Regions 1- and 2-containing fragments of these chips were isolated. Samples prepared from these chips were used as controls to determine background contamination. The average yields of axon and whole-cell proteins prepared from a chip were calculated to be 1.00 ± 0.12 (mean ± S.D., n = 3) and 27.31 ± 3.55 (mean ± S.D., n = 3) µg, respectively.

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Figure 1. Schematic outlining the approach to study the proteome of the rat cortical axons. Rat cortical neurons were cultured on the surface of glass chips containing a poly-L-lysinecoated micro-pattern on the surface. At DIV 15, the chip was cleaved along the grooves on the backside into fragments containing axons-occupied Region 2 and whole-cells-occupied Region 1. Proteins were extracted from these chip fragments, followed by reduction and alkylation, then digested exhaustively with trypsin. Resultant peptides were subjected to 2D-LC-MS/MS analysis and referred to as label-free experiments. The whole-cell and axon peptide samples were labeled with light and heavy formaldehyde, respectively, then mixed, and subjected to 2D-LC-MS/MS analysis, and referred to as dimethyl-labeling experiments. Alternatively, the whole-cell and axon peptides were labeled with heavy and light formaldehyde, then subjected to 2D-LC-MS/MS analysis, and referred to as swap experiments (indicated by broken lines). The mass spectrometry-based data of selected proteins were validated by immunofluorescence staining and Western blotting.

Immunofluorescence staining

Immunofluorescence staining of DIV 15 cultured rat

cortical neurons growing on the surface of glass chips was performed according to the procedures of Cheng et al.25 Neurons were fixed with 4% (v/v) paraformaldehyde in PBS at

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37°C for 30 min, then permeabilized with 0.05% (v/v) Triton X-100 in PBS for 15 min, and finally incubated with primary antibodies at 4°C overnight. Immunofluorescence staining of the axonal growth cones of DIV 9 neurons was performed by fixing the samples with 3.75% (v/v) paraformaldehyde plus 0.25% (v/v) glutaraldehyde in PBS at 37°C for 30 min. The samples were then reacted with 50 mM glycine at room temperature for 30 min to reduce the autofluorescence, followed by incubating with primary antibodies at 4°C overnight. Subsequently, samples were incubated with secondary antibodies at 37°C for 1.5 hr. The primary antibodies used in this study included those against neurofilament (NF) heavy polypeptide (SMI312), MAP2 (microtubule-associated protein 2), histone H3.3, histone H2A.Z, histone H2B, GFAP (glial fibrillary acidic protein), βIII-tubulin, pre-immune rabbit IgG, and pre-immune mouse IgG. The secondary antibodies used in this study included goat anti-mouse Alexa Fluor 488 and goat anti-rabbit Alexa Fluor 647 (the manufacturers and dilution factors of the primary and secondary antibodies used in the experiments are shown in Table S-1). Factin was labeled with Alexa Fluor 546 phalloidin at room temperature for 1.5 hr, and nuclei were labeled with DAPI (4', 6-diamidino-2-phenylindole; Sigma-Aldrich) at room temperature for 15 min. The resultant cells were washed three times with PBS, mounted with Prolong Antifade (Molecular Probes, Inc.), and then examined with a confocal microscope (LSM 510, Carl Zeiss MicroImaging, Inc., Thornwood, NY, USA).

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Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis

SDS-PAGE analyses were performed in a mini-gel apparatus (Mini-

Protean 3; Bio-Rad Laboratories Inc., Hercules, CA) according to the procedure reported previously by Laemmli et al.26 Western blotting was performed according to the procedure of Wu et al.27 Proteins from whole-cell, axon and control samples in the polyacrylamide gels were either visualized by silver-staining28 or electrotransferred to PVDF membranes (0.2 μm, PerkinElmer) for Western blotting analysis. Proteins on PVDF blots were probed with antibodies against CaMKIIα (calcium, calmodulin-dependent protein kinase II α-subunit), GAP43 (growth associated protein 43), BSA (bovine serum albumin), pan-actin, GluR-2 (glutamate ionotropic receptor AMPA type subunit 2), βIII-tubulin, histone H2B, histone H3.3, histone H2A.Z, and pre-immune rabbit and mouse IgGs (Table S-1). Blots were then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG or HRP-conjugated goat anti-rabbit IgG (Table S-1) and visualized with Western lighting pro-ECL (Enhanced chemiluminescence) reagents (PerkinElmer Inc., MA, USA). The relative intensities of immune-stained bands on PVDF blots were quantified by using the ImageJ (version 1.49v; National Institutes of Health, USA) system. Protein concentrations of the axon and whole-cell samples were determined with the Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA).

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Label-free experiments

The axon and whole-cell samples were homogenized with a

sonicator, centrifuged at 15,700 X g at 4℃ for 20 min to remove cell debris, freeze-dried, and finally re-suspended in ddH2O. The resultant samples containing equal amounts of proteins (4.6 μg) were then reduced with DL-dithiothreitol, followed by alkylating with iodoacetamide, and finally exhaustively digested with trypsin. The digested samples were desalted on reverse phase (RP) micro-columns packed with SOURCE 15RPC resin (GE Healthcare, Uppsala, Sweden). The resultant axon and whole-cell peptides were subjected to 2D-LC-MS/MS analyses as described below (Fig. 1). For generating the “possible contamination” database, the culture medium used in this study was subjected to the same treatments as described above and subjected to 2D-LC-MS/MS analysis.

Dimethyl labeling experiments

The whole-cell and axon peptides prepared as described

above were labeled respectively with light formaldehyde (12CH2O) and heavy formaldehyde (13CD2O) according to procedures reported previously22 with modifications (Fig. 1). Briefly, the whole-cell peptides (from samples containing 4.6 ug whole-cell proteins) in 84 µl sodium acetate buffer (200 mM, pH 5.1) were incubated with 4 µl light formaldehyde (20%, v/v) for 2 hrs, followed by reduction with 12 µl freshly prepared NaCNBH3. Axon peptides (from samples containing 4.6 µg axon proteins) were treated similarly except that heavy formaldehyde was used instead. The resultant dimethylated whole-cell and axon peptides were

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acidified by adding 5 µl formic acid (FA), mixed together, and then dried. The dried sample was dissolved in 200 µl FA (1% in ddH2O, v/v), desalted with an RP micro-column (10 µl Source 15RPC, GE Healthcare), and finally subjected to 2D-LC-MS/MS analysis. The above experiments were designated as labeling experiments. Alternatively, the whole-cell and axon peptides were dimethylated with heavy and light formaldehyde, respectively. These latter experiments were referred to as swap experiments. The labeling and swap experiments are considered as technical replicates here.

2D-LC-MS/MS analysis

Peptide separation was performed by using an online two

dimensional (2D)-strong cation exchange (SCX)/RP-liquid chromatography (LC) system (Ultimate 3000; Thermo Fisher Scientific/Dionex, Germering, Germany). Peptides were first dissolved in 50% (v/v) acetonitrile (ACN) containing 0.1% (v/v) FA and then loaded onto the SCX column (0.38 × 100 mm, packed with Luna-SCX particles, Phenomenex, Torrance, CA) and eluted with a gradient of 0–0.5 M NH4Cl containing 30% ACN and 0.1% FA at a flow rate of 0.5 µl/min over 1,430 min. The separation was divided into 22 segments of 65 min each. The eluate from each segment was continuously diluted 60 folds with 0.1% FA before passing through an RP trapping column. The peptides-loaded trapping column was then connected to a second-dimensional RP column (0.075 × 130 mm, packed with Synergi Hydro-RP particles from Phenomenex) and eluted with a 57-min ACN gradient (5-40% in 50 min, 40-100% in 4

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min, and 100% for 3 min). Two RP trapping columns were installed on a two position/ten-port valve of the LC system, which enabled the trapping and separation processes to be operated simultaneously and alternately. The eluate from the RP-analytical column was analyzed on an Orbitrap Elite Hybrid Ion Trap-Orbitrap mass spectrometer (Thermo Electron, Bremen, Germany) equipped with a Nanospray Flex Ion Source. The instrument was operated in positive ion mode with an orbitrap resolution of 60,000 at m/z 400 to gain the full-scan MS spectra between m/z 400–m/z 2,000. The cyclosiloxane peaks at m/z 445.1200, 462.1466, and 536.1654 were used for mass calibration. MS/MS spectrum acquisition was performed in a data-dependent manner. From each full-scan MS spectrum, the top fifteen intense ions with ion intensities greater than 5,000 were selected for collision-induced fragmentation (CID) in the linear trap quadrupole (LTQ) analyzer to obtain MS/MS spectra. Each of the selected precursor ions was sequenced once with exclusion duration of 40 s.

Analysis of MS data in label-free experiments

Peptide identification and quantification of

the MS data were performed by using MaxQuant software suite (version 1.5.2.8).29 Extracted MS/MS peak lists were subjected to database search against the Rattus database (Swiss-Prot database version 2014_04; 7,911 proteins) by the Andromeda search engine (part of the MaxQuant package) built by Cox et al.30 Possible contaminants (including trypsin, human

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keratins and the 94 proteins (Table S-2) identified in the culture medium here) were also included in MaxQuant database. The raw data files of axon and whole-cell samples in all labelfree experiments were uploaded together into MaxQuant software for comparative analysis. The type “match between runs” was set to 0.7 and 20 min for the match and alignment time windows, respectively. Acetyl (protein N-term), gln->pyro-glu (N-term Q) and oxidation (M) were set as variable modifications. Carbamidomethyl (C) was set as a fixed modification. The maximum missed cleavages by trypsin were set to 2. In addition, the mode of decoy database was set to “randomize” in sequence, and the false discovery rate (FDR) was set to 0.01 for both peptide and protein levels. The minimum peptide length was set to 7 amino acids. The peptides for quantification were set as “unique + razor”. The iBAQ option was selected for protein quantification. Mass tolerance for precursor ions in the main search was set to 4.5 ppm, and mass tolerance for fragment ions was set to 0.5 Da. The top eight fragment peaks per 100 Da m/z window of each MS/MS spectrum were collected for database search. The minimum number of unique peptides required for a protein was set to 1. In the data processing, proteins matched to the “reverse” and “potential contaminant” databases were removed. Protein identifications based only on modified peptides were also removed. The minimum number of razor and unique peptide identifications required for each protein was set to 2. The “leading razor proteins” were selected for further correlation and classification analyses.

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Analysis of MS data in dimethyl labeling experiment

Peptide identification and

quantification of MS spectrum data in dimethyl labeling experiments were also performed with the MaxQuant software package using parameters as those in analyzing the data from labelfree experiments with the following exceptions. The raw data files of dimethyl labeling experiments were analyzed individually. In “Group-specific parameters” option, multiplicity option was set as 2; maximum labeled amino acids was 4; labels in light labels (12C21H4) were “dimethyl Lys0” and “dimethyl Nter0”; labels in heavy labels (13C2D4) were “dimethyl Lys6” and “dimethyl Nter6”. The minimum ratio count was set to one. In the data processing, peptides with “Razor + unique peptides” numbers greater than 2, ratio counts smaller than 2, and variabilities (%) of heavy/light ratio smaller than 100% were selected. For peptides with ratio counts larger than 2, no limitation of the variability (%) was used. The “leading razor proteins” were subjected to further correlation and classification analyses. The mass spectrometry proteomics data were deposited to the ProteomeXchange Consortium via the PRIDE31 partner repository with the dataset identifier PXD005527.

Correlation, Gene Ontology, and statistical analyses

Protein abundances were log-

transformed and then subjected to Spearman’s rank correlation analysis by using SigmaPlot 12.5 (SyStat Software Inc.). Gene Ontology (GO) analyses were performed by using the functional annotation tool DAVID (Database for Annotation, Visualization and Integrated

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Discovery; Bioinformatics Resources 6.8, NIAID/NIH). Statistical analysis was performed using the Statistical Package for the Social Sciences (Prism version 6.00 for Windows, GraphPad Software; www.graphpad. com; RRID:SCR_002798). Data were expressed as mean ± S.D. (standard deviation).

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RESULTS AND DISCUSSION Preparation and characterization of axon and whole-cell samples from cultured rat cortical neurons Axons of rat cortical neurons grew on the surface of micro-patterned chips were isolated and used in this study (Fig. 2A). Immunofluorescence staining showed that Region 1 of the chip surface was occupied by numerous neurons exhibiting MAP2 (a dendritic marker)positive cell bodies, MAP2-positive dendrites, SMI312 (an antibody recognizes an axon neurofilament marker)-positive axons, and DAPI (a nuclear marker)-labeled nuclei. A few GFAP (an astroglial marker)-positive astrocytes were also found in Region 1. On the other hand, only SMI312-positive axons were found in Region 2. Most importantly, MAP2 and GFAP immunoreactivity or DAPI labeling were not observed in Region 2. These results ascertained that axons were isolated in Region 2.24 Chips were broken into fragments containing only Regions 1 or 2. Proteins extracted from the Region 2-containing fragments were designated as axon samples. The Region 1-containing fragments contained the cell bodies, axons that never crossed the border, and dendrites of neurons, few astroglial cells, and short axon stretches found on the PLL-coated lines. Proteins extracted from Region 1-containing fragments were designated as whole-cell samples. Western blotting analysis indicated that the axon sample contained GAP43 (an axon marker), while the amount of CaMKIIα (a dendritic marker) in the sample was below detection limit (bottom rows in Fig. 2B). Both CaMKIIα and GAP43 were detected in the whole-cell 16


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sample. Again, these observations supported the notion that the axon sample was free of contaminations from the cell bodies and dendrites. However, proteins were carried over from the culture medium, as demonstrated by the presence of BSA (bovine serum albumin) in controls which were prepared from chips without seeded cells (Fig. 2B).

Figure 2. (A) Characterization of cellular structures occupying the different parts of the micropatterned glass chip and (B) biochemical characterization of whole-cell and axon samples. A. left panels: at DIV15, the cells on the chip surface were immunofluorescence stained with antibodies against SMI312 (an axon marker, green) and MAP2 (a dendritic marker, red) and then labeled by DAPI (labeling nuclei, blue). right panels: DIV 15 cells on chip surface were immunofluorescence stained with antibodies against SMI312 (green) and GFAP (an astroglial marker, red) and then labeled with DAPI (blue). Scale bars: 100 µm. B. top panel: SDS-PAGE analysis of axon (0.5 μg protein) and whole-cell (0.5 μg protein) samples prepared from DIV 15 neurons grew on micro-patterned glass chips. Axon and whole-cell control samples prepared from the same numbers of chips without seeded neurons, indicated as axon control and wholecell control, were also subjected to the same analysis. Molecular size markers are shown to the left. Bottom panels: Western blotting analysis of the axon, whole-cell, axon control, and wholecell control samples as those described in the top panel by using antibodies against BSA, CaMKIIα and GAP43.

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Studying axon proteome by label-free experiment Axon samples were subjected to the label-free experiment (Fig. 1) and analysis with the MaxQuant software package. For comparison, a parallel study of whole-cell samples was also performed. Proteins were quantified by the iBAQ tools of MaxQuant software. The iBAQ values of proteins were reported to correlate well with protein amounts.32,33 Here, protein abundance was defined as the fraction of the iBAQ value of a protein in a sample relative to the summed iBAQ values of all proteins found in the same sample. The axon and whole-cell samples prepared from two batches of neuronal cultures were subjected to the label-free experiment independently. From these two replicates, we identified 47,226 and 37,282 peptide sequences and assigned them to 3,733 and 3,469 unique proteins, respectively (Supporting Files S-1 and S-2). Among these proteins, 2,548 of them were identified in both replicates of the axon sample, 2,752 proteins were identified in both replicates of the whole-cell samples (Table 1). For samples prepared from the same batch of culture, >99% of the proteins found in the axon sample were also detected in the whole-cell sample, while >89% of the proteins found in the whole-cell sample were also detected in the axon sample. Strong correlations between the abundances of proteins identified in the two replicate axon samples (rs= 0.90) and between the abundances of proteins identified in the two replicate whole-cell samples (rs= 0.93) (see Supporting Information Fig. S-1). The results imply a good reproducibility of the protein abundances obtained here. A strong correlation was also found between the abundances of

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proteins in the axon and whole-cell samples (rs= ~0.9) regardless whether the axon and wholecell samples were prepared from the same batch of culture or not (Fig. S-2). The complete lists of the proteins identified in the axon and whole-cell samples and their abundances were summarized in Supporting File S-3. The top 100 most abundant proteins identified in the axon sample are listed in Table 2. A comparison between the axon proteome as described above and the axonal growth cone proteomes reported earlier13,14 was made. More than 50% of the proteins identified in axonal growth cones were also found in the axon proteome. We found a moderate correlation (rs= ~0.5) between the abundances of proteins present in both growth cones and axons. As axons consist of axonal shafts and a small portion of growth cones, the differences between axon and growth cone proteomes may show differential localization of proteins in the two axonal subdomains. We further found very weak correlations between the abundances of the proteins and the levels of the mRNAs9 encoding the same proteins in cortical axons (rs= ~0) and between the abundances of proteins in rat cortical axons and the levels of the mRNAs34 encoding the same proteins in the projections of human stem cell derived neurons (rs= ~0.1). These results are consistent with the poor correlations generally reported in the literature between protein and mRNA expression in different organisms.35 A Gene Ontology (GO) analysis of the proteins identified in cortical axons revealed that rat cortical axons contain the protein constituents of organelles (including mitochondria,

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ribosome, synaptic vesicle, endoplasmic reticulum, lysosome, proteasome, endosome, synaptic vesicle, and peroxisome) and cytoskeleton (including microtubule, actin and intermediate filament) (Fig. 3 and Supporting File S-4). These structures were well documented in earlier electron microscopic (EM) studies of cortical axons in rat brains.23 Sixty-six of the 75 rat ribosomal proteins with known complete amino acid sequences36 and 70 of the 78 ribosomal proteins found in the Rattus database were identified in axons with an average abundance of 0.07 % (the range was from 0.38% for P1 to 0.01% for L29). Two ribosomal proteins, P1 and L22, are among the top 100 most abundant proteins identified in axons (Table 2). Cortical axons contain two cytoplasmic actin isoforms, γ- and β-actin. γ-Actin is among the top 100 most abundant protein in axons. The abundance of β-actin is ~250 fold lower than that of γ-actin. This difference seems to be consistent with their different localization in axons, with γ-actin being uniformly distributed in axons and β-actin being confined to growth cones.37,38 Although EM studies indicated that neurofilaments are abundant in cortical axons, the abundance of neurofilament proteins was low in our analysis. Most likely, this discrepancy is related to the exceptionally high arginine (R) and lysine (K) contents in neurofilament subunits and the use of trypsin in our protein digestion. Many tryptic peptides derived from neurofilament subunits might possibly be too short to be included in the analysis here. Although the axon lacks conventional Golgi apparatus,23 we found 241 Golgi proteins in cortical axons, including two widely used Golgi markers, syntaxin-6 and GM130 (cis-Golgi

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matrix protein). Proteins previously identified from Golgi-enriched fractions and in Golgiderived COP1 vesicles39-41 were also detected. The result appears to agree with earlier reports in detecting Golgi proteins in peripheral axons and central axonal growth cones by immunofluorescence staining.14,42,43 EM studies indicated that cortical axons also lack nucleus.23 However, 840 proteins were categorized into the nuclear proteins groups according to their known functions in the nucleus without taking into consideration the extranuclear functions that some of them44-48 may play. Furthermore, 7 histones, brain acid soluble protein 1, and nucleophosmin are among the top 100 most abundant axon proteins in this study (Table 2). It has been reported in the literature that nuclear proteins such as histones H2B type 3-A, H3f and H2A.Z, transcription factors, and nuclear structure proteins were observed in axonal growth cones.13,14 Our data also support the previous findings that various nuclear proteins, such as lamin B2, HMGB1, RNA-binding proteins, and transcription factors, are synthesized locally in the axon.44-46,48-53

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Figure 3. Functional annotation (Cellular Component) of the 2,548 axonal proteins identified in duplicate label-free experiments. Using DAVID informatics tool, these proteins are classified into 18 groups under the indicated GO terms with enrichment P values < 0.01. The 535 proteins that cannot be classified are grouped under “others”. The fractions of proteins under different GO terms relative to the total number of proteins are shown as blue bars (% Count). The fractions of the summed abundances of proteins under different GO terms relative to the summed abundances of all proteins are shown as orange bars (% Group abundance). The means of the abundances of individual proteins as measured from two label-free experiments were used in the calculation. The numbers in parentheses are the accession numbers of GO terms. The numbers in square brackets are the protein counts under GO terms. Enrichment P values are shown on the right.

Identification of axon-enriched proteins by dimethyl labeling experiments For detecting proteins enriched in axons, the axon and whole-cell samples prepared from

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two batches of culture were subjected to dimethyl labeling experiments separately (Fig. 1). To verify the reproducibility of the resultant data, these pairs of axon and whole-cell samples were also subjected to the swap experiment (Fig. 1, indicated by broken lines). A total of 22,796 peptides and 12,718 peptides were identified in the 1st labeling experiment and 1st swap experiment, respectively. A total of 22,100 peptides and 20,009 peptides were identified in the 2nd labeling experiment and 2nd swap experiment, respectively (Supporting File S-1). The peptides identified in the 1st labeling and swap experiments were assigned to 2,852 and 2,031 proteins, respectively; the peptides in the 2nd labeling and swap experiments were assigned to 2,650 and 2,520 proteins, respectively (Supporting File S-2). The ratios between the abundances of proteins in the axon sample and the abundances of their counterparts in the whole-cell sample, designated as A/W ratios, were calculated. In the two replicate labeling experiments, the A/W ratios of 2,091 and 1,921 proteins were calculated; in the two replicate swap experiments, the A/W ratios of 1,356 and 1,850 proteins were calculated (Table 1). The A/W ratios of 1,208 proteins were found in all 4 sets of data. For the axon and whole-cell samples prepared from the same batch of culture, the correlation between the A/W ratios measured in the labeling and swap experiments is high (rs= ~0.8), indicating the good reproducibility of these data (Figs. S-3A and S-3B). Conversely, the correlations between the A/W ratios calculated from samples prepared from different batches of culture was moderate (rs=~0.4) (Figs. S-3C and S-3D). This latter observation might reflect the dynamic nature of

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biomolecules localized in the axon.54 Furthermore, the correlation between the A/W ratios and the abundances of axonal proteins was weak (rs=~0.2). The A/W ratios of more than 70% of axon proteins were lower than 1, and the median of A/W ratios of all axon proteins was ~0.5 (Fig. S-4). It was noted that ~4% of axonal proteins had A/W ratios > 1 and ~6% of axonal proteins had A/W ratios < 0.2. The results further indicate that comparing to the whole neurons, certain proteins in axons are enriched, while others are depleted. This observation suggests that the protein repertoire of the axon is shaped by multiple mechanisms. The barriers found in the axonal initial segment might hinder the protein flow from the cell body to the axon.55-57 There may also be a protein-specific mechanism, utilizing adaptors, motor proteins, and cytoskeleton11,58,59 to regulate the localization of some axonal proteins. Furthermore, local protein synthesis and degradation might also influence the protein composition of the axon. In each of the 4 data sets from the 2 labeling and 2 swap experiments, 400-500 proteins with A/W ratios > 1 were found (Table 1). Only 103 proteins with A/W ratios > 1 in all four data sets were found (Table 3), and these proteins were designated as axon-enriched proteins. A Gene Ontology analysis revealed that the majority of the axon-enriched proteins are those with known functions in the nucleus and extracellular matrix proteins (Fig. 4 and Supporting File S-5). Again, proteins were classified into the nuclear GO category without taking their possible extranuclear functions into account.

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Figure 4. Functional annotation (Cellular components) of the 103 axon-enriched proteins. These proteins are classified into 4 groups under the indicated GO terms with enrichment P values < 0.05. The 23 proteins that cannot be classified into subcellular components are grouped under the term of “others”. The fractions of proteins under different GO terms of the 103 proteins are shown as blue bars (% Count). The fractions of the summed abundances of proteins under different GO terms relative to the summed abundances of all proteins are shown as orange bars (% Group abundance). The means of the abundances of individual proteins as measured from two replicate dimethyl-labeling and swap experiments were used in the calculation. The numbers in parentheses are the accession numbers of GO terms. The numbers in square brackets are the protein counts under GO terms. Enrichment P values are shown on the right.

Validating the A/W ratios of selected proteins by Western blotting analysis To validate the A/W ratios as described above, the A/W ratios of 7 proteins were affirmed by Western blotting analysis. Four well-characterized antibodies against CaMKIIα, GluR-2, pan-actin and βIII-tubulin60 and three antibodies against histones H2A.Z, H3.3 and H2B were used. The specificity of these 3 anti-histone antibodies was first confirmed by their recognizing proteins with the molecular sizes of histones in brain homogenates (Fig. 5A). The axon and 25



whole-cell samples containing 1 µg of protein were subjected to Western blotting analysis using these 7 antibodies (left panel of Fig. 5B). The ratios between the intensities of stained bands in the axon and whole-cell samples were determined (right panel of Fig. 5B). The resultant A/W ratios were in good agreement with those calculated in dimethyl labeling experiments (right panel of Fig. 5B), except for that of CaMKIIα in axon sample, which was below the detection limit.

Figure 5. Western blotting analyses of selected axonal proteins. (A) Western blotting analyses of E18 rat brain homogenates (containing 2 and 1 μg of protein, as indicated) with antibodies against histone subunits H2A.Z, H3.3 and H2B (right 3 strips). The same blots were also subjected to staining with different combinations of HRP-conjugated anti-rabbit, HRPconjugated anti-mouse IgG, pre-immune rabbit IgG, and pre-immune mouse IgG (left 4 strips, as indicated on top). Molecular size markers are shown to the left. Arrows indicate immunostained histone bands. (B) left panel: Western blotting analyses of axon and whole-cell samples (containing 1 µg protein) and axon control and whole-cell control samples (prepared from the same numbers of control chip fragments containing Regions 2 and 1 as those used for preparing axon and whole-cell samples containing 1 µg protein) with antibodies against CaMKIIα, GluR-2, pan-actin, βIII-tubulin and histone subunits H2A.Z, H3.3 and H2B. Right panel: the ratios between the intensities of immunostained bands in the axon and whole-cell samples in Western blotting (WB) analyses. Results are the mean ± S.D. from 4 blots. The A/W ratios of these proteins as determined by mass spectrometry (MS) are also included for comparison.

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Validating the presence of histones in the shafts and growth cones of axons by immunofluorescence staining The presence of histones in the axon was further verified by immunofluorescence staining. DIV 15 neurons growing on glass chips were immuno-stained using antibodies specific to histones H2A.Z, H3.3 and H2B. Images taken from regions where axons were localized (indicated by the squares a and b in Fig. 6B) revealed the presence of histone immunoreactivity in axons (Fig. 6A). As controls, antibodies to these histones were omitted or replaced with preimmune IgGs in the immuno-staining experiments. No stained structure could be observed in area b (Fig. 6D). Histone H2A.Z, H3.3 and H2B immunoreactivities were also detected in the growth cones, which were recognized as finger-like and F-actin (phalloidin)-enriched structures at the ends of axons, of DIV 9 neurons (Fig. 6C). By omitting these anti-histone antibodies or replacing these antibodies with pre-immune IgGs in the same staining experiment, histone immunoreactivity was not detected in phalloidin-positive axonal shafts and growth cones (Fig. 6E). The results support the presence of histones in the growth cones and shafts of the axons of cultured rat cortical neurons. As histone transcripts were reported in earlier axonal RNA profiles,9,34,61-64 it is likely that histones can be synthesized locally in the axon as well as being transported from the cell body. In the nucleus, histones play critical roles in chromatin assembly and transcript regulation; in various extranuclear locations of different cells, histones also play

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a multitude of functions in cellular signaling and innate immunity.65 The role(s) that histones play in axons is unclear presently. It is possible that histones may contribute to the growth and maintenance of axons as several other nuclear proteins, such as nucleolin,44 RNA-binding protein La,45 lamin B,46 HMGN5,47 amphoterin,48 do in the same subcellular compartment. It is also possible that histones may play a role in retrograde signaling as some locally synthesized transcription factors do.49,50,52 More effort is needed in the future to elucidate the functional role(s) of histones in axons.

Figure 6. Verification of the presence of histones in rat cortical axons by immunofluorescence staining. (A) Double immunofluorescence staining of the axons of DIV 15 rat cortical neurons in areas a and b on the chip surface (the squares indicated by a and b in (B)) with antibodies 28


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against histones H2A.Z, H3.3 or H2B (top row images) in combination with the SMI312 antibodies or anti-βIII-tubulin antibodies (middle row images). (B) Localizations of areas a and b on the surface of the glass chip. (C) Double immunofluorescence staining of axonal growth cones of DIV 9 rat cortical neurons in area b on the chip surface with antibodies against histones H2A.Z (top row images), H3.3 (middle row images) or H2B (bottom row images) in combination with antibodies against βIII-tubulin (4th column in blue) and labeling with phalloidin which interacts with F-actin (3rd column in red). Images of the growth cones enclosed by the white squares in the images in the 1st and 5th columns are shown at a higher magnification in the 2nd and 6th column, respectively. (D) Immunofluorescence staining the axons of DIV 15 rat cortical neurons in area b (as indicated in (B)) with Cy5-conjugated goat anti-rabbit IgG and DyLight 488-conjugated goat anti-mouse IgG and labeling with phalloidin (left column). Immunofluorescence staining the axons of DIV 15 in area b with pre-immune rabbit and mouse IgGs as the primary antibodies and Cy5-conjugated goat anti-rabbit IgG and DyLight 488-conjugated goat anti-mouse IgG as the secondary antibodies and labeling with phalloidin (right column). (E) Immunofluorescence staining the axonal growth cones of DIV 9 rat cortical neurons in area b (as indicated in (B)) with Cy5-conjugated goat anti-rabbit IgG and DyLight 488-conjugated goat anti-mouse IgG and labeling with phalloidin (left column). Immunofluorescence staining the axonal growth cones of DIV 9 in area b by using pre-immune rabbit and mouse IgGs as primary antibodies and Cy5-conjugated goat anti-rabbit IgG and DyLight 488-conjugated goat anti-mouse IgG as secondary antibodies and labeling with phalloidin (right column). Scales bars: 10 µm.

Conclusions Here, we report the proteome of rat cortical axons. The results show the protein basis of the structure and function of the axon. The results confirm the presence of organelles, cytoskeleton, and other cellular structures in cortical axons, as documented in earlier EM studies. However, the results also demonstrate the existence of proteins known to associate with the Golgi apparatus and nucleus, two organelles not detected in axons by EM. The identification of Golgi proteins in cortical axons supports the notion that the axon contains an equivalent of Golgi apparatus with a structure not yet identified.14,42,43 The various Golgi 29



proteins identified in the cortical axons here could be used as targets for future biological manipulations and/or as markers in studying how the Golgi apparatus in the axon participates in the local synthesis and sorting of membrane and secretory proteins.42,43,66-68 Our understanding of the functional roles played by nuclear proteins in the axon is far from complete. Some nuclear proteins in axons may act as “growth cone-to-nucleus” signals for adjusting the nuclear expression program when the axon is injured or stimulated.50,53,69,70 Yet, some nuclear proteins in axons may play supporting roles in axon growth and maintenance.46,53,69 Future studies targeting at the axon-enriched nuclear proteins will furnish more mechanistic details of the functional roles played by nuclear proteins in the axon. In the future, the long list of axon-enriched proteins as reported here will be useful in studying the mechanisms underlying the polarized protein localization in axons with proteintargeted methods, such as multiple reaction monitoring mass spectrometric assay15 and fluorescent tagging.71 The axon proteome reported here is also a useful database for the future studies of how individual proteins contribute to various axonal functions, such as growth, branching, collapse, turning behavior, and responses to local stimulation and injury.

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Supporting Information: The following files are available free of charge at ACS website http://pubs.acs.org. Supporting Information (PDF) Table S-1. Antibodies and fluorescent labeling reagents used in current study. Table S-2. Proteins detected in culture medium. Figure S-1. Correlation analyses between the abundances of axon proteins and between the abundances of whole-cell proteins prepared from two biological replicates (as indicated by 1 and 2). Figure S-2. Correlation analyses between the abundances of the axon and whole-cell proteins prepared from the same batch of culture (A and B) and between the abundances of the axon and whole-cell proteins prepared from different batches of culture (C and D). Figure S-3. Correlation analyses between the A/W ratios calculated from the labeling and swap experiments with samples prepared from the same batch of culture (A and B) and between the A/W ratios calculated from the labeling and swap experiments with samples prepared from different batches of culture (C and D). Figure S-4. Distribution of A/W ratios obtained in two labeling (A and C) and two swap (B and D) experiments.

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Supporting Files (XLSX) Supporting File S-1. Peptides identified in label-free experiments and dimethyl labeling experiments. Supporting File S-2. Proteins identified in label-free experiments and dimethyl labeling experiments. Supporting File S-3. The lists of the proteins identified in the axon and whole-cell samples. Supporting File S-4. Classification of axonal proteins identified in two independent label-free experiments. Supporting File S-5. Classification of the 103 proteins consistently enriched in the axon.

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FOOTNOTES Author Contributions: Y.-C. C. and C.-F. C. conceived and designed the research and wrote the paper; C.-F. C. performed and analyzed all experiments; C.-E. K. helped the sample preparation and protein classification; B.-W. H. performed part of the experiment shown in Fig. 5A and contributed to proteins classification; K.-Y. C. performed the stable isotope dimethyl labeling reaction and MS analyses.

ACKNOWLEDGEMENTS This work is supported in part by grants to Y.-C. C. from Ministry of Science and Technology of the Republic of China, Taiwan (102-2311-B-007-005 and 103-2311-B-007-009MY3) and to K.-Y. C. from Chang Gung Memorial Hospital, Taiwan (CLRPD190016). Technical support of mass spectrometric works is from the Proteomics Core Laboratory, Chang Gung University, Taoyuan City, Taiwan.

CONFLICT OF INTEREST DISCLOSURE The authors declare no competing financial interest.

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TABLES

Table 1. Proteins identified in label-free and dimethyl labeling experiments.

Label-free

Expt. 1

Expt. 2

Expt. 1

Expt. 2

Axon

Axon

Whole cell

Whole cell

3035

2673

3100

2944

Proteins identified

experiments Proteins identified in

2548

2752

Expt. 1

Expt. 2

both trials

Proteins identified Dimethyl

Proteins identified in

labeling

both trials

Labeling

Swap

Labeling

Swap

2091

1356

1921

1850

1314

1690

experiments Proteins also identified in axon sample from

2039a / 1959b

1339a / 1321b

1898a / 1854b

1828a / 1795b

451

405

435

517

label-free experiments Proteins with A/W > 1

a b

Number of proteins also identified in the axon sample in the label-free experiment 1. Number of proteins also identified in the axon sample in the label-free experiment 2.

41



Page 42 of 56

Table 2. Top 100 most abundant proteins identified in the axon. Rank

GN1

Description

Abun. (%)

*1

Hist1h4b

Histone H4

7.61

2

H2afj

Histone H2A.J

5.29

3

Tubb2a

Tubulin beta-2A chain

3.82

Histone H2B type 1

3.69

*4 *5

H3f3b

Histone H3.3

3.32

6

Tuba1a

Tubulin alpha-1A chain

2.91

7

Actg1

Actin, cytoplasmic 2

2.53

8

Tmsb4x

Thymosin beta-4

1.45

*9

H2afz

Histone H2A.Z

1.21

10

Cfl1

Cofilin-1

1.05

11

Calm1

Calmodulin

0.97

12

Ywhaz

14-3-3 protein zeta/delta

0.94

13

Dpysl2

Dihydropyrimidinase-related protein 2

0.89

14

Atp6v0c

V-type proton ATPase 16 kDa proteolipid subunit

0.86

15

Ppia

Peptidyl-prolyl cis-trans isomerase A

0.85

16

Tubb4b

Tubulin beta-4B chain

0.82

17

Gapdh

Glyceraldehyde-3-phosphate dehydrogenase

0.78

18

Tubb3

Tubulin beta-3 chain

0.77

19

Uchl1

Ubiquitin carboxyl-terminal hydrolase isozyme L1

0.72

20

Dpysl3


0.68

21

Atp5b

ATP synthase subunit beta, mitochondrial

0.64

22

Fabp5

Fatty acid-binding protein, epidermal

0.64

23

Tubb5

Tubulin beta-5 chain

0.61

24

Vamp2

Vesicle-associated membrane protein 2

0.55

25

Gdi1

Rab GDP dissociation inhibitor alpha

0.55

26

Dpysl5


0.45

27

Hspa8

Heat shock cognate 71 kDa protein

0.43

28

Ywhag

14-3-3 protein gamma

0.41

29

Eno1

Alpha-enolase

0.41

30

Ywhae

14-3-3 protein epsilon

0.41

31

Crmp1


0.40

32

Tubb2b

Tubulin beta-2B chain

0.39

*33

Arf5

ADP-ribosylation factor 5

0.39

34

Rplp1

60S acidic ribosomal protein P1

0.38

42


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*35

Npm1

Nucleophosmin

0.37

36

Ckb

Creatine kinase B-type

0.37

37

Basp1

Brain acid soluble protein 1

0.36

38

Atp5a1

ATP synthase subunit alpha, mitochondrial

0.36

39

Fkbp1a

Peptidyl-prolyl cis-trans isomerase FKBP1A

0.35

40

Gnao1

Guanine nucleotide-binding protein G(o) subunit alpha

0.35

41

Ldha

L-lactate dehydrogenase A chain

0.35

42

Snap25

Synaptosomal-associated protein 25

0.33

43

Atp1a3

Sodium/potassium-transporting ATPase subunit alpha-3

0.33

44

Gnb1

Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1

0.33

45

Gap43

Neuromodulin

0.32

Histone H2A type 1

0.31

46 47

Pebp1

Phosphatidylethanolamine-binding protein 1

0.31

48

Prdx2

Peroxiredoxin-2

0.31

49

Hsp90ab1

Heat shock protein HSP 90-beta

0.30

50

Stxbp1

Syntaxin-binding protein 1

0.30

51

Vdac1

Voltage-dependent anion-selective channel protein 1

0.30

52

Nme2

Nucleoside diphosphate kinase B

0.29

53

Ncam1

Neural cell adhesion molecule 1

0.28

54

Aldoa

Fructose-bisphosphate aldolase A

0.28

55

Mif

Macrophage migration inhibitory factor

0.27

56

Arhgdia

Rho GDP-dissociation inhibitor 1

0.27

57

Sncb

Beta-synuclein

0.25

58

Eef1a1

Elongation factor 1-alpha 1

0.25

59

Hspe1

10 kDa heat shock protein, mitochondrial

0.25

60

Atp1b1

Sodium/potassium-transporting ATPase subunit beta-1

0.25

61

Stmn1

Stathmin

0.24

*62

Set

Protein SET

0.24

63

Pkm

Pyruvate kinase PKM

0.24

64

Mdh1

Malate dehydrogenase, cytoplasmic

0.23

65

Snca

Alpha-synuclein

0.22

66

Gpm6a

Neuronal membrane glycoprotein M6-a

0.22

*67

Hnrnpa1

Heterogeneous nuclear ribonucleoprotein A1

0.22

*68

Hist1h1e

Histone H1.4

0.22

69

Rac1

Ras-related C3 botulinum toxin substrate 1

0.21

70

Slc25a5

ADP/ATP translocase 2

0.21

43



71

Syp

Synaptophysin

0.20

72

Tpi1

Triosephosphate isomerase

0.20

73

Mdh2

Malate dehydrogenase, mitochondrial

0.19

74

Ywhaq

14-3-3 protein theta

0.19

75

Tpm3

Tropomyosin alpha-3 chain

0.19

76

Atp5i

ATP synthase subunit e, mitochondrial

0.19

77

Pgam1

Phosphoglycerate mutase 1

0.18

*78

Hnrnpk

Heterogeneous nuclear ribonucleoprotein K

0.18

*79

Hnrnpa3


0.18

80

Hint1

Histidine triad nucleotide-binding protein 1

0.18

81

Pgk1

Phosphoglycerate kinase 1

0.18

82

Sod1

Superoxide dismutase [Cu-Zn]

0.18

83

Mapre1

Microtubule-associated protein RP/EB family member 1

0.18

84

Atp5d

ATP synthase subunit delta, mitochondrial

0.18

85

Gdi2

Rab GDP dissociation inhibitor beta

0.17

86

Acot7

Cytosolic acyl coenzyme A thioester hydrolase

0.17

*87

H2afy

Core histone macro-H2A.1

0.17

88

Slc25a4

ADP/ATP translocase 1

0.17

89

Hspd1

60 kDa heat shock protein, mitochondrial

0.17

90

Ppib

Peptidyl-prolyl cis-trans isomerase B

0.17

91

Mtco2

Cytochrome c oxidase subunit 2

0.17

92

Nedd8

NEDD8

0.17

93

Ywhab

14-3-3 protein beta/alpha

0.17

94

Rab6a

Ras-related protein Rab-6A

0.16

95

Cltc

Clathrin heavy chain 1

0.16

96

Ywhah

14-3-3 protein eta

0.16

97

Map1lc3b

Microtubule-associated proteins 1A/1B light chain 3B

0.16

98

Rpl22

60S ribosomal protein L22

0.16

99

Ldhb

L-lactate dehydrogenase B chain

0.16

*100

Ptn

Pleiotrophin

0.16

*Proteins consistently found to be enriched in the axon in Table 3. 1

Page 44 of 56

Gene name

44


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Table 3. The 103 axon-enriched proteins. Label-1 Swap-1 Label-2 Swap-2 A/W A/W A/W A/W

Rank

GN

Description

1

Fn1

Fibronectin

20.25

14.16

17.55

6.96

2

Hmgb2

High mobility group protein B2

17.86

24.12

17.77

22.29

*3

Hist1h4b

Histone H4

13.60

9.05

4.05

5.30

4

Lpl

Lipoprotein lipase

10.42

5.63

10.81

7.62

*5

Hist1h1e

Histone H1.4

9.49

18.20

3.56

4.14

6

Pygl

Glycogen phosphorylase, liver form

8.47

3.55

1.55

4.96

*7

H2afz

Histone H2A.Z

7.74

12.61

5.31

6.52

8

C3

Complement C3

7.65

8.54

9.20

12.16

9

Rpl27


7.42

5.97

1.41

2.04

10

Ssr1

Translocon-associated protein subunit alpha

7.40

5.56

4.31

2.46

11

Bhmt

Betaine--homocysteine S-methyltransferase 1

6.66

6.07

19.91

37.85

12

Rplp2


6.49

6.04

8.74

9.25

13

Slit1

Slit homolog 1 protein

6.46

6.07

4.37

8.01

14

Top2a

DNA topoisomerase 2-alpha

6.02

3.39

2.49

1.53

15

Hist1h1b

Histone H1.5

5.87

5.06

2.88

3.26

16

Sf3b4

Splicing factor 3B subunit 4

5.13

3.43

3.82

4.60

17

Rpl15


5.12

1.55

2.14

2.55

18

Hist2h2aa3

Histone H2A type 2-A

4.87

3.61

2.77

3.18

*19

Ptn

Pleiotrophin

4.81

10.74

28.10

30.90

*20

Npm1

Nucleophosmin

4.34

7.68

2.35

2.82

21

Rpl23a

60S ribosomal protein L23a

4.24

3.13

2.29

2.01

22

Ctgf

Connective tissue growth factor

4.13

5.58

3.24

4.19

23

Bzw2

3.97

2.32

2.44

1.80

Histone H2B type 1

3.93

5.35

3.56

3.95

High mobility group protein B1

3.88

5.83

3.04

5.51

3.87

3.98

1.19

1.03

3.86

8.64

4.55

4.34

Basic leucine zipper and W2 domain-containing protein 2 *24 25

Hmgb1

26

Nudt21

Cleavage and polyadenylation specificity factor subunit 5 Carbamoyl-phosphate synthase [ammonia], 27

Cps1 mitochondrial

28

Rpl38


3.84

5.01

1.71

2.10

29

Plg

Plasminogen

3.81

3.49

1.42

2.04

30

Aldh1a7

Aldehyde dehydrogenase, cytosolic 1

3.77

4.40

6.41

8.44

31

Hdgfrp2

Hepatoma-derived growth factor-related protein 2

3.75

4.14

3.72

3.72

45



32

Olfm1

33

Rbmxrtl

Noelin

Page 46 of 56

3.58

7.03

4.98

4.86

3.51

2.80

4.38

4.69

RNA-binding motif protein, X chromosome retrogene-like 34

Ncl

Nucleolin

3.50

5.17

1.40

2.45

*35

H3f3b

Histone H3.3

3.49

4.49

3.14

4.06

36

Serpine2

Glia-derived nexin

3.44

6.72

4.49

6.34

37

Ssb

Lupus La protein homolog

3.41

3.87

1.08

1.54

38

Khdrbs3

3.20

2.30

1.64

1.98

KH domain-containing, RNA-binding, signal transduction-associated protein 3 39

Eif4a3

Eukaryotic initiation factor 4A-III

3.10

4.91

1.53

1.60

40

Pspc1

Paraspeckle component 1

3.06

3.13

1.24

1.31

41

Sub1

3.06

4.19

2.26

4.33

Activated RNA polymerase II transcriptional coactivator p15 42

Rbm8a

RNA-binding protein 8A

2.97

2.14

5.06

1.38

43

Nova1

RNA-binding protein Nova-1 (Fragment)

2.96

2.03

1.14

1.20

*44

H2afy

Core histone macro-H2A.1

2.92

4.03

2.17

2.50

45

Ncan

Neurocan core protein

2.81

5.66

8.93

12.09

46

Rpn2

2.72

3.55

1.29

1.82

2.71

5.13

1.99

2.19

2.66

3.33

1.67

2.19

Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 2 47

Rpl35

48

Smarce1

60S ribosomal protein L35 SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily E member 1

49

Rps10

40S ribosomal protein S10

2.66

2.92

2.36

2.62

50

Ilf3

Interleukin enhancer-binding factor 3

2.63

3.33

1.56

1.18

51

Eif1a

Eukaryotic translation initiation factor 1A

2.62

3.77

1.59

2.77

52

Kdelr2

ER lumen protein retaining receptor 2

2.61

1.93

1.03

1.10

53

Khdrbs1

2.60

3.22

2.32

1.23

KH domain-containing, RNA-binding, signal transduction-associated protein 1 54

Hnrnpa2b1

Heterogeneous nuclear ribonucleoproteins A2/B1

2.60

3.04

3.83

3.10

55

Rbfox2

RNA binding protein fox-1 homolog 2

2.57

2.80

1.27

1.72

56

Hp1bp3

Heterochromatin protein 1-binding protein 3

2.45

2.00

1.72

1.60

57

Hnrnpd

Heterogeneous nuclear ribonucleoprotein D0

2.43

3.30

1.47

1.85

58

Matr3

Matrin-3

2.40

2.27

1.96

2.78

59

Magoh

Protein mago nashi homolog

2.39

2.92

1.18

2.22

60

Dnmt3a

DNA (cytosine-5)-methyltransferase 3A

2.36

2.57

1.74

1.82

*61

Hnrnpk

Heterogeneous nuclear ribonucleoprotein K

2.35

2.72

1.59

1.80

62

Mecp2

Methyl-CpG-binding protein 2

2.33

3.15

1.15

1.26

46


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63

Nop58

Nucleolar protein 58

2.32

3.02

1.68

2.23

64

Serpinh1

Serpin H1

2.31

3.13

1.60

1.54

65

Puf60

Poly(U)-binding-splicing factor PUF60

2.30

2.57

1.48

1.50

66

Lmna

Prelamin-A/C

2.29

3.79

1.50

1.75

67

Smu1

WD40 repeat-containing protein SMU1

2.29

2.39

1.30

1.20

*68

Set

Protein SET

2.23

3.76

1.52

2.08

69

Top1

DNA topoisomerase 1

2.22

2.93

3.38

2.66

70

Zfr

Zinc finger RNA-binding protein

2.19

3.32

1.34

2.50

*71

Hnrnpa3


2.14

2.30

1.28

1.49

72

Anp32a

2.13

1.23

2.46

1.90

Acidic leucine-rich nuclear phosphoprotein 32 family member A 73

Rplp0


2.08

2.38

1.04

1.22

74

Ptbp2

Polypyrimidine tract-binding protein 2

2.05

2.71

1.23

1.19

75

Cdc5l

Cell division cycle 5-like protein

2.03

2.65

1.21

1.99

76

Adcyap1

Pituitary adenylate cyclase-activating polypeptide

2.00

1.72

4.25

3.53

77

Lmnb1

Lamin-B1

1.97

2.84

1.50

2.17

*78

Hnrnpa1


1.96

2.41

1.33

1.37

79

Tm9sf2

Transmembrane 9 superfamily member 2

1.94

1.09

1.22

1.56

80

Ylpm1

YLP motif-containing protein 1

1.86

3.77

1.20

1.55

*81

Arf5

ADP-ribosylation factor 5

1.74

1.13

3.67

3.46

82

Clstn1

Calsyntenin-1

1.71

3.37

5.89

6.24

83

Eef1a2

Elongation factor 1-alpha 2

1.67

1.00

4.24

2.87

84

Prpf19

Pre-mRNA-processing factor 19

1.67

1.96

1.08

1.19

85

Ik

Protein Red

1.67

2.73

1.09

1.34

86

Ddx21

Nucleolar RNA helicase 2

1.63

2.76

1.46

1.43

87

Snrnp200

U5 small nuclear ribonucleoprotein 200 kDa helicase

1.56

2.43

1.19

1.22

88

Safb

Scaffold attachment factor B1

1.52

2.62

1.14

1.30

89

Actc1

Actin, alpha cardiac muscle 1

1.45

1.94

3.45

5.54

90

Ugdh

UDP-glucose 6-dehydrogenase

1.45

10.64

9.11

9.86

91

Wbp11

WW domain-binding protein 11

1.40

2.51

1.06

1.31

92

Rtn1

Reticulon-1

1.40

1.46

1.17

1.42

93

Rpl7a

60S ribosomal protein L7a

1.39

1.56

1.93

1.97

94

Ubqln1

Ubiquilin-1

1.31

1.18

1.74

1.96

95

Tra2b

Transformer-2 protein homolog beta

1.28

1.43

1.61

1.50

96

Psmd11

26S proteasome non-ATPase regulatory subunit 11

1.25

1.51

1.59

1.73

97

Rnmt

mRNA cap guanine-N7 methyltransferase

1.25

1.36

1.34

1.39

98

Cirbp

Cold-inducible RNA-binding protein

1.23

3.07

2.32

2.45

47



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99

Dkc1

H/ACA ribonucleoprotein complex subunit 4

1.15

2.39

1.37

2.78

100

Ftl1

Ferritin light chain 1

1.15

3.28

5.62

4.11

101

Tagln2

Transgelin-2

1.09

12.99

30.82

1.52

102

Rab10

Ras-related protein Rab-10

1.05

1.21

1.28

1.26

103

Tceb2

Transcription elongation factor B polypeptide 2

1.04

1.82

2.33

1.62

*Proteins among the top 100 most abundant axonal proteins listed in Table 2.

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for TOC only

82.5x78.3mm (600 x 600 DPI)

49



FIGURES

Figure 1. Schematic outlining the approach to study the proteome of the rat cortical axons. Rat cortical neurons were cultured on the surface of glass chips containing a poly-L-lysinecoated micro-pattern on the surface. At DIV 15, the chip was cleaved along the grooves on the backside into fragments containing axons-occupied Region 2 and whole-cells-occupied Region 1. Proteins were extracted from these chip fragments, followed by reduction and alkylation, then digested exhaustively with trypsin. Resultant peptides were subjected to 2D-LC-MS/MS analysis and referred to as label-free experiments. The whole-cell and axon peptide samples were labeled with light and heavy formaldehyde, respectively, then mixed, and subjected to 2D-LC-MS/MS analysis, and referred to as dimethyl-labeling experiments. Alternatively, the whole-cell and axon peptides were labeled with heavy and light formaldehyde, then subjected to 2D-LC-MS/MS analysis, and referred to as swap experiments (indicated by broken lines). The mass spectrometry-based data of selected proteins were validated by immunofluorescence staining and Western blotting. 83x102.1mm (600 x 600 DPI)

50


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Figure 2. (A) Characterization of cellular structures occupying the different parts of the micropatterned glass chip and (B) biochemical characterization of whole-cell and axon samples. A. left panels: at DIV15, the cells on the chip surface were immunofluorescence stained with antibodies against SMI312 (an axon marker, green) and MAP2 (a dendritic marker, red) and then labeled by DAPI (labeling nuclei, blue). right panels: DIV 15 cells on chip surface were immunofluorescence stained with antibodies against SMI312 (green) and GFAP (an astroglial marker, red) and then labeled with DAPI (blue). Scale bars: 100 µm. B. top panel: SDS-PAGE analysis of axon (0.5 μg protein) and whole-cell (0.5 μg protein) samples prepared from DIV 15 neurons grew on micro-patterned glass chips. Axon and whole-cell control samples prepared from the same numbers of chips without seeded neurons, indicated as axon control and wholecell control, were also subjected to the same analysis. Molecular size markers are shown to the left. Bottom panels: Western blotting analysis of the axon, whole-cell, axon control, and wholecell control samples as those described in the top panel by using antibodies against BSA, CaMKIIα and GAP43. 170x84.2mm (600 x 600 DPI)

51



Figure 3. Functional annotation (Cellular Component) of the 2,548 axonal proteins identified in duplicate label-free experiments. Using DAVID informatics tool, these proteins are classified into 18 groups under the indicated GO terms with enrichment P values < 0.01. The 535 proteins that cannot be classified are grouped under “others”. The fractions of proteins under different GO terms relative to the total number of proteins are shown as blue bars (% Count). The fractions of the summed abundances of proteins under different GO terms relative to the summed abundances of all proteins are shown as orange bars (% Group abundance). The means of the abundances of individual proteins as measured from two label-free experiments were used in the calculation. The numbers in parentheses are the accession numbers of GO terms. The numbers in square brackets are the protein counts under GO terms. Enrichment P values are shown on the right. 167.2x134.6mm (300 x 300 DPI)

52


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Figure 4. Functional annotation (Cellular components) of the 103 axon-enriched proteins. These proteins are classified into 4 groups under the indicated GO terms with enrichment P values < 0.05. The 23 proteins that cannot be classified into subcellular components are grouped under the term of “others”. The fractions of proteins under different GO terms of the 103 proteins are shown as blue bars (% Count). The fractions of the summed abundances of proteins under different GO terms relative to the summed abundances of all proteins are shown as orange bars (% Group abundance). The means of the abundances of individual proteins as measured from two replicate dimethyl-labeling and swap experiments were used in the calculation. The numbers in parentheses are the accession numbers of GO terms. The numbers in square brackets are the protein counts under GO terms. Enrichment P values are shown on the right. 170.5x93.3mm (300 x 300 DPI)

53



Figure 5. Western blotting analyses of selected axonal proteins. (A) Western blotting analyses of E18 rat brain homogenates (containing 2 and 1 μg of protein, as indicated) with antibodies against histone subunits H2A.Z, H3.3 and H2B (right 3 strips). The same blots were also subjected to staining with different combinations of HRP-conjugated anti-rabbit, HRPconjugated anti-mouse IgG, pre-immune rabbit IgG, and pre-immune mouse IgG (left 4 strips, as indicated on top). Molecular size markers are shown to the left. Arrows indicate immunostained histone bands. (B) left panel: Western blotting analyses of axon and whole-cell samples (containing 1 µg protein) and axon control and whole-cell control samples (prepared from the same numbers of control chip fragments containing Regions 2 and 1 as those used for preparing axon and whole-cell samples containing 1 µg protein) with antibodies against CaMKIIα, GluR-2, pan-actin, βIII-tubulin and histone subunits H2A.Z, H3.3 and H2B. Right panel: the ratios between the intensities of immunostained bands in the axon and whole-cell samples in Western blotting (WB) analyses. Results are the mean ± S.D. from 4 blots. The A/W ratios of these proteins as determined by mass spectrometry (MS) are also included for comparison. 170x61.3mm (600 x 600 DPI)

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Figure 6. Verification of the presence of histones in rat cortical axons by immunofluorescence staining. (A) Double immunofluorescence staining of the axons of DIV 15 rat cortical neurons in areas a and b on the chip surface (the squares indicated by a and b in (B)) with antibodies against histones H2A.Z, H3.3 or H2B (top row images) in combination with the SMI312 antibodies or anti-βIII-tubulin antibodies (middle row images). (B) Localizations of areas a and b on the surface of the glass chip. (C) Double immunofluorescence staining of axonal growth cones of DIV 9 rat cortical neurons in area b on the chip surface with antibodies against histones H2A.Z (top row images), H3.3 (middle row images) or H2B (bottom row images) in combination with antibodies against βIII-tubulin (4th column in blue) and labeling with phalloidin which interacts with F-actin (3rd column in red). Images of the growth cones enclosed by the white squares in the images in the 1st and 5th columns are shown at a higher magnification in the 2nd and 6th column, respectively. (D) Immunofluorescence staining the axons of DIV 15 rat cortical neurons in area b (as indicated in (B)) with Cy5-conjugated goat anti-rabbit IgG and DyLight 488-conjugated goat anti-mouse IgG and labeling with phalloidin (left column). Immunofluorescence staining the axons of DIV 15 in area b with pre-immune rabbit and mouse IgGs as the primary antibodies and Cy5-conjugated goat anti-rabbit IgG and 55



DyLight 488-conjugated goat anti-mouse IgG as the secondary antibodies and labeling with phalloidin (right column). (E) Immunofluorescence staining the axonal growth cones of DIV 9 rat cortical neurons in area b (as indicated in (B)) with Cy5-conjugated goat anti-rabbit IgG and DyLight 488-conjugated goat anti-mouse IgG and labeling with phalloidin (left column). Immunofluorescence staining the axonal growth cones of DIV 9 in area b by using pre-immune rabbit and mouse IgGs as primary antibodies and Cy5-conjugated goat anti-rabbit IgG and DyLight 488-conjugated goat anti-mouse IgG as secondary antibodies and labeling with phalloidin (right column). Scales bars: 10 µm. 170x139.9mm (600 x 600 DPI)

56


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unbiased proteomic study of the axons of cultured rat cortical neurons

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